EPA/630/P-02/002F Decem ber 2002 Final Report



## A REVIEW OF THE REFERENCE DOSE AND REFERENCE CONCENTRATION PROCESSES

Prepared for the Risk Assessm ent Forum U.S. Environm ental Protection Agency W a shington, DC

Reference Dose/Reference Concentrat ion (RfD/RfC) Technical Panel

Bob Benson (OPRA/Region 8) Edward Ohanian (OST/OW ) Gary Foure m an (NCEA/ORD) Jennifer Or m e -Zavaleta (NHEERL/ORD) Lee Hof m ann ( PARMS /OS W E R) Deborah Rice (NCEA/ORD) Carole Ki m m e l (NCEA/ORD)* Jennifer Seed (OPPT/OPPTS) Gary Kim m e l ( NCEA/ORD) Hugh Tilson (NHEERL/ORD) Susan Makris (OPP/OPPTS) Vanessa Vu (SAB St aff Offi ce, formerl y Deirdre Murphy (OAQPS/OAR) OSCP/OPPTS and NCEA/ORD)

*Technical Panel Chair

Technical Advisors

Am y Mills, IRIS Director, NCEA/ORD Bill W ood, RAF Director, NCEA/ORD

Risk Assessm ent Forum U.S. Environm ental Protection Agency W a shington, DC 20460DISCLAIMER

This docum ent has been reviewed in accordance with U.S. Environm ental Protection Agency policy and approved for publication. Mention of trade nam e s or com m e rcial products
does not constitute endorsem e nt or recom m e ndation for use.

iiCONTENTS

L I S T O F T A B L E S

............................................................ v i L I S T O F F I G U R E S

.......................................................... v i i LIST OF ABBREVIATIONS AND ACRONYMS

.................................. v i i i P R E F A C E ...................................................................x E X E C U T I V E S U M M A R Y

.................................................... x i i i

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 R E F E R E N C E V A L U E S

............................................... 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 Program s (OPP) Procedures for Setting A c u t e a n d I n t e r m e d i a t e R f D s

..................................... 2

- 5 2.1.4. Office of W a ter (OW ) Health Advisories (HAs)

...................... 2

- 6 2.1.5. Agency for Toxic Substances and Disease Registry (ATSDR) M i n i m a l R i s k L e v e l s ( M R L s )

..................................... 2

- 9 2.2. SUMMARY OF CURRENT METHODS FOR SETTING ACUTE, SHORT-TERM, AND LONGER-TERM REFERENCE VALUES

............. 2

- 9 2.3. RECOMMENDATION

.............................................. 2

- 1 1

3. REVIEW OF TESTING GUIDELINES W I TH RESPECT TO LIFE S T A G E A S S E S S M E N T

................................................... 3

- 1 3.1. EVALUATION OF CURRENT GUIDELINE T E S T I N G P R O T O C O L S

.............................................. 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

- 1 0 3.1.2.1. Overview of Tests

..................................... 3

- 1 0 3.1.2.2. Gaps in Life Stage of Assessm ent

......................... 3

- 1 3 3.1.2.3. Gaps in Assessm ent Endpoints

........................... 3

- 1 4 3.1.2.4. Gaps in Duration/Latency Assessm ent

..................... 3

- 1 4 3.1.3. Exposures and Endpoints Related to Evaluation of Neurotoxicit y........ 3

- 1 5 3.1.3.1. Overview of Tests

..................................... 3

- 1 5

iiiCONTENTS (continued)

3.1.3.2. Gaps in Life Stage of Assessm ent

......................... 3

- 1 7 3.1.3.3. Gaps in Assessm ent Endpoints

........................... 3

- 1 8 3.1.3.4. Gaps in Duration/Latency Assessm ent

..................... 3

- 1 8 3.1.4. Exposures and Endpoints Related to Evaluation of Im m unotoxicity

............................................ 3

- 1 9 3.1.4.1. Overview of Tests

..................................... 3

- 1 9 3.1.4.2. Gaps in Life Stage of Assessm ent

......................... 3

- 2 2 3.1.4.3. Gaps in Assessm ent Endpoints

........................... 3

- 2 2 3.1.4.4. Gaps in Duration/Latency Assessm ent

..................... 3

- 2 3 3.1.5. Exposures and Endpoints Related to Eval uation of Cardiovascular Toxicity 3-23 3.1.5.1. Overview of Tests

..................................... 3

- 2 3 3.1.5.2. Gaps in Life Stage of Assessm ent

......................... 3

- 2 3 3.1.5.3. Gaps in Assessm ent Endpoints

........................... 3

- 2 5 3.2. CONCLUSIONS AND RECOMMENDATIONS

.......................... 3

- 2 6 3.2.1. Conclusions

.................................................. 3

- 2 7 3.2.2. Recom m e ndations

............................................. 3

- 2 9 3.3. OPTIONS FOR ALTERNATIVE TESTING APPROACHES

................ 3

- 3 1 3.3.1. Alternative Acute Toxicity Testing Protocol

........................ 3

- 3 1 3.3.2. Alternative Chronic Toxicity Testing Protocols

...................... 3

- 3 3 3.3.2.1. The Expanded Chronic/Carcinogenicity Study

............... 3

- 3 3 3.3.2.2. The Unified Screening Study

............................. 3

- 3 5

4. FRAMEW ORK 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 f o r R i s k A s s e s s m e n t

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

- 1 1 4.3.2.1. The W e ight-of-Evidence Approach

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

- 1 1 4.3.2.2. Use of Hum a n and Anim al Data in Risk Assessm ent

.......... 4

- 1 2 4.3.2.3. Characterization of Effects in Potentially Susceptible Subpopulations

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

- 1 5 4.3.3. Characterization of the Extent of the Database

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

- 1 7 4.3.3.1. Extent of the Database

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

- 2 0 4.4. DERIVATION OF REFERENCE VALUES

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

- 2 1

ivCONTENTS (continued)

4.4.1. Sam p le Reference Values and Selection of Endpoints to U s e a s t he P O D f or R e f e r e nc e V a l ue s

............................. 4- 22 4.4.2. Dose Adjustm e nt for Duration of Exposure

......................... 4- 24 4.4.2.1. Duration Adjustm e nt Procedures for Inhalation Exposures to Continuous-Exposure Scenarios

............... 4- 24 4.4.2.2. Duration Adjustm e nt for Inhalation Developm ental T oxi c i t y S t udi e s —A C ur r e nt E xc e pt i on

.................... 4- 25 4.4.2.3. Duration Adjust m e nt for Acute Reference Values—Discontinuous S c e na r i os of 24 H our s or L e s s

............................ 4- 26 4.4.3. Derivation of a HEC or a HED

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

- 2 9 4.4.3.1. PBPK Models and Derivation of HEDs

a nd H E C s : E s t i m a t i ng I nt e r na l D os e

....................... 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 Derm al Exposure—Use of BW 3/4 a s a C r os s

- S pe c i e s D A F

......................... 4- 35 4.4.4. Other Issues

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

- 3 7 4.4.5. Application of Uncertainty/Variability Factors

...................... 4- 38 4.4.5.1. Recom m e ndations 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. Chem ical-Specific Adjustm e nt Factors (CSAFs)

............. 4- 47 4.4.6.2. Probabilistic Approaches

................................ 4- 48 4.4.7. Sum m a ry of Key Points from the Case Studies

...................... 4- 49

5. RECOMMENDATIONS

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

A P P E N D I X A : I S S U E S R A I S E D B Y T H E 10X T A S K F O R C E

................... A

- 1

APPENDIX B: CASE STUDIES—EVALUATING AND SELECTING HEALTH E N D P O I N T S F O R D E R I V I N G R E F E R E N C E V A L U E S .............B

- 1

G L O S S A R Y

.............................................................. G

- 1

R E F E R E N C E S .............................................................R

- 1

vLIST OF TABLES

T a bl e 2- 1. D ur a t i on de f i ni t i ons us e d f or va r i ous r e f e r e nc e va l ue s

.................. 2- 10

T a bl e 2- 2. U nc e r t a i nt y/ s a f e t y f a c t or s f or va r i ous r e f e r e nc e va l ue s

................. 2- 12

T a bl e 3- 1. A ppr oxi m a t e a ge a t e qui va l e nt l i f e s t a ge s i n s e ve r a l s pe c i e s

.............. 3- 3

Table 3-2. System s/endpoints evaluated by routin e toxicity guideline testing protocols

. . 3 -5

Table 4-1. Factors for evaluation of the weight of evidence regarding the likelihood of e f f e c t s i n hu m a ns

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

- 1 4

Table 4-2. Factors for evaluating of evidence regarding identification and characterization of susceptible subpopulations

..................... 4- 17

Table 4-3. DAFs based on BW 3/4 f or va r i ous s pe c i e s

............................ 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

viLIST 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 im m unotoxicity evaluations

............... 3- 20

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

........... 3- 24

F i gur e 3- 6. A l t e r na t i ve a c ut e t oxi c i t y pr ot oc ol

................................. 3- 32

Figure 3-7. Expanded chronic/carcinogenicity study

............................. 3- 34

F i gur e 3- 8. U ni f i e d s c r e e ni ng s t udy

.......................................... 3- 36

Figure 4-1. Concentration-by-duration plot showing the effect of the exponent in the C n x T = k on e xt r a pol a t i on a c r os s t i m e

. ........................ 4- 27

Figure 4-2. Current and proposed generalized procedures for deriving HECs or HEDs from ani m al exposures

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

- 3 1

viiLIST OF ABBREVIATIONS AND ACRONYMS

ACE II Angiotensin converting enzym e II ADI Acceptable Daily Intake AEGL Acute exposure guideline level ARE A cute reference exposure ATSDR A gency for Toxic Substances and Disease Registry AUC Area under the curve BMC B enchm a rk concentration BMCL Benchm ark concentration lower confidence lim it BMD B enchm a rk dose BMDL Benchm ark dose lower confidence level BMR B enchm a rk response CatReg Categorical Regression (software) CFSAN C enter for Food Safety and Nutrition CNS C entral nervous system CSAF Chem ical-specific adjustm e nt factor DAF Dosim e tric adjustm e nt f actor DNT Developm ental neurotoxicity ECE-1 E ndothelin-converting enzym e -1 ELISA Enzym e -linked im m unosorbent assay FDA F ood and Drug Adm i nistration FQPA Food Quality Protection Act FIFRA F ederal Insecticide, Fungicide, and Rodenticide Act GD Gestational day GLP G ood Laboratory Practices HA Health Advisory HEC H um an equivalent concentration HED H um an equivalent dose IPCS International Program m e on Chem ical Safety IRIS Integrated Risk Inform ation System LOAEL L owest-observed-adverse-effect-level MF Modifying factor MOE M argin of exposure MRL M inim al risk level NAAQS National Am bient Air Quality Standards NCEA National Center for Environm ental Assessm ent NK Natural Killer NOAEL N o-observed-adverse-effect-level OAR Office of Air and Radiation OECD Organisation for Econom ic Cooperation and Developm ent OPP O ffice of Pesticide Program s

viiiLIST OF ABBREVIATIONS AND ACRONYMS (continued)

OPPTS O ffice of Prevention, Pesticides, and Toxic Substances OSW E R O ffice of Solid W a ste and Em ergency Response OW Office of W a ter P P arental PAD P opulation adjusted dose PBPK Physiologically-based pharm acokinetic m odel PFC Plaque-form i ng cell PND P ostnatal day POD P oint of departure PRA P lasm a renin activity RDDR Regional deposited dose ratio RGDR Regional gas dose ratio RfC R eference concentration RfD R eference dose SAB S cience Advisory Board SPF Specific pathogen free SRBC Sheep Red Blood Cells TSCA Toxic Substance Control Act TW A T im e-weighted average UF Uncertainty f actor

ixPREFACE

The U.S. Environm ental Protection Agency (EPA) Risk Assessm ent Forum was established to prom ote scientific consensus on risk assessm ent issues and to ensure that this consensus is incorporated into appropriate risk assessm ent guidance. To accom p lish this, the
Forum assem b les experts throughout EPA in a form al process to study and report on these issues
f r om an Agency-wide perspective. For m a jor risk assessm ent activities, the Forum has established Technical Panels to conduct scientific reviews and analyses. Mem b ers are chosen to ensure that necessary technical expertise is available. The RfD/RfC Technical Panel (hereafter the Technical Panel) was established by the
Risk Assessm ent Forum in early 1999 in response to a request from the Agency’s 10X Task
Force 1 to the Science Policy Council and the Forum

. In the process of developing a strategy for
im plem enting 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 requirem e nts (U.S. EPA, 1999c) that were used by the Office of Pesticide Program s to develop a policy docum ent for im plem entation of the FQPA safety factor (U.S. EPA, 2002b). The draft 10X toxicology report (U.S. EPA, 1999b) raised a num ber of issues that relate to the derivation of the oral reference dose (RfD) and inhalation reference concentration (RfC).
Exam ples of these issues include the following. (1) Appropriate application of a database uncertainty factor (UF) or m odifying factor fo r studies that are considered necessary but are absent or judged inadequate that m a y show children to be significantly m o re sensitive or susceptible than adults. Addressing this issue also im plicates aspects of other UFs that relate to children’s health, including the factor for inter-individual variability in hum ans (e.g., response of the aged vs. response of the younger adult or child), and the interspecies UF (e.g., young anim als vs. young hum ans). (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 hum ans within the context of dose, r oute, duration, and tim ing of exposure. (3) The

1 The 10X Task Force was created by the EPA Adm i nistrator to explore the adequacy of current testing approaches for pesticides for protecting children’s health and to recom m e nd approaches for im plem enting the additional 10X safety factor m a ndated by the 1996 Food Quality Protection Act.

xuse of developm ental 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 developm ental 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 benchm ark dose from developm ental toxicity data with inhalation exposures using a concentration-tim es-tim e (C x t) adjustm e nt, 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 recom m e nded by the 10X Task Force, when such protocols should be required, and how the data should be interpreted for risk assessm ent

purposes. These additional protocols include (1) collection of toxicokinetic data, both in adults and at different developm ental 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) developm ental im m unotoxicity testing and appropriate triggers; (5) advanced developm ental neurotoxicity testing, in particular, cognitive testing that is m o re sim ilar
to that used in hum ans; and (6) exposure assessm ents that are m o re com p atible with the doseresponse assessm ent. (See Appendix A for m o re a detailed discussion of the issues raised by the 10X Task Force.) The Science Policy Council and the Risk Assessm ent Forum agreed that these issues
should be exam ined—with input from various program offices within the Agency and from the outside scientific/policy com m unity—on a broader scale than just for pesticides. This charge was expanded by the Forum to include a m o re in-depth review of a num ber of issues related to the RfD/RfC process, in part because of several other Forum activities that were underway. These activities included developm ent 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

num ber of years, and several scientific issues concerning children’s health, for exam ple, neurotoxicity and im m unotoxicity, have becom e increasingly im portant in risk assessm ent. These various but related activities have prom pted the need to re-exam i ne the Rf D/Rf C process
and to coordinate these ef f o rts with other related activities. In particular, it was im portant that
efforts continue to focus on m oving toward the goal of harm onization of risk assessm ent

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), m i nim a l risk level (MRL), or other sim ilar values.

xiapproaches for all health endpoints. This docum ent represents the review and deliberations of the RfD and RfC processes by the Risk Assessm ent Forum Technical Panel.

xiiEXECUTIVE SUMMARY

This docum ent sum m a rizes the review and deliberations of the Risk Assessm ent Forum ’ s RfD/RfC Technical Panel and its recom m e ndati ons for im provem e nts in oral reference dose/inhalation reference concentration (RfD/RfC) process as well as additional efforts that are
needed. It discusses revisions to the fram ework for the derivation of reference values. The
docum ent is a review, not guidance, but it does m a ke recom m e ndations that should be
considered in the im plem entation of changes in the current process and/or developm ent of needed guidance.
The Technical Panel reviewed m o st of the issues relating to hazard characterization for developing reference values and the need for de veloping 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 inform ation becom e s available and new scientific and risk assessm ent approaches are developed. This does not m ean th at 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 docum ent reviews and discusses a num ber of issues and provides conclusions and

recom m e ndations that are intended to im prove the RfD/RfC process. The Technical Panel has provided specific recom m e ndations for the developm ent of guidance in som e cases and m o re general conclusions and recom m e ndations in others. In the latter cases, the Technical Panel felt that developm ent of specific recom m e ndations was beyond the scope of its efforts or that policies needed to be f u rther developed bef o re specif i c guidance could be written to im plem ent the recom m e ndations. The docum ent 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-lifetim e values from briefings to the Technical Panel and a colloquium held August 2, 2000, and includes discussions of the proposed Acute Reference Exposure (ARE) m e thodology for acute inhalation exposures, the Acute Exposure Guideline Level (AEGL) Program , the Office of Pesticide Program s’ procedures for setting acute and longer-term duration RfDs, the Office of W a ter’s Health Advisories, and the Agency for Toxic Substances and Disease Registry’s Minim a l Risk Levels.

xiiiOn 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 recom m e ndation of the 10X Task Force that such values should be set, where possible, and that they should be incorporated into the Integrated Risk Inform ation System (IRIS) database. In addition, the
Technical Panel recom m e nded that this process be done in a consistent m a nner 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 fram ework for deriving these additional values is presented in Chapter 4. Chapter 3 reviews the current Office of Prevention, Pesticides and Toxic Substances’
harm onized health effects testing guidelines for the purpose of determ ining 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 chem ical in order to fill in the inform ation gaps identified for those organ system s evaluated. Nor is it suggested that alternative testing protocols that are discussed in this chapter should be conducted for every chem ical or becom e part of current toxicology testing requirem e nts or that these alternative protocols are the only options available. Rather, it is the goal of this docum ent to provide a basis for the developm ent of innovative alternative testing approaches and the use of such data in risk assessm ent, and to
then illustrate som e aspects of this concept with a f e w exam ples. In reviewing the current testing protocols, target organs/system s that are evaluated were reviewed as was the thoroughness of testing with respect to life stage assessm ent, endpoint assessm ent, route, tim ing and duration of exposure, and latency to response. These issues were all considered im portant in evaluating potentially susceptible subpopulations, including life stages. The testing guideline protocols were reviewed overall for these issues; in addition, four biological system s were evaluated in depth, two that are fairly thoroughly evaluated (the reproductive and nervous system s) and two
that are evaluated to a m o re lim ited extent (the im m une and cardiovascular system s). In each case, an overview of the tests for the particular system is given, as well as a m o re specific discussion of gaps in life stage of assessm ent, gaps in assessm ent endpoints, and gaps in duration and latency assessm ent. The Technical Panel has m a de a num ber of recom m e ndations concerning toxicity testing, including developm ent of a strategy for approaches to toxicity testing, with guidance on how and when to use existing and newly recom m e nded guidelines; developm ent of guidelines or guideline study protocols that will provide m o re system atic inform ation on toxicokinetics and toxicodynam i cs (i.e., m echanism or m ode of action), including at different life stages; developm ent of protocols for acute and short-term studies that provide m o re com p rehensive data

xivfor setting reference values; m odification of existing guideline study protocols to provide m o re

com p rehensive coverage of life stages for both exposure and outcom e s; collection of m o re inform ation from less-than-lifetim e exposure to evaluate latency to effect and reversibility of effect; developm ent of guidelines or guideline study protocols to assess im m unotoxicity, carcinogenicity, and cardiovascular toxicity at different life stages; and exploration of the
f easibility of setting derm al ref e rence values f o r direct toxicity at the portal of entry, including
sensitization. A prim ary goal of this review was to provide the basis for recom m e ndations for the developm ent of a strategy for approaches to toxicity testing and for innovative alternative testing approaches to provide data for risk assessm ent. The Technical Panel is suggesting that alternative strategies and guidance for testing approaches be developed that incorporate
inform ation on toxicokinetics and m ode of action early in the process, thus allowing a m o re targeted testing approach. In addition, alternative protocols are discussed that are aim e d at m o re efficient use of anim als and resources in com b ined studies that would provide m o re extensive data on life stages, endpoints, and other factors not well characterized in current testing approaches. Recom m e ndations are also m a de about research areas that should be encouraged to aid in better study design and interpretation of data for risk assessm ent. Finally, an exam ple of an alternative testing protocol for acute exposure and evaluation that incorporates the types of endpoints and evaluations optim al for setting acute reference
values is discussed. Two sam p le alternative protocols are presented for chronic exposures and options are discussed for com b ining studies and evaluations to include a wider array of life stage and endpoint assessm ents. Chapter 4 discusses a num ber of m odifications to the existing fram ework 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 assessm ents of any type of health effect known or assum e d to be produced through a nonlinear and/or threshold m ode of action (which m a y include U-shaped
or other nonm onotonic dose-response curves as well as thresholds). Thus, the Technical Panel

recom m e nds m oving away from the dichotom y between “cancer” and “noncancer.” The term
“noncancer” has been rem oved from the reference value definition, denoting the m ove toward defining approaches for low-dose estim ation or extrapolation based on m ode of action. Two
case studies that illustrate m a ny of the concepts discussed in this chapter are presented in m o re detail in Appendix B. The Technical Panel recom m e nds including the acute, short-term , longerterm , and chronic reference values derived on the basis of the recom m e ndations in this report in

xvIRIS 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 lim itations f o r an individual
chem ical that m a y preclude developm ent of all four reference values, and it is aware that tim e and resources need to be considered when im plem enting these recom m e ndations. The IRIS

program has begun to im plem ent a pilot program to test whether developm ent of the expanded array of reference values is practical and can be accom p lished without unduly delaying the

com p letion of an IRIS f ile. As a part of the pilot, the IRIS program will need to identif y the

m e thods to be used in deriving these additional values.
The Technical Panel recom m e nds that endpoint-specific reference values should not be developed, including the reference dose for developm ental toxicity, RfD DT . Rather, a sam p le 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 type s 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 recom m e nded; 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 W o rking Group of the 10X Task Force have been incorporated into this approach.
In the context of this fram ework, the Technical Panel recom m e nds a som e what different approach to characterizing the extent of the database for reference values. Instead of specifying particular studies, this approach em phasizes the types of data needed (both in term s of hum an and anim al data) for deriving reference values, and it recom m e nds the use of a narrative description of the extent of the database rath er than a single confidence ranking of high, m e dium
, or low. To characterize the database, the Technical Panel has developed a description of a
“m inim al” 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 judgem e nt in the process of sum m a rizing the extent of the database, including its strengths and
lim itations. The narrative approach is intended to em phasize the types of data available (both hum an and anim al) as well as the data gaps that could im prove the derivation of reference values. This approach should encourage the use of a wider range of inform ation in deriving reference values,

xvitaking into consideration the issues of duration, tim ing, and route of exposure; the types and extent of endpoint assessm ents (i.e., structure a nd function); the life stages evaluated; and the potential f o r latent ef f ects and/or reversibility of ef f ects. Dosim e tric adjustm e nt of values for deriving a hum an equivalent concentration (HEC) for inhalation exposure is discussed, as is the derivation of a hum an equivalent dose (HED) for
oral or derm al exposure. The Technical Pane l recom m e nds that duration adjustm e nt procedures to continuous exposures based on concentration tim es tim e (C x t) be used as a default procedure for inhalation developm ental toxicity studies as for other health effects from inhalation

exposures. In addition, further evaluation of current dosim etric adjustm e nts for deriving HECs should be pursued to confirm or assess the relevance for population subgroups (particularly for children).
Because of the recom m e ndation for deriving several duration reference values, the
Technical Panel recom m e nds that the data for the point of departure (POD) be evaluated on the
basis of a com p arison of all relevant endpoints carried through the derivation of sam p le reference values, with selection of the lim iting value(s) as the final step rather than on the basis of selection of a single “critical study” and “critical eff ect.” To aid in this evaluation, the use of an exposure-response array is recom m e nded as a visual display of all relevant and appropriate endpoints and durations of exposure in order to determ ine the range of num erical values for each
reference value.
The Technical Panel m a kes a num ber of recom m e ndations concerning the application of uncertainty factors (UFs) for reference value derivation. In particular, it is im perative that the
IRIS docum entation contain a justification for th e individual factors selected for each chem ical

or assessm ent because rigid application of UFs could lead to an illogical set of reference values. Although default factors of 10 are recom m e nded, with 3 used in place of half-power values (i.e.,
10 0.5 ) when occurring singly, the exact value of the UF chosen should depend on the quality of the studies available, the extent of the data base, and scientific judgm ent. Sound scientific judgm ent 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 recom m e nds that if there is uncertainty in m o re 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 determ ine whether the derivation of a reference value is appropriate. In addition, the Technical Panel

recom m e nds lim iting the total UF applied to a chronic reference value for any particular

xviichem ical to 3000. This m a xim u m of 3000 applies only to the UFs and does not include the various adjustm e nt factors discussed in Chapter 4. The intraspecies UF is applied to account for variations in susceptibility within the
hum an population (interhum an 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 hum an population that are m o st sensitive to the health hazards of the chem ical 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
reaffirm s the im portance of this UF, recom m e nding that reduction of the intraspecies UF from adefault of 10 be considered only if data are sufficiently representative of the exposure/doseresponse data for the m o st susceptible subpopulation(s). At the other extrem e, a 10-fold factor

m a y som e tim es be too sm all because of factors that can influence large differences in susceptibility, such as genetic polym orphism s. The Technical Panel urges the developm ent of data to support the selection of the appropriate si ze 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 sim ilarities 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 developm ent 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 incom p lete characterization of the chem ical’s toxicity. In addition to the identification of toxicity inform ation that is lacking, review of existing data m a y also suggest
that a lower reference value m i ght 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 m a gnitude, the assessor should consider both the data lacking and the data available for particular organ system s as well as life stages. The Panel considers the purpose of
the m odifying factor (MF) to be sufficiently subsum ed in the general database UF, and

recom m e nds 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 assum e d to overlap to som e extent, the Technical Panel agrees with the 10X Task Force Toxicology W o rking Group that the current interspecies, intraspecies, and database deficiency UFs, if appropriately applied using the

xviiiapproaches recom m e nded in this review, will be adequate in m o st cases to cover concerns and uncertainties regarding the potential for pre- and postnatal toxicity and the com p leteness of the toxicology database. In other words, an additional uncertainty factor is not needed in the
RfC/RfD m e thodology 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 m a y be decreased or increased from the default value of 10). The approach to using chem ical-sp ecific data for toxicokinetic and toxicodynam i c

com ponents of the interspecies UF is part of the current RfC m e thodology. The Technical Panel encourages the Agency to develop its own guidance for chem ical-specific adjustm e nt factors (CSAFs) on the basis of som e of the available m e thodologies (e.g., the International Program m e on Chem ical Safety [IPCS]). Several other issues discussed by the Technical Panel were considered m o re appropriate for deliberation by other panels/com m ittees, for exam ple, further consideration of the use of
BMD m odeling approaches for deriving reference values; harm onization of the approaches for HEC and HED derivation for all types of health e ffects; further evaluation of approaches such as probabilistic analysis f o r characterizing variability and uncertainty in toxicity ref e rence values; further evaluation of appropriate adjustm e nt of doses for duration of exposure for acute toxicity data; and further evaluation of duration adjustm e nt for short-term and longer-term reference values analogous to the subchronic-to-chronic duration UF for chronic reference values. Chapter 5 sum m a rizes the recom m e ndations of the Technical Panel.

xix1. INTRODUCTION, PURPOSE, AND SCOPE

The RfD/RfC Technical Panel (hereafter the Technical Panel) was established by the
U.S. Environm ental Protection Agency’s (EPA’s, or the Agency’s) Risk Assessm ent 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 becom e m o re im portant and of greater concern in risk assessm ent; and to raise issues that should be explored or developed further for application in the RfD/RfC process. This docum ent sum m a rizes the
review and deliberations of the Technical Panel and its recom m e ndations for im provem e nts in the process as well as additional efforts that are needed. It discusses revisions to the fram ework
for the derivation of RfDs and RfCs. The docum ent is a review, not guidance, but it does m a ke

recom m e ndations that should be considered in the im plem entation of changes in the current process and/or developm ent of needed guidance. Many of the recom m e ndations m a de in this report are consistent with the Agency’s

com m itm ent to harm onization of health risk assessm ent procedures, including the harm onization of approaches for noncancer and cancer endpoints, and to m a king efficient use of anim al testing to achieve this goal. As noted in several places in the docum ent, 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 f o r revised or f u rther guidance on this issue. Although m i xtures or m u ltiple chem ical exposures are not specifically discussed in this review, m o st of the recom m e ndations are applicable to the approach to risk assessm ent of

m i xtures. The Agency’s m i xtures risk assessm ent guidelines should be consulted for issues specific to the evaluation of m i xtures (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 m u ltiple stressors and their overall im pacts on exposure-effect relationships. The risk assessm ent approaches discussed within this fram ework are likely to be the subject of further guidance as well.
The Technical Panel attem p ted to review m o st 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 pro cess 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 benchm ark dose [BMD] guidance docum ent

1-1and 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 inform ation becom e s available and
new scientific and risk assessm ent 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 som e cases, no change in the current value m a y result. This report provides conclusions and recom m e ndations that are intended to im prove the RfD/RfC process. The audience for this review is prim arily the Integrated Risk Inform ation System (IRIS) program , IRIS chem ical m a nagers, a nd other scientists within the Agency who are involved in developing the RfDs and RfCs, as we ll as IRIS users and the program offices within EPA that develop RfDs and RfCs or sim ilar values (see Chapter 2), particularly resource

m a nagers who m a y be im pacted by the potential for additional workload due to several of the

recom m e ndations. The Technical Panel has provided specific recom m e ndations for guidance in

som e cases and m o re general conclusions and recom m e ndations in others. In the latter cases, the Technical Panel felt that developm ent of specific recom m e ndations was beyond the scope of its
ef

f o rts or that policies needed to be f u rther developed bef o re specif i c guidance could be written
to im plem ent the recom m e ndations.
The m e thodology recom m e nded in the RfD docum ent 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 em phasis in this docum ent is on the calculation of RfDs and RfCs, the sam e processes and considerations are applicable to the

m a rgin of exposure (MOE), as discussed in th e draft cancer risk assessm ent guidelines (U.S. EPA, 1999a).
The Technical Panel discussed a num ber of issues concerning a revised fram ework for the RfD/RfC process, with particular em phasis on the extent to which children and other potentially susceptible subpopulations are considered. The next three chapters sum m a rize these issues, and several recom m e ndations are m a de. 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 th e current testing guidelines with respect to life stage assessm ent and discusses the gaps in life stage assessm ent, endpoint assessm ent, and

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

com b ining studies and evaluations are discussed.

1-2Chapter 4 provides constructive com m e ntary on the current fram ework used in deriving
ref e rence values and on the need and possibilities f o r calculating ref e rence values f o r dif f e rent 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
dosim etric adjustm e nt, the application of uncerta inty factors (UFs), and derivation of sam p le 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) sum m a rizes all of the recom m e ndations of the Technical
Panel. Two case studies that illustrate several of the recom m e nded changes are also included as Appendix B.

1-32. REVIEW OF THE CURRENT USE OF ACUTE, SHORT-TERM,
AND LONGER-TERM REFERENCE VALUES

The Technical Panel considered the recom m e ndation of the 10X Task Force that acute,
short-term , and longer-term reference values as well as chronic reference values should be set for
environm ental agents (see Appendix A). It is likely that the endpoints critical for setting acute,
short-term , and longer-term reference values m a y 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 m a ny types of effects could be derived are not often available. Data on
acute and short-term health effects m u st 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 chem ical types and

m e dia. For exam ple, acute and chronic oral RfDs are set for pesticides, with som e interm ediate 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 W a ste and Em ergency 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 em ergenc y response planning. The National Center for
Environm ental Assessm ent (NCEA) is currently developing the acute reference exposure (ARE)

m e thodology for acute inhalation exposures. These developm ents are reviewed in m o re 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

m e thods currently used to set various less-than-lifetim e reference values. Subsequently, on August 2, 2000, a Risk Assessm ent Forum colloquium was held on this topic (CDM Group, Inc., 2000). Each of the m e thods was presented and discussed. In addition, a recom m e ndation by the Technical Panel to begin deriving acute, short-term , and longer-term reference values as well as
chronic values and to standardize the definiti ons for each duration was presented and discussed.
Each m e thod presented is sum m a rized below.

2.1.1. Acute Reference Exposure (ARE) Methodology
The ARE m e thodology is being developed at the request of the EPA’s Office of Air and
Radiation. It is intended for developm ent of reference values for acute inhalation exposures of

2-124 hours or less. The criteria air pollutants are not included, because they are assessed within the
National Am bient 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 interm ittent 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 Am endm ents. The ARE m e thodology is
described in a 1998 EPA external review draft docum ent (U.S. EPA, 1998a). The m e thod builds on the procedures of the RfC m e thodology.
The ARE m e thodology includes three approaches in order to accom m odate the varying types of data available for acute exposure. The first two approaches, the no-observed-adverseef

f ect level (NOAEL) and the benchm ark concentration (BMC) are f a m iliar. The third approach, categorical regression (CatReg), is newer. Th e NOAEL approach is useful for chem icals that
have lim ited available data and for which no or lim ited dose-response relationships have been
established. The BMC approach is suitable for analysis of studies that establish dose-response relationships. The CatReg approach requires m u ltiple studies that report not only dose and response, but also duration; it is m o st applicable for data-rich chem icals. A feature of the CatReg approach is that effects data are grouped into severity categories (e.g., m ild or severe to lethal) to which sophisticated regression procedures are then applied. Adjustm e nts for deriving ARE values of di fferent durations (e.g., 15 m i nutes or 8 hours)

are m a de differently for the CatReg approach than for the NOAEL and BMC approaches. For any approach, the preferred adjustm e nt procedure is to use a pharm acokinetic m odel, if
available. W h en the NOAEL or BMD approach is used, the default procedure is to use the

m

u ltiple of concentration tim es tim e (C x t) (C n x t = k; ten Berge et al., 1986) to extrapolate
from short to long duration and to use the sam e concentration as obtained for long duration to extrapolate from long to short duration. W h en m o re than one duration is available, interpolation is perform e d. W h en 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 m o re f u lly and illustrated in Chapter 4.
A m i nim a l 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-e ffect 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 m a tter, ozone, carbon m onoxide, nitrogen oxides, sulfur dioxide, and lead.

2-2default 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 m e thodology
docum ent and m a de a num ber of com m e nts that addressed, am ong other things, issues about the NOAEL and BMC approaches, the need for addressing protection of children, the dosim etry adjustm e nt and duration extrapolation, and the CatReg approach. The SAB discussed the fact that the CatReg m odel, 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 lim its around the m a xim u m likelihood estim ate and about the appropriateness of the approach used to accom m odate group versus individual data. This

m e thodology has since (March 2001) undergone an Agency review by the Risk Assessm ent
Forum

. The principal com m e nts from this review concerned reevaluation of whether CatReg
should rem a in as an approach in the ARE m e thodology and further evaluation of the procedures for cross-species dosim etry adjustm e nt. Revision of the ARE m e thodology is currently underway. In addition to revising the ARE m e thodology and CatReg software docum ents, NCEA-Research Triangle Park will develop a f r am ework f o r adding AREs to the IRIS database.

2.1.2. Acute Exposure Guidelines (AEGL) Program The prim ary purpose of the AEGL programis to develop guideline levels for once-in-alifetim e short-term exposures to airborne concentrations of acutely toxic chem icals (NRC, 2000). AEGLs are needed for a wide variety of em ergency planning, response, and prevention applications. AEGLs represent threshold exposure lim its for the general public and are applicable to em ergency exposure periods ranging from 10 m i nutes to 8 hours. Specific values
are set for 10 m i nutes, 30 m i nutes, 1 hour, 4 hours, and 8 hours. It is believed that the

recom m e nded exposure levels are applicable to the general population, including infants and children and other individuals (e.g., asthm a tics) who m a y be sensitive or susceptible. It is recognized that certain individuals who m a y 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. W ith increasing air borne concentrations above each AEGL level there is a progressive increase in the likelihood of occurrence and the severity of effects described for
each level.

2-3AEGL-1 is the airborne concentration of a substance above which it is predicted that the general population, including susceptible indivi duals, could experience notable discom fort, irritation, or certain asym ptom atic, 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 im paired 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

m ild and progressively increasing odor, taste, and sensory irritation or certain asym ptom atic, nonsensory effects. UFs are used for extrapolations. If there are no appropriate hum an 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 m echanism of action, (3) the range of response in the species tested, (4) the variability of response am ong the species tested, and (5) pharm acokinetic differences am ong the species
tested. An intraspecies UF of 1, 3, or 10 is al so used. The factors considered when assigning a specific value include (1) the toxicological endpoint observed and the likely m e chanism of action, (2) the range of response am ong hum ans and subpopulations, and (3) pharm acokinetic
differences am ong individuals. Individual factors of 3 are often used to ensure that the final values are not overly conservative. Adjustm e nt for duration is conducted using the equation C n 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-m i nute value is usually assigned equal to the 30-m i nute 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 m e ntioned above, this procedure is further explained and illustrated in Chapter 4.

2-42.1.3. Office of Pesticide Programs (OPP) Procedures for Setting Acute and
Intermediate RfDs OPP developed m e thodologies for acute dietary as well as occupational and residential
risk assessm ents during the process of re-registration following the 1988 revision to the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). In 1998, a guidance docum ent, Toxicology Endpoint Selection Process (U.S. EPA, 1998b), was presented to the FIFRA Scientific Advisory Panel for review and com m e nt. This docum ent, which provided the basis for procedures that are still in place, describes toxicology endpoint selection for less-than-lifetim e dietary and occupational/residential risk assessm ents for pesticides. It includes guidance on evaluating toxicity studies that are relevant fo r use, selecting appropriate endpoints for hazard identification, the process of hazard identificati on, the influence of derm al absorption in hazard identification, the criteria for the use of the NOAEL and the LOAEL, and the use of MOEs in
risk assessm ents. Since this guidance was first issued, som e changes have evolved, such as the replacem ent of the acute MOE with the acute RfD and the addition of standard consideration of
short- and interm ediate-term incidental nondietary ingestion exposures for toddlers. Toxicology Endpoint Selection Process (U.S. EPA, 1998b) describes the types of studies
that are m o st likely to provide appropriate e ndpoints for the various exposure durations and risk

assessm ents that will be conducted for each pesticide. OPP can rely on the availability of a wide variety of standard guideline toxicity studies fr om which to select endpoints because such studies are required by regulation for any pesticide regi stration (40 CFR Part 158). Additionally, OPP considers other sources of toxicology data, such as studies published in the open literature, as appropriate.
For the establishm ent 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, th e prenatal developm ental toxicity study, and the developm ental neurotoxicity (DNT) study.
Acute effects from subchronic and chronic dietary studies are also used in the
establishm ent of the acute dietary RfD. Careful scrutiny of toxicological data from early in the
first week of treatm e nt can som e tim es identify e ffects that can be described as acute. However,
for a num ber of reasons, this option has not often been used. These reasons include the absence
of

detailed toxicological observations other than m o rbidity and m o rtality checks in subchronic
and chronic studies before the end of the first w eek of treatm e nt (i.e., after 7 days of treatm e nt), the nature of the dietary exposure (i.e., each daily exposure results from an extended period of nightly f eeding rather than f r om a discrete acute dose), and the possibility that apparent adverse

2-5ef f ects during the f i rst week of treatm e nt m a y be related to palatability issues as the anim als adjust to treated feed.
OPP does not calculate short- or interm ediate-term references doses. However, risk

assessm ents 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 hom e or other nonoccupational sources such as schools, parks, and golf courses. The post-application
risk assessm ent considers or accounts prim arily for incidental ingestion of (1) the dry pesticide

m a terials (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 norm a l m outhing activities, and (3) pesticide residues that are transf erred to the skin of toddlers playing in treated areas and are subsequently ingested as a result of hand-to-m outh
transfer. These risk assessm ents consider short-term (1 day to 1 m onth) and interm ediate-term (1–6 m onths) 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 chem ical-specific field data are unavailable) on assum p tions.
OPP also conducts short-term , interm ediate, and long-term (longer than 6 m onths) derm al
and inhalation risk assessm ents 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 assessm ents is that derm al absorption and inhalation toxicity data are often not available for food-use pesticides; in that case, appropriate assum p tions are applied, and the available oral toxicity data are converted for use in derm al and inhalation
risk assessm ent. Toxicology Endpoint Selection Process (U.S. EPA, 1998b) does not address the use of UFs in acute dietary risk assessm ent. In practice, however, the sam e 10-fold inter- and intraspecies UFs are used in calculating the acute dietary RfD as are used for the chronic RfD.
Other standard UFs m a y be used when a ppropriate (e.g., the LOAEL-to-NOAEL threefold factor). Others are not appropriate for an acute risk assessm ent, for exam ple, the threefold
subchronic-to-chronic factor. However, no standa rd set of “core” studies has been defined for acute dietary risk assessm ent; therefore, a database UF is not used. If appropriate endpoints and doses cannot be selected for acute dietary risk assessm ent 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

contam inants found in drinking water. Since th en, HAs have also been developed for regulated

2-6contam inants. HAs are derived for contam inants that are known to or are likely to occur in drinking water and that m a y cause adverse, noncarcinogenic health effects (Orm e and Ohanian, 1991). The approach for developing HAs is based on recom m e ndations from the National
Academ y of Sciences (NAS, 1977). HAs are deve loped for specific exposure durations (1 day, 10 days, longer-term
, and lifetim e) that reflect different em ergency contam ination situations. HAs are not legally enforceable, but they do serve as technical guidance to assist in em ergency spills or contam ination situations or for determ ining unreasonable risks to health under sections 1415 and 1416 of the Safe Drinking W a ter Act. They also are issued at the request of State or local governm e nts or to fill a need for criteria, guidelines, or standards. HAs undergo scientific peer review and can function as a prelim inary risk assessm ent, if necessary. The following assum p tions are used in setting the various HAs. The 1-day HA represents
a concentration of the contam inant 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-lifetim e HAs because a child of this size is likely to receive a greater dose on a

m g /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 fr om experim e ntal 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 m a y be based on experim e ntal studies of 30-day
duration or less. The longer-term HA, which is based on subc hronic exposure studies covering 10% of an
anim al’s lifetim e, is considered protective of an exposure period in hum ans of up to 7 years (i.e.,
10% of an individual’s lifetim e). The longer-ter m HA is developed to protect both a 10 kg child and a 70 kg adult.
The lifetim e HA is considered protective of lifetim e exposures and is usually based on
chronic or subchronic or other m o re relevant experim e ntal data. The Lifetim e 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 exam ple, 20% of the toxicant represented by
intake of water. HA levels are generally based on available, well-conducted studies that involve hum ans or anim als. 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 consum ption by the protected

2-7individual. For a child, the assum e d water consum ption level is 1 L/day; for an adult, 2 L/day is
used.
W h en data are absent for setting a 1-day or a 10-day HA, OW uses scientific judgm ent 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 m a y be used to develop a less-thanlifetim e HA. Given the pressure under which HAs need to be calculated, m a ny assessm ents are based on whatever toxicological data are available and on scientific judgm ent. Although this

m a y be an overly conservative approach, OW considers the error to be protective of public
health.
OW applies the sam e f actors f o r m i nim u m data as those outlined in the Agency’s Rf D

m e thodology. For exam ple, in em ergency situations, m i ssing data are accounted for by applying
another factor of 3 or 10. Or, for instance, wher e inhalation data m i ght be applied to estim ate a HA based on water consum ption, a factor m a y be applied to account for differences in absorption. Judgm ents based on toxicokinetic and toxicodynam i c considerations are reached through intensive consultation. Calculation of HAs is straightforward a nd fam iliar, and in m o st cases the NOAEL/UF approach is used. For each of the less-than-lifetim e HA values, it is assum e d that all of an individual’s exposure to a contam inant com e s from a drinking water source. The calculation of
the lif etim e HA dif f e rs f r om that of the less-than-lif etim e values in that a relative source
contribution factor is included. This factor ad justs the exposure to reflect the portion that is likely to be contributed from drinking water. Un less actual exposure data are available, a default
factor of 20% is used to reflect the assum e d c ontribution to exposure from drinking water. Also,
in cases where there is lim ited evidence suggesting a carcinogenic potential of a contam inant, an additional “policy” factor of 10 is applied in calculating the lifetim e HA.
The m e thodology 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 W a ter Hotline and the W a ter 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.

2-82.1.5. Agency for Toxic Substances and Diseas e 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 estim ates intended to be screening levels in identifying contam inants and potential health effects that m a y be of concern; they do not define clean-up or action levels. The derivation procedures for MRLs have m a ny sim ilarities and parallels to the derivation of Rf Ds and Rf Cs; MRLs are based on caref u l
scientific consideration of noncancer health eff ects only, not on consideration of cancer effects. A list of various procedural specifics em ploye d 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 rout es of concern for MRL derivation are oral and inhalation (but not derm al). The EPA procedures and m e thodologies 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 thr ee different duration categories: acute = < 14 days,
interm ediate = 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 contam inant 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

m odifying factor (MF) can include database consid erations, that is, deficiencies in the data or
overestim ates from bioaccum u lative chem icals.

2.2. SUMMARY OF CURRENT METHODS FOR SETTING ACUTE, SHORT-TERM,
AND LONGER-TERM REFERENCE VALUES
In sum m a ry, several m e thods are used by various EPA program s for setting acute, shortterm , and longer-term reference values. The definitions for each of the durations used for the

m e thods reviewed are included in Table 2-1. Because there are som e differences in these

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

Reference value duration D efinition

Acute

ARE I nhalation single continuous exposure values for durations

< 24 hrs (to be protective of interm ittent exposures)

AEGL 10 and 30 m i n; 1, 4, and 8 hrs

OPP acute RfD M axim um 1-day dietary exposure

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

ATSDR acute MRL < 14 days

Standardized def i nition a 24 hrs or less

Short-term

ARE N A

AEGL NA

OPP short-term RfD 1 day–1 m onth

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

ATSDR MRL N A

Standardized def i nition a >24 hrs up to 30 days
Longer-term

ARE N A

AEGL NA

OPP interm ediate RfD 1–6 m onths

OW longer-term HA Approxim a tely 10% of life span in hum ans (90 days to 1 year in test species)

ATSDR interm ediate MRL 15–364 days

Standardized def i nition a >30 days up to approxim a tely 10% of the life span in hum ans (>30–90 days in typically used laboratory species)

a See Chapter 4 for further discussion of these definitions.

2-10definitions, standardized definitions were di scussed at the Risk Assessm ent Forum Colloquium (CDM Group, 2000), and these are shown in Table 2-1. Definitions for durations are further discussed in Chapter 4.
A com p arison of the UFs applied for various reference values is shown in Table 2-2. Although there is som e variation in the UFs applied, those for anim al-to-hum an extrapolation (U A ), f o r within-hum an variability (U H ), and for LOAEL-to-NOAEL (U L ) are f a irly consistent. Less consistent is the way in which database deficiencies (U D ) 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. Som e type of duration adjustm e nt of the NOAEL or the BMD is done for the ARE and the AEGL m e thods, and there appears to be consistency in the use of C n x t for extrapolating from shorter to longer exposures but in using the sam e value (i.e., no duration adjustm e nt) 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 recom m e ndation 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

recom m e nded that these values be set in a consistent m a nner, 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 schem e for deriving these additional values is presented in Chapter 4.

2-11Table 2-2. Uncertainty/safety factors for various reference values

Reference value UF a

U A U H U L U D FQPA b

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

AEGL 1, 3, 10 1, 3, 10 3 c ND d NA

OPP acute and interm ediate Rf Ds 10 10 3, 10 ND e 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 ND d NA

a Uncertainty factors: U A = anim al-to-hum an; U H = within-hum an variability; U L = LOAEL-to-NOAEL; U D = database deficiency.

b Additional safety factor required under FQPA.

c Endpoint = lethality, not really a L OAEL-to-NOAEL adjustm e nt in this case.

d Database deficiencies considered, and a factor m a y be included for interm ediate RfDs if, for

exam ple, 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

2-123. REVIEW OF TESTING GUIDELINES WITH RESPECT TO
LIFE STAGE ASSESSMENT

As a first step in determ ining 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 guidelines 2 to determ ine what inf o rm ation is gathered in these studies. The intent of this review is not to suggest that additional testing be conducted for each and every chem ical in order to fill in the inform ation gaps identified for those organ system s evaluated. Nor is it suggested that the alternative testing protocols that are discussed in this chapter should be conducted for every chem ical or becom e part of current toxicology testing requirem e nts or that these alternative protocols are the only options available. Rather, it is the goal of this docum ent to provide a basis for the developm ent of innovative alternative testing approaches and the use of such data in risk assessm ent and to then illustrate

som e aspects of this concept with a few exam ples. Developm ent of a toxicology testing paradigm that is based not on rigid conform a nce to a list of required guideline screening studies but rather on the application of knowledge about the
chem ical is encouraged. Under such a paradigm , both the selection of studies that would be required as well as the design of the tests them selves could be influenced by other substantive
and reliable inform ation about the chem ical. For exam ple, the incorporation of toxicokinetic and

m ode-of-action data early in the developm ent of the testing strategy for a chem ical would provide particularly valuable direction for developm ent 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

exam ine age-related sensitivity or susceptibility to chem ical exposure, and inform ation on potential or actual exposure to hum ans. These data could be used to inform a m o re targeted approach in the design of individual studies or of an overall testing strategy and m i ght in som e cases result in a reduction in the num ber of anim als 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 system s are evaluated in current testing protocols and how thorough the testing protocols are with respect to life stage assessm ent; endpoint assessm ent; route, tim ing, and

2 The guidelines are available on the OPPTS web page (http://www.epa.gov/docs/ OPPTS_Harm onized/870_Health_Effects_Test _Guidelines/Series/).

3-1duration of exposure; reversibility; and latency to response. These issues were all considered of
im portance 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 system s, provide a m o re in-depth analysis as to whether and how current protocols address these issues. The organs/functional system s that were exam ined in greater detail included the reproductive and the nervous system s, which were selected to represent system s that are thought to be rather well-evaluated. The im m une and the cardiovascular system s were selected for review because the current evaluation of these system s is lim ited. 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 num ber of gaps in life stage assessm ent, endpoint assessm ent, tim ing and duration of exposure, reversibility, and latency to response were noted for each organ system that is reviewed in depth. The last section provides recom m e ndations for alternative testing approaches that are designed to m a ke m o re efficient use of anim als and resources in com b ined studies that would provide m o re 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 sum m a rize the exposures and endpoints covered in current testing guidelines, what is covered for each organ system /endpoint m easured, and the relative depth of evaluation for each system /endpoint. In addition, the life stages covered by exposures and outcom e s are illustrated. The discussions that correspond to the figures give an
overview of the tests that are currently availa ble and the gaps in assessm ent of life stages, endpoints, tim ing and duration of exposure, and latency to response. Together, these analyses provide a clear picture of the testing guidelines currently available, the system s/endpoints

m easured, the life stages during which exposures and outcom e s are m easured, the tim ing and duration of exposures included, and the degree of detail covered for both structural and
functional outcom e s. In order to m a ke com p arisons am ong laboratory anim al species and hum ans in term s of life stages covered, the approxim a te 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 fe w cases, no data could be found on appropriate ages corresponding to particular life stages. In particular, the ages for m a ture adults and older adults often were not available, and there is som e cont roversy about what constitutes old age in today’s

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

3-3RatMouseRabbitBeagle dogHumanLife stageAgeLife stageAgeLife stageAgeLife stageAgeLife stageAgeEmbryonicGD 0–16EmbryonicGD 0–15EmbryonicGD 0–19EmbryonicGD 0–30?EmbryonicGD 0–58FetalaGD 16–22(22–23 days)FetalGD 15–20(18–22 days)FetalGD 19–32(30–32 days)FetalGD 30–63(53–71 days)FetalGD 58–267NeonatebPND 0–14NeonatePND 0–14NeonatePND 0–21?NeonatePND 0–21NeonatePND 0–30WeaningcPND 21WeaningPND 21(19–28)WeaningPND 42(42–56)WeaningPND 42InfancyPND 30–1 yrToddler2–3 yrsYoungPND 22–35YoungPND 21–35YoungPND 42–?Young1.5–5 mosPreschool3–6 yrsElementaryschool age6–12 yrsPubertyPND 35–60PubertyPND 35–?Puberty3–8 mosPuberty5–7 mosAdolescence12–21 yrsSexualmaturity2.5–3 mosBreedingage1.5–2 mosBreedingage6–9 mosBreedingage12 mosYoung adult21–40 yrsMatureadult5–18 mosMatureadultMatureadultMatureadultMature adult40–65 yrs?Old adult18 mos–2yrs+Old adultOld adultOld adult-15 yrsOld adult>65 yrs?aRange of gestation length in parentheses.bSomeneonatal events in rodents occur in utero in humans.cRange of weaning ages in parentheses.GD = gestation dayPND = postnatal daypopulation. A background paper on aging com m i ssioned as part of this review discusses this
issue to som e extent (Versar Inc., 2001a). In anim al studies, the use of dietary restriction has
been shown to af f ect aging and lif e span to a signif i cant extent, so the issue of what constitutes
an older anim al is also som e what controversial.

3.1.1. Exposures and Endpoints Related to General Toxicity Testing Table 3-2 provides an overview of the biol ogical system s 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 m o st of the test designs that focus on specific biological functions. The acute and subchronic studies are intended to give general inform ation on the potential toxicity of an agent by screening the m a jor organ system s, in particular, the liver, the kidney, and the gastrointestinal tract. This inform ation can be used to
determ ine where to look in m o re detail at specific organ system structure and function. The chronic studies, which are usually done in com b ination with a carcinogenicity study, evaluate general toxicity in all m a jor organ system s. Several testing guidelines have been developed with the idea that certain system s should be evaluated frequently in m o re 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 developm ental toxicity studies). More detailed inform ation about specific aspects of guideline test designs for certain system s (e.g., life stages covered, exposure periods, outcom e s m easured, etc.) is included in the figures.
Table 3-2 is shaded and m a rked to indicate the extent of the evaluation of a particular system /endpoint within a particular test design. indicates that the system /endpoint is a prim ary focus of the particular test design and that detailed assessm ent of the dose-response relationship of an exposure is carried out within som e defined life stage and exposure period for

m a jor elem ents of the system /endpoint. indicates those system s/endpoints for which

som e histopathology or clinical m easure of system f

unction is carried out. X indicates those system s/endpoints that are assessed in som e observational or gross m a nner. “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 syst em s/endpoints are exam ined in any significant
detail. The system s/endpoints under the acute test designs are for the m o st part observational in

nature. The acute inhalation toxicity w ith histopathology guideline (40 CFR 799.9135) was developed under the Toxic Substances Control Act for characterizing the exposure-response

3-43-5Table 3-2.by routine toxicity guideline testing protocolsaGuidelinebSystemsOtherendpointsLungrespiratoryCardio-vascularHema-tologicMusculo-skeletalSkinEyeGastro-intest-inalKidney-urinaryLiverImmuno-logicalRepro-ductiveNeuro-logicalEndocrino-logicalPharmaco-kinetic-metabolicMutagenicCancerImmediatedeathShortlifespanAcute, oralXXXXXXXX0XX0Acute, inhalationXXXXXXXXXX0XX0Acute, dermalXXXXXXXXXX0XX0Subchronic, oralXXXXXXXXXXXXXXXXXXXXXXXXX0XXXSubchronic,inhalationXXXXXXXXXXXXXXXXXXXXXXXXX0XXXSubchronic,dermalXXXXXXXXXXXXXXXXXXXXXXXXXX0XXX21-day,dermalXXXXXXXXXXXXXXXXXXXXXXXXXX0XX0Chronic,oralXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXChronic,inhalationXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXChronic,dermalXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXPrenatal developmentaltoxicityXXXXXXXXXXXXXXXX0X0Reproduction and fertilityeffectsXXXXXXXXXXXXXXXNeurotoxicity, acuteXXXXNeurotoxicity, subchronicXXXXNeurotoxicity, acute-delayedXXXXNeurotoxicity, subchronic-delayedXXXXNeurotoxicity, chronicXXXXDevelopmental neurotoxicityXXXXXOperant behaviorXXXXPeripheral nerve functionXXXXSensoryevoked potentialXXXXEye irritation, primaryXX00X0Dermal irritation, primaryXX00X0Dermal, sensitizationXX000X0Dermal, penetrationXXX0X0Metabolism/pharmacokineticsXXX0X0Genetic toxicity0XXX00ImmunotoxicityXXX00X0aXeobservational or gross endpoints are included;indicates level X plus histopathology orsomeclinical measure of systemfunction.ental toxicitystudy includes a more in-depth structural evaluation.indicates the major focus of the evaluation.0 indicates that this endpoint cannot be included as a majoraspect in this protocol. A blankindicates that an aspect is not routinely included but could be.bA Series 870 guideline(s) exists for conducting each of the above tests.Systems/endpoints evaluatedXXXXX0000X0indicates that somThe prenatal developmrelationship for sensitive endpoints following acu te inhalation exposure and the toxicologic response following acute high exposures (see further discussion in section 3.1.1.1). Acute toxicity inform ation 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 m o st endpoints with som e what greater detail than do the acute test designs. Although the histopathology and/or clinical m easures of system function are screening in nature, there is greater confidence that with this leve l of exam ination the dose-response relationship will
be m o re clearly defined. Nevertheless, it shoul d be recognized that m o st system s/endpoints are evaluated at a screening level, and detailed an alyses of pathology and function are generally not
carried out. Even in those test designs that do incorporate detailed analyses, these analyses are
lim ited in regard to the life stages, exposure periods, and m easures that are assessed. Figure 3-1 shows the study designs that are used for general toxicity testing superim posed on a tim e line that indicates the life stages during which exposure occurs (hatched bars) and endpoints are m easured (indicated in the boxes). The guideline studies shown represent the m i nim u m requirem e nt for derivation of a chronic oral RfD. Sim ilar studies are required for the chronic inhalation RfC, with appropriate endpoints for inhalation exposure and toxicity included. In som e cases, only a 90-day subchronic study is available instead of the
chronic studies shown. Because the relative le ngth of tim e between life stages varies am ong species, the placem ent 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. Sim ilar figures related to sp ecific 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 prim ary purpose of the guideline acute toxicity tests (870.1100 acute oral; 870.1200 acute derm al; 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 exposur e studies, provide a basis for classification and

3-63-7PreconceptionEmb/FetalJuvenileAdulthoodOldAgePrenatal DevelopmentalToxicityStudyReproductionandFertilityEffectsStudy C o n c e p t i o n B i r t h W

e a n i n g M at

u r i t y S e n e s c e n c e D e a t hAdolescence

P u b e r t yPF1Rodent CombinedChronic/CarcinogenicityStudyNB/PreweaningF1 sexualmaturationF1 &F2litter size,sex ratio, F1&F2survival, weight,gross structure,brain,spleen, thymusweightsLife StagesGuideline StudyDesigns:Satellite groupfollowed 28daysFetal survival, weight,gross structureDog Chronic StudySatellite groupfollowed 28daysClinical observations,viability,bodyweight,food&waterconsumption,clinicalpathology(every6months)Opthalmological exam,grossstructure &histopathologyNeurotoxicityevaluationNeurotoxicityevaluationP&F1 estrous cycles, spermmeasures, fertility, pregnancymaintenance, parturition,organweight, grossstructure,histologyPreconceptionEmb/FetalJuvenileAdulthoodOldAgePrenatal DevelopmentalToxicityStudyReproductionandFertilityEffectsStudy C o n c e p t i o n B i r t h W

e a n i n g M at

u r i t y S e n e s c e n c e D e a t hAdolescence

P u b e r t yPF1Rodent CombinedChronic/CarcinogenicityStudyNB/PreweaningF1 sexualmaturationF1 &F2litter size,sex ratio, F1&F2survival, weight,gross structure,brain,spleen, thymusweightsLife StagesGuideline StudyDesigns:Satellite groupfollowed 28daysFetal survival, weight,gross structureDog Chronic StudySatellite groupfollowed 28daysClinical observations,viability,bodyweight,food&waterconsumption,clinicalpathology(every6months)Opthalmological exam,grossstructure &histopathologyNeurotoxicityevaluationNeurotoxicityevaluationP&F1 estrous cycles, spermmeasures, fertility, pregnancymaintenance, parturition,organweight, grossstructure,histologyEmb =EmbryonicFigure 3-1.Exposures and endpoints related to general toxicity evaluations.Endpoints shown are fororal exposures; endpoints specific to inhalationand dermal exposure are included for studies bythose routes of exposure.F1 and F2 = 1st and 2nd filial generationsP = ParentalNB = Newbornlabeling, and enable the selection of exposure ranges for longer-term studies. 3 Acute guideline studies are conducted in young adult anim als, with a 14-day post-exposure observation period.
Other than m o rtality, the endpoints include cage-si de observations, body weight at the end of the observation period, gross pathology changes at necropsy, and histopathological exam ination of organs showing evidence of gross pathology in anim als surviving 24 hours or m o re. Two other available guideline studies cover acute exposures followed by extensive assessm ent of a specific organ system

. The first is the acute inhala tion toxicity study with histopathology (40 CFR 799.9135), which was developed for hazardous air pollutants. This study includes assessm ents of liver, kidney, and broncho alveolar lavage sam p les for several indicators of cellular dam a ge (e.g., total protein, cell count, percent leukocytes) and a phagocytosis assay to determ ine

m acrophage activity. For the respiratory tr act 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 de veloped for the evaluation of neurotoxic
chem icals and includes assessm ents of functional behavior and m o tor activity at the tim e of peak effect and again at 14-days post-treatm e nt a nd histopathology of the central and peripheral nervous system s at 14-days post-treatm e nt. The prenatal developm ental 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 assessm ent because m a ternal observations are often recorded daily and there is a presum ption that effects during developm ent m a y 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 anim als and in young adults. No direct inform ation is available from any of these
studies on acute or short-term exposure in postweaning young anim als or aged anim als.

3 Alternative test protocols have been adopted by the Organization for Econom ic Cooperation and Developm ent for acute toxicity testing for oral, derm al, and inhalation exposure, including the fixed-dose procedure, th e acute toxic class m e thod, and the up-and-down procedure. All are designed to m i nim i ze anim al usage and provide m i nim a l hazard and doseresponse inform ation for classification, labeling, and dose selection. In the future, EPA plans to put prim ary reliance on the up-and-down procedure for testing of technical grade pesticides, although the other tests m a y be acceptable in som e circum stances, e.g., testing of pesticidal products. These studies are not designed to provide inform ation for use in less-than-lifetim e risk

assessm ents.

3-83.1.1.1.3. Gaps in assessment endpoints. Data on only a lim ited num ber 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., hem a tological, clinical, histology of

m o re organs), are collected. Som e data from anim als exam ined at early tim es m i ght be available in guideline subchronic or chronic studies. These data could augm ent 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 developm ental toxicity studies in rats and rabbits and the DNT study include repeated dosing of m a ternal anim als for periods of less than 25 days. Because of the post-exposure observation period in acute guideline studies and in the DNT study, som e inform ation on latency to effect and reversibility of effect m a y 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 com b ined chronic and carcinogeni city study with a 2-year exposure) provide an in-depth look at a num ber of organ system s, and in som e cases they evaluate both structure and function (see Figure 3-1). The chronic study in nonrodents, usually dogs, involves a 12-m onth exposure with sim ilar endpoints assessed as in rodents. The prenatal developm ental 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 anim als, with exposure in the chronic/carcinogenicity study continuing into old

age. No inform ation is available from chronic studies in pre- or postnatal anim als. Exposures in subchronic study protocols do not include pre- or postnatal developm ent, although the reproduction and fertility effects study does provide data on subchronic exposures in anim als that are exposed before birth, through prenatal and postnatal developm ent up to m a ting of the F1

3-9m a les and fem a les and through pregnancy (F1 young adult fem a les). No subchronic toxicity evaluations are conducted in aged anim als. No chronic studies are conducted in pre- or postnatal
anim als, although aged anim als 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, im m unotoxicity testing in adults, and m o re thorough toxicokinetics in anim als at various life stages. Gaps in assessm ent endpoints during prenatal and postnatal developm ent are discussed in the next section. Assessm ent endpoints for routine toxicity testing in old age are com p letely lacking, as is background inform ation 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 m onths in rodents do assess latency to response for a brief period of tim e (28 days or m o re).

3.1.2. Exposures and Endpoints Related to Evaluation of Reproductive Toxicity
3.1.2.1. Overview of Tests The reproductive organs are exam ined structurally in a num ber 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 developm ental toxicity st udy (OPPTS 870.3700), and the reproduction and fertility effects study (OPPTS 870.3800), which is a two- generation reproduction study. In addition,
extensive assessm ent of num erous functional aspects of the reproductive system is conducted in the reproduction and fertility effects study. Specific functional effects on the reproductive system of m a le anim als can also be assessed in the rodent dom inant lethal assay (OPPTS 870.5450). As illustrated in Figure 3-2, these studies include a variety of both structural and
functional assessm ents of the reproductive system over a wide sam p ling of life stages. In guideline subchronic and chronic/carcinogeni city studies, gross structural evaluation and general qualitative histopathology are conducted on reproductive organs and tissues. The
anim als in these studies are adults, but at the tim e of organ assessm ent they m a y be young (e.g., rats 45 days to 5 m onths of age from a subchronic study), m a ture (e.g., rats 5–18 m onths of age
from a reproduction study), or old anim als (e.g., rats 18 m onths to 2 years of age from a chronic study), depending on the protocol.

3-103-11Life StagesPreconceptionEmb/FetalNB/PreweaningJuvenileAdulthoodOld AgeGuideline StudyDesigns:Prenatal DevelopmentalToxicityStudyReproduction andFertilityEffects Study C o n cep

t i o n B i r t h W

e a n i n g M

a t u r i t y S e n e s c e n c e D e a t hDevelopmental NeurotoxicityStudyF1 sexualmaturationAdolescence

P u b e r t yPF1F1&F2litter size,sexratio,survival,weight, gross structure,brain,spleen,thymus weightsEmbryonic deathRodent DominantLethal AssayLittersize, sexratio,survival,weight,gross structure,developmentallandmarks includingsexualmaturationLittersize,sexratio, fetalsurvival,weight, gross structureP& F1 Estrous cycles,spermmeasures,fertility,pregnancymaintenance,parturition,organweight,gross structure,histologySubchronic orChronicToxicityStudyOrgan weight,histologyLife StagesPreconceptionEmb/FetalNB/PreweaningJuvenileAdulthoodOld AgeGuideline StudyDesigns:Prenatal DevelopmentalToxicityStudyReproduction andFertilityEffects Study C o n c e p t i o n B i r t h W

e a n i n g M

a t u r i t y S e n e s c e n c e D e a t hDevelopmental NeurotoxicityStudyF1 sexualmaturationAdolescence

P u b e r t yPF1F1&F2litter size,sexratio,survival,weight, gross structure,brain,spleen,thymus weightsEmbryonic deathRodent DominantLethal AssayLittersize, sexratio,survival,weight,gross structure,developmentallandmarks includingsexualmaturationLittersize,sexratio, fetalsurvival,weight, gross structureP& F1 Estrous cycles,spermmeasures,fertility,pregnancymaintenance,parturition,organweight,gross structure,histologySubchronic orChronicToxicityStudyOrgan weight,histologyEmb =EmbryonicFigure 3-2.F1 and F2 = 1st and 2nd filial generationsP = ParentalNB = NewbornExposures and endpoints related to reproductive evaluations.Standard guideline prenatal developm ental toxicity studies are designed to evaluate the potential effects of the test substance on the developing fetus. Observations on the reproductive capacity of the m a ternal anim als in this study generally consist only of clinical observations (including any abnorm a lities of pregnancy m a intenance) and gross necropsy data (including uterine). Selected fetuses are exam ined for gross structural changes to the internal reproductive

organs. In studies that em ploy m e thods of se rial sectioning in the process of soft tissue

exam ination, a lim ited m acroscopic evaluation of the internal structure and integrity of the reproductive organs is perform e d; however, the fetal tissues are not exam ined m i croscopically. Additionally, there are no assessm ents 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 anim als are young adults of approxim a tely 6–9 weeks of age. Daily exposure continues during all phases of developm ent and reproductive function. Adult anim als of both generations are killed as m a ture adults, generally prior to reaching reproductive senescence (that is, the cessation of norm a l reproductive function) or an age that would be considered geriatric in that species. Assessm ents of reproductive capability and function are conducted at least once in each generation. These

assessm ents include direct evaluation of the age of sexual m a turation, estrous cyclicity (im m e diately prior to m a ting), sperm m easures (at term ination), m a ting success, f e rtility and fecundity, im plantation, pregnancy m a intenance, gestation duration, parturition, and success of lactation (e.g., m a ternal nurturing and nesting behavior).
Indirect assessm ents of som e reproductive functions are also evaluated. These
observations are based on evidence of norm a lity in a structure, function, or process that is dependent on norm a l functioning of the com ponent parts, including, for exam ple, horm onal
hom eostasis, ejaculation, accessory gland function, placental function, m ilk production, pup nursing behavior or ability, and, to som e extent, reproductive senescence (although the adult
anim als are term inated at the end of each generation, when they are only around 6 m onths of age; therefore, there are no assessm ents conducted in older rats). Gross structural assessm ents of
the whole anim al are conducted on adult and im m a ture anim als throughout the course of the study; gross internal (organ) structural assessm ents are conducted on offspring that are killed at litter standardization (postnatal day [PND] 4), weaning (PND 21), and term ination of each generation (m ature adults). Histopathological ev aluation of the reproductive organs (gonads and accessory structures) is conducted only in the m a ture parental adult anim als that are killed at the

term ination of each generation. The guideline specifies a very focused pathological exam ination of the reproductive organs in this study.

3-12The dom inant lethal assay is not conducted for every chem ical, but it m a y be conducted in response to a concern raised by other developm ental or reproductive toxicity findings in the
database. In this study, sexually m a ture adult m a les are treated with the test substance to
determ ine whether there is an effect in the germ inal tissue that does not cause dysfunction in the

gam e te but is lethal to the fertilized egg or developing em bryo. Exposed m a les are m a ted with
untreated fem a les, and uterine contents are eval uated. Evidence of pre- and/or postim plantation loss is generally thought to be indicative of treatm e nt-related chrom o som a l dam a ge in germ inal
tissue.

3.1.2.2. Gaps in Life Stage of Assessment
Determ ination of gaps in the assessm ent of potential effects of any chem ical across all life stages requires consideration of both the exposure period and the tim e of assessm ent. In the prenatal developm ental toxicity study, anim als are exposed from im plantation through gestation. The reproductive organs are exam ined for gross structural changes, but no m i croscopic

exam ination is conducted. There is no follow-up of the anim als to determ ine the functional consequences of prenatal exposure. In the reproduction and fertility effects study, the F1
anim als are exposed from preconception throughout prenatal and postnatal developm ent until
after m a ting. The reproductive organs are exam ined m acroscopically at weaning and adulthood.
The m a turation of the reproductive system is assessed, as is its function. Thus, the study provides a fairly thorough assessm ent of struct ure and function following exposure during m a ny critical periods of developm ent. In the parental generation, the anim als are exposed as young adults, and the structure and function of the reproductive organs are assessed.
The dom inant lethal study, when conducted, assesses a single aspect of the function of the reproductive system for one sex, although a detailed structural assessm ent is not conducted. In the subchronic and chronic studies, the anim als are exposed beginning as young adults, and the structure—but not the function—of the reproductive organs is assessed. Therefore, the m a jor gaps include (1) the lack of functional assessm en t (particularly the age of onset of reproductive senescence) in older adult anim als following adult-only exposures, and (2) the lack of structural
and functional assessm ents in older adult anim als following developm ental exposures. The onset of reproductive senescence can be m a rked by findings such as altered
horm onal hom eostasis, disruption of estrous cyclicity, dim i nished sperm m easures (num ber,

m o tility, or m o rphology), or gonadal atrophy. Studies in rodents have dem onstrated the adverse
effects of a num ber of agents (e.g., ionizing radiation, chem otherapeutic agents, polycyclic

arom atic hydrocarbons, and agents that form epoxides, such as 1, 3-butadiene and 4- vinylcyclohexene) on reproductive senescence (reviewed by Hoyer and Sipes, 1996).

3-13In hum ans, prem ature reproductive senescence has been associated with cigarette

sm oking (Jick et al., 1977). In addition to potentia lly dim i nishing fertility in individuals who are only slightly past prim e reproductive age, early reproductive senescence can adversely affect the general health of the aged hum an. For exam ple, horm onal alterations that are associated with early senescence have been linked to abnorm a lities of cardiovascular function, osteoporosis, and even a predisposition to early m o rtality.

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 assessm ent of functional endpoints in older anim als following adult exposur es, and there are no structural or functional endpoints assessed in older anim als following developm ental exposures, including reproductive

senescence. In addition, concerns have recently been raised about the ability to detect rare

m a lform a tions of the reproductive organs and abnorm a lities in the m a turation of the reproductive system in the two-generation reproductive toxicity study. This concern relates particularly to
endocrine-active chem icals. In the current guideline, three pups/sex/litter are exam ined

m acroscopically at weaning. Questions have been raised about whether these weanlings should be retained until day 45 (fem ales) or day 60 (m ales) to ensure that any later-appearing gross or functional changes are
detected. This issue is currently being exam ined 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 developm ent of the reproductive system

. As indicated above, it has been suggested that anim als 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.

3-143.1.3. Exposures and Endpoints Related to Evaluation of Neurotoxicity
3.1.3.1. Overview of Tests
Observation of the anim als for signs of overt toxicity and routine gross pathological

assessm ent 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, derm al, or inhalation exposure). Motor activity, grip strength, and sensory reactivity and neuropathology are

m easured in the rodent oral study, the derm al 21–28- and 90-day subchronic studies, and the 90- day inhalation study. In rodent subchronic st udies, specific assessm ent for neurotoxicity is perform e d at or near the end of the study, although observations of the anim als, including those for detection of overt neurotoxicity, are m a de routinely throughout the study. No specific functional tests for neurotoxicity are requi red for nonrodent subchronic studies, although observation and neuropathology are required. Chronic toxicity studies (oral, derm al, inhala tion) are to be perform e d in two species (one rodent) over a 12-m onth 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 11 th m onth. Clinical observation is perform e d weekly throughout the study and would presum ably detect gross neurological abnorm a lity. In current practice, the chronic study is often com b ined with the carcinogenicity test, in which dosing extends for 24 m onths in rats and 18 m onths in m i ce (OPPTS 870.4300). Motor activity would be perform e d at 11–12 m onths 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 (Fi gure 3-3). The endpoints exam ined 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 abnorm a lities, as well as forelim b and hindlim b grip strength, landing foot splay, sensorim otor reactivity to sensory stim uli, and pain reception. Motor activity
and a m o re detailed neuropathological observation are also required in this battery. For acute studies, assessm ents are m a de before initiation of dosing, at the estim ated peak of activity within 8 hours of dosing, and at 7 and 14 days post-dos ing. For subchronic studies, assessm ents are perform e d pre-exposure and at 7, 8, and 13 weeks of exposure. For chronic studies, assessm ent is at pre-exposure and every 3 m onths post-expos ure. There is no specific guidance regarding

3-153-16Life StagesPreconceptionEmb/FetalNB/PreweaningJuvenileAdulthoodOldAgeGuideline StudyDesigns:PrenatalDevelopmentalToxicityStudy C o n c e p t i o n B i r t h W

e a n i n g M

a t u r i t y S e n e s c e n c e D e a t hDevelopmental NeurotoxicityStudyAdolescence

P u b e r t yGross structure ofthe CNSMotoractivityAuditorystartle,learning & memoryNeuropathologyDelayedNeurotoxicityofOrganophosphorousPesticides(adulthen)acute28-dayNeuropathytargetesterase(NTE),neuropathology,grossobservation, motor function,acetylcholinesterase(AChE)Schedule-controlledoperantbehavior,peripheralnerve function,sensoryevokedpotentialsMayberequested/requiredNeurotoxicityScreeningBatteryacutesubchronic(90 days)chronic(12 months)Chronic/carcinogenicity(24 months)NeuropathologyMotor activity,FOB(includesgripstrength,landing footsplay,grosssensorimotorreactivity,painperception)???Life StagesPreconceptionEmb/FetalNB/PreweaningJuvenileAdulthoodOldAgeGuideline StudyDesigns:PrenatalDevelopmentalToxicityStudy C o n c e p t i o n B i r t h W

e a n i n g M

a t u r i t y S e n e s c e n c e D e a t hDevelopmental NeurotoxicityStudyAdolescence

P u b e r t yGross structure ofthe CNSMotoractivityAuditorystartle,learning & memoryNeuropathologyDelayedNeurotoxicityofOrganophosphorousPesticides(adulthen)acute28-dayNeuropathytargetesterase(NTE),neuropathology,grossobservation, motor function,acetylcholinesterase(AChE)Schedule-controlledoperantbehavior,peripheralnerve function,sensoryevokedpotentialsMayberequested/requiredNeurotoxicityScreeningBatteryacutesubchronic(90 days)chronic(12 months)Chronic/carcinogenicity(24 months)NeuropathologyMotor activity,FOB(includesgripstrength,landing footsplay,grosssensorimotorreactivity,painperception)???Emb = EmbryonicFigure 3-3.?FOB = Functional observation batteryNB = NewbornExposures and endpoints for neurotoxicity evaluations.the assessm ent schedule for the com b ined chronic/carcinogenicity study, but presum ably the schedule required for the chronic study would be m a intained. The DNT study protocol (870.6300) currently requires dosing of the dam s from gestational day (GD) 6 through PND 10, although the requirem e nt m a y soon be extended to PND 21 (i.e., until weaning). Motor activity is m easured at PNDs 13, 17, 21, and 60. Auditory
startle is m easured around weaning and at PND 60, as is a test of learning and m e m o ry, which

m a y be the sam e test or different tests at th e two tim e points. Cage-side observation of both
dam s and pups is required, and neuropathology in the pups is required at PND 11 and at the

term ination of the study (usually PND 60). The prenatal developm ental toxicity study (870.3700) requires dosing of the dam s 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 exam inations conducted in this study.

3.1.3.2. Gaps in Life Stage of Assessment
One of the m o st significant gaps revealed by Figure 3-3 is the lack of exposure or

assessm ent under any protocol during old age. For exam ple, following acute exposure,

assessm ent is for 14 days in juvenile or young adult anim als. The chronic exposure protocol extends exposure into adulthood and the com b ined chronic/carcinogenicity protocol extends exposure up to approxim a tely the aged period in the rat, but neurotoxicology assessm ents are not perform e d in aged anim als. Thus, none of the protocols assess potential effects of chem icals on aging as a function of exposure during developm en t. This m a y be im portant, because studies in
anim als have shown that developm ental exposure to agents that cause neurotoxicity, such as
trim ethyl tin, can accelerate the onset of cognitive deficits m easured later in life. Other studies
with m e thyl m e rcury have docum ented early-onset sensory dysfunction in m onkeys exposed during developm ent. Furtherm ore, current tes ting protocols do not provide inform ation collected at different life stages—that is, com p arison of effects of exposure during infancy, adulthood, or old age. This is im portant, because life stage-dependent differences in pharm acokinetic, and possibly toxicodynam i c, param e ters could result in quantitatively or qualitatively different effects at different life stages. Under the DNT protocol, there currently is no requirem e nt to perform kinetic studies to ascertain either in utero or postnatal exposure. There is no m echanism to guarantee exposure postnatally (i.e., direct dosing of pups) because the com pound m a y not be excreted into breast

m ilk or it m a y be excreted only at very low concentrations. This is of particular im portance, because the early postnatal period in the rodent is equivalent to a prenatal life stage in hum ans.

3-17There is no long-term follow-up assessm ent to detect delayed neurotoxic effects, a situation that is arguably m o re worrisom e for developm ental exposure than for exposure later in life.

3.1.3.3. Gaps in Assessment Endpoints The nervous system is one of the m o st fully assessed organ system s in the EPA/OPPTS 870 guidelines. Nonetheless, m o st of the endpoint assessm ents are designed to be screening procedures rather than sensitive assessm ents of nervous system function. In addition, the

assessm ents required are different in the neurotoxicity screening battery than in the DNT study. The adult neurotoxicity screening battery does not require assessm ent of learning and m e m o ry or auditory startle. The lack of assessm ent of cognitive f unction in the neurotoxicity screening battery constitutes a significant om ission that should be addressed.
It m a y also be pointed out that even in the developm ental protocol, the tests that are used to assess learning and m e m o ry m a y be very sim p le, potentially revealing only relatively gross
deficits. In addition, although potentially m o re sensitive cognitive, sensory, and m o tor tests are available (Figure 3-3), there is no guidance as to what would trigger a requirem e nt for these

assessm ents. Except for the protocol for delayed neurotoxicity for organophosphorous pesticides in the hen, there is no assessm ent of neurochem i cal endpoints. Additionally, the required neuropathological assessm ents m a y also be considered screening.
Minim a l m o rphom etric analysis, consisting of the thickness of “representative” layers in the neocortex, hippocam pus, and cerebellum
, is required in the DNT study. No m o rphom etric analyses are required in the adult neurotoxicity testing protocols. Although m o re sophisticated tests would presum ably not be perform e d on all agents, m o re sophisticated m easures could be triggered by results f r om screening tests. It also m a y be advisable to require m o re sensitive tests in instances of particular concern, for exam ple, adding m o re extensive m o rphom etric analysis to the DNT protocol.
In sum m a ry, although the nervous system is one of the m o st thoroughly assessed system s in the 870 test guideline studies, it m u st be kept well in m i nd when interpreting the results that these are screening tests. Positive f i ndings m u st 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 assessm ent 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 acrylam ide. For DNT, it is assum e d that a single exposure

3-18to a chem ical during a critical period of developm ent could result in an adverse effect on the developing nervous system

. There are, however, few data that com p are the effects of a single exposure to a chem ical with the effects of the sam e chem ical given m u ltiple tim es during developm ent.

3.1.4. Exposures and Endpoints Related to Evaluation of Immunotoxicity
3.1.4.1. Overview of Tests
Exam ination of the m acro- and/or m i crosc opic structural anatom y of im m une system organs and tissues is perform e d in a num ber of general guideline screening studies, including the acute inhalation toxicity with histopa thology 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 developm ental toxicity study (OPPTS 870.3700), and the two-generation reproduction study (OPPTS 870.3800). In addition,
functional assessm ents of the im m une system are evaluated in the skin sensitization study (OPPTS 870.2600) and the im m unotoxicity testing guideline (OPPTS 870.7800) (see Figure 3- 4). In the guideline im m unotoxicity study, young adult rats (6–8 weeks of age) are exposed to the test substance for 28 days, at which tim e they are term inated. The spleen and thym us are

exam ined m acroscopically, and organ weights are recorded; a histopathological evaluation is not perform e d. Assessm ents of im m une system functi on 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-form i ng cell (PFC) assay or an im m unoglobulin quantification by enzym e -linked im m unosorbent assay (ELISA). In addition, an assessm ent of natural killer (NK) cell activity and/or enum eration of splenic or peripheral blood total B cells, total T cells, and T cell subpopulations m a y be required on a case-by-case basis. The skin sensitization study has been generally conducted in guinea pigs as a Guinea Pig
Maxim i zation Test (GPMT) or a Buehler test. In a recent review by the FIFRA Science Advisory Panel (U.S. EPA, 2001a), it was recom m e nded that in the future, skin sensitization

m e thods should preferentially include the local lym ph node assay (LLNA), which uses young
adult m i ce. The skin sensitization test involves an initial intraderm a l (GPMT) and/or epiderm a l (Buehler, LLNA) exposure of the test anim al to a substance, followed by a challenge exposure approxim a tely 1 week later. In the guinea pig tests, sensitization is determ ined by exam ining the reaction to the challenge exposure and com p aring this reaction with that of the initial induction

3-193-20Life StagesPreconceptionEmb/FetalNB/PreweaningJuvenileAdulthoodOld AgeGuideline StudyDesigns:Prenatal DevelopmentalToxicityStudyReproduction andFertilityEffects Study C o n cep

t i o n B i r t h W

e a n i n g M

a t u r i t y S e n e s c e n c e D e a t hAdolescence

P u b e r t yPF1Organweights,grossstructure,histologyGross thymus/spleen structureChronic/carcinogenicityStudySubchronic StudySpleen/thymusweightsClinicalpathology(differentialWBC,serumimmunoglobulinevery6monthsReactionto initialinduction orchallengedoseImmunotoxicityStudySkin Sensitization StudyGross structure &weightsof thymus/spleen,responseto Tcell-dependent antigen,SRBC(PFCor ELISA).Case-by-case -NKcell activity,totalB cells,TcellsandT cellsubpopulationsGrossstructure,thymus/spleenweights,spleen/thymus/lymph node(2 sites) histologyLife StagesPreconceptionEmb/FetalNB/PreweaningJuvenileAdulthoodOld AgeGuideline StudyDesigns:Prenatal DevelopmentalToxicityStudyReproduction andFertilityEffects Study C o n c e p t i o n B i r t h W

e a n i n g M

a t u r i t y S e n e s c e n c e D e a t hAdolescence

P u b e r t yPF1Organweights,grossstructure,histologyGross thymus/spleen structureChronic/carcinogenicityStudySubchronic StudySpleen/thymusweightsClinicalpathology(differentialWBC,serumimmunoglobulinevery6monthsReactionto initialinduction orchallengedoseImmunotoxicityStudySkin Sensitization StudyGross structure &weightsof thymus/spleen,responseto Tcell-dependent antigen,SRBC(PFCor ELISA).Case-by-case -NKcell activity,totalB cells,TcellsandT cellsubpopulationsGrossstructure,thymus/spleenweights,spleen/thymus/lymph node(2 sites) histologyEmb = EmbryonicF1= 1st filial generationPFC = Placque-forming cellse-linked immunosorbent assayWBC = White blood cellsFigure 3-4.P = ParentalNB = NewbornSRBC = Sheep red blood cellELISA = EnzymNK = Natural killerExposures and endpoints for immunotoxicity evaluations.exposure. In the LLNA, proliferation of lym phocytes is m easured (as a function of in vivo radioisotope incorporation into cellular DNA) in draining lym ph nodes proxim a l to the application site. Hence, although the GPMT and Buehler tests result in a qualitative assessm ent of hypersensitivity, the LLNA provides a quantitative dose-response evaluation. Histopathological evaluation of the skin is not required with any of these m e thods, but it m a y be
conducted. No other im m une system endpoints or organs are evaluated in this study. In guideline subchronic and chronic/carcinogenicity studies, an evaluation of

m acroscopic structure and general qualitative histopathology are conducted on only a few
im m une system tissues. In studies that include young adult anim als (e.g., rats 45 days to 5

m onths of age from a subchronic study), the spleen, thym us, and lym ph nodes from two locations (one near to and the other distant from the site of adm i nistration) are exam ined; the spleen and thym us are weighed. In chronic and carcinogenicity study guidelines, there is no requirem e nt that the thym us be exam ined and/or weighed. For rodents (e.g., rats or m i ce 18

m onths to 2 years of age), it is reasonable to assum e that the thym us would have undergone

norm a l age-related atrophy by study term ination. However, the thym us m i ght be present at early
interim sacrifices of rodents (e.g., at 6 m onths or 12 m onths of study) during a long-term study, and it would certainly be present at study term ination in a canine chronic study (at which point the dogs are young adults of only approxim a tely 1.5 years of age). Differential white cell counts in the circulating blood are exam ined at study term ination in the subchronic study and at approxim a tely 6- m onth intervals in long-term studies. Serum
im m unoglobulin levels m a y be m easured at the sam e intervals. Perturbations m a y indicate
increased im m une system response to som e unspecified initiator, but this inform ation does not address the adequacy of im m une system function. In the sam e m a nner, histopathological evaluation of other organ system s in the subc hronic and chronic/carcinogenicity studies m a y identify cellular alterations that are nonspecific i ndicators of an effect on im m une response, for

exam ple, the presence of increased num bers of m acrophages in lung tissue or an increased
incidence of inflam m a tory derm al lesions. In the reproduction and fertility effects study in rats, a m acroscopic evaluation of all organ system s is conducted in a sam p le of offspring at weaning and in the m a ture adult parental
anim als at the term ination of each generation. Additionally, the spleen and thym us are weighed for those pups that are necropsied at weaning; these m easurem ents are intended to provide
inform ation on the need for further evaluation of the im m unotoxic potential of a chem ical to the
im m a ture anim al. In the prenatal developm ental toxicity study, an evaluation of the m acroscopic structure of the thym us and spleen is conducted in at least half of the fetuses from each litter.

3-213.1.4.2. Gaps in Life Stage of Assessment In the available guideline studies, assessm ents of organs with im m une system function are conducted in fetuses following prenatal exposure, in weanling anim als following pre- and postnatal exposure, and in young and/or m a ture adult anim als at a variety of tim e points. W ith prenatal exposures and evaluation at early life stages, these assessm ents consist entirely of the
evaluation of m acroscopic changes, with no m i croscopic exam ination. Toxicokinetic data that characterize the exposure in the young (i.e., exposure of the fetus to the chem ical or its

m e tabolites via the placenta or of the neonate via breast m ilk) are not routinely required and are
seldom available.
Som e detailed structural assessm ent (histopathology) is conducted in m a ture or older
adult anim als. Indirect assessm ent of im m une system function is conducted in adult anim als of various ages via the evaluation of peripheral blood cells and chem istry. Direct functional

assessm ents of the im m une system are conducted only in young adult anim als; generally this age group is selected for assessm ent because of the anticipated robustness of the im m une response. There is no guideline that exam ines potential perturbation of im m une system function following early pre- and/or postnatal exposure (often referred to as a developm ental
im m unotoxicity study). Com p arisons of im m une effects following exposure at various life stages (i.e., during in utero or postnatal devel opm ent, adulthood, or old age), including data that analyze whether these effects are m o re severe in one age group or whether the effects are persistent, are not required. To achieve even a m i nim a l assessm ent of im m une system structure and function, a broad variety of studies would need to be conducted and assessed; yet there could still be relatively low conf idence in the ability of the results of these com b ined studies to predict
the outcom e of age-specific insults to the im m une system

.

3.1.4.3. Gaps in Assessment Endpoints There are identifiable gaps in the endpoints that are used to assess im m unotoxicity in guideline studies. For exam ple, for fetuses, im m a ture anim als, and old anim als (rodents),

assessm ents are com posed entirely of the evaluation of m acroscopic structural changes, with no histopathological or functional evaluations. In m a ture adult anim als, thorough m acroscopic and

m i croscopic structural assessm ents, as well as routine hem a tological testing (e.g., blood cell counts), are perform e d; however, those assessm ents are generally very lim ited in young anim als, and guideline requirem e nts do not consider species differences. The only assessm ents of functional integrity of the im m une system are provided by the guideline sensitization study and the 28-day im m unotoxicity study. These studies are conducted only in young adult anim als, and

3-22they include only a few exam ples of potential im m une system response (e.g., hypersensitivity,
hum oral im m unity, or nonspecific cell-m e diated im m unity). In very young and very old
anim als, there is no direct assessm ent of im m unological function. No assessm ent of autoim m une effects is conducted in any of the current guideline protocols.

3.1.4.4. Gaps in Duration/Latency Assessment
Latent effects on im m une function that result from early lifetim e exposure are not assessed; these can include effects in aged anim als that result fromin utero, neonatal, or youngadult exposure. Exacerbation of effects in relation to aging and response to subsequent
im m unological challenge are not routinely or syst em atically assessed to any extent. The twogeneration reproduction study offers an opportunity to evaluate im m unotoxic response in
adulthood that resulted from prenatal or early postn atal exposure. In the chronic toxicity studies in rodents, aged anim als are available for evaluation. However, in both cases there is little focus
on the evaluation of the im m une system

. Only indirect evidence of perturbation of the im m une system m a y be observed through m acroscopic and m i croscopic evaluation of various organs; corollary functional assessm ent is not perform e d. Response to an im m unological challenge is

exam ined only in the hypersensitization study, and even when the results from this study are positive, no f u rther specif i c assessm ent of the im m une 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 m a jor vessels augm ented by conditional standard pathology is m e ntioned in m o st applicable OPPT S Series 870 health effect guidelines (Figure 3- 5).

3.1.5.2. Gaps in Life Stage of Assessment The period from birth to m a turity is essentially without toxicological m onitoring of cardiovascular endpoints for both repeated chronic and single acute-exposure regim e s.

3-233-24 D e a t hLife StagesPreconceptionEmb/FetalNB/PreweaningJuvenileAdulthoodOld AgeGuideline StudyDesigns:Prenatal DevelopmentalToxicityStudy Co

n c ep t i o n B i r t h W

ea

n i n g M

a t u r i t y S e n e s c e n c eAdolescence

P u b e r t yGross structureCombined ChronicToxicity/CarcinogenicityTestSubchronic ToxicityTestAcute ToxicityTestGrosspathology,nosectioninginstructionsstipulated D e a t hLife StagesPreconceptionEmb/FetalNB/PreweaningJuvenileAdulthoodOld AgeGuideline StudyDesigns:Prenatal DevelopmentalToxicityStudy C o n c e p t i o n B i r t h W

e a n i n g M

a t u r i t y S e n e s c e n c eAdolescence

P u b e r t yGross structureCombined ChronicToxicity/CarcinogenicityTestSubchronic ToxicityTestAcute ToxicityTestGrosspathology,nosectioninginstructionsstipulatedEmb = EmbryonicFigure 3-5.NB = NewbornExposures and endpoints related to cardiovascular evaluations.3.1.5.3. Gaps in Assessment Endpoints Gross observation only of the heart is provided for in m o st OPPTS guidelines. Functional clinical or histopathological cardiac exam ination is not currently part of any testing guideline. Even gross pathology could be im proved and brought into line with current cardiovascular evaluation by separating, weighing, a nd constructing right and left ventricle-tobody weight ratios to give an evaluation of cardiac hypertrophy. Also, guidelines regarding sectioning procedures for the heart, either num ber or plane, could be provided.
No sim p le cardiac functional evaluation is currently available, including even systolic or diastolic blood pressures. It should be noted that telem e tric in-dwelling echocardiogram s (ultrasound exam inations of the heart) can be used to detect occlusions and atherosclerosis and to detect alterations in cardiac output. Com b ination echocardiogram s and electrocardiogram analysis can detect cardiac wave f o rm s 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

autonom ic nervous system ). Heart rate variability m a y be critical in explaining toxicity, as was shown in recent work associating exposures to fine particulate m a tter with decreases in heart rate variability in elderly hum ans (Creason et al., 2001). Both echocardiogram s and electrocardiogram s can be done on rats down to 100 g, well within the size range of juvenile and
adolescent rats.
Chem icals can produce degenerative and/or inflam m a tory changes in the peripheral blood vessels as a consequence of an excessive pharm acologic effect or by an interaction with a
vascular structural or functional m acrom olecule. As a result of sustained arterial vasoconstriction, peripheral arterial lesions consisting of intim al proliferation and m e dial degenerative changes could result in gangrene. Also, chem icals can induce or enhance atherom a form ation, which is characterized by endothelial dam a ge with increased perm eability, m onocyte adhesion, and endothelial proliferation. Selected representative techniques to study the peripheral vascular system consist of flow

m easurem ent techniques (Sm ith et al., 1994), su ch as electrom a gnetic flowm e try, pulsed Doppler
flowm e try, transit tim e flowm e try, laser Doppler fluxm etry, and laser scanner m e thods. These techniques allow investigation of blood flow in vessels as large as the aorta and as sm all as the capillary, determ ination of the level of perf usion in tissues, and calculation of the derived
hem odynam i c variable of resistance.
The two m a jor noninvasive techniques for determ ining m i crovascular velocity are the flying spot technique and the dual-slit technique. External ultrasound m a y be used to exam ine
internal vascular dim e nsions. A noninvasive asse ssm ent of arterial flow in rodents and m onkeys can be perform e d using Doppler spectrum anal ysis (duplex ultrasound technology) (Leopold et

3-25al., 1997). This test detects arterial com p rom i se in extrem ities, functional severity, and the
hem odynam i c significance of vascular lesions. In m o st cases, the locations in the arteries involved can be designated. Inform ation regarding the extent and effectiveness of collateral circulation can also be gained. This testing is a valuable tool for m onitoring early flow

com p rom i se secondary to chronic reoccurrence of anastom o tic or distal disease. Several blood/plasm a tests for clinical assessm ent are in active use in cardiovascular
research. In general, these are tests that m a y be used to docum ent a cardiovascular accident (within 48–96 hours). Their utility for risk assessm ent has yet to be evaluated. Specific

enzym e s currently being used by the research com m unity for these purposes include LDH-I, creatinine kinase-II, and troponin. Other enzym e s useful as prognostic indicators of risk of a cardiovascular accident include angiotensin c onverting enzym e II, plasm a renin activity, endothelin-converting enzym e -1, and catecholam ines (epinephrine and norepinephrine).

3.2. CONCLUSIONS AND RECOMMENDATIONS A review of current testing guidelines was conducted to determ ine 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 system s, (3) tim ing 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 assessm ent, endpoints assessed, tim ing and duration of exposures included in guideline studies, reversibility, and latency to response. Although a num ber of areas of toxicity testing have been discussed, this review should not be considered exhaustive, and
other health effects m a y be as or m o re im portant for particular chem icals 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 asthm a and other respiratory tract toxicity. For both children and the elderly, renal and liver function can be a m a jor factor in the disposition, m e tabolism
, and
excretion of chem icals and, therefore, their toxi city. Thus, the evaluation of toxicity and the interpretation of data in term s of its com p lete ness will always require scientific judgm ent about whether or not adequate data have been collected on effects of im portance at the appropriate life stages and tim ing and duration of exposure, for exam ple, for a given agent.
Effects seen at the term ination of a chronic study m a y be due to cum u lative dam a ge from a continued repeated chem ical insult, but they could also be a latent response from an earlier

3-26single or short-term m u ltiple exposure. Thus, late nt effects m i ght 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 inform ation on the latency of a response would follow only from a clearer understanding of the m echanism 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

m

u ch longer period of tim e. It thus follows that any chem ical database that does not have exposure-response studies of lifetim e duration or any specific exposure-latency protocols would not cover the possibility of latent ef f ects. Effects that persist throughout a designated post-exposure period m a y be considered irreversible; those that do not are reversible. For chronic lifetim e exposures, designation of an
effect as irreversible or reversible is academ ic, as exposure is presum ed to be lifetim e (i.e., there is no post-exposure period). For shorter-term values (e.g., acute, short-term ) where an appreciable period of tim e post-exposure is anticipated, designation of an effect as reversible or
irreversible becom e s m o re 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 im portant 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 exam ple, low birth weight m a y be “reversible” through cat ch-up growth postnatally, but it also m a y be related to developm ental delays or other health outcom e s that result from prenatal growth
reduction/retardation.

3.2.1. Conclusions
From this review, the Technical Panel reached the following m a jor conclusions:

1. There are a num ber of gaps in life stages covered in current guideline testing protocols, particularly in term s of the exposure periods included. In particular, there is

m i nim a l evaluation of aged anim als, especially after exposures that include early developm ent.

2. There are a num ber of gaps in the evaluation of endpoints included for certain system s; for exam ple, the evaluations of the cardiovascular and im m une system s in various guideline studies were reviewed as exam ples of system s that are m i nim a lly
covered. Other system s, for exam ple, the reproductive and nervous system s, are evaluated in m o re detail, but even in these system s there are gaps that need to be

3-27considered; notably, functional evaluations are not always included or integrated with structural evaluations of particular system s.

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 m a jor im pact on hazard characterization, especially in designing
acute and short-term test guideline protocols and ultim ately on the risk m a nagem e nt options that can be used for intervention or prevention.

5. Although not m o re specifically discussed, it is clear that there is a lack of inform ation
on toxicokinetics. The available data are generally lim ited to studies that are conducted in young adult anim als, but there are no guideline protocols for toxicokinetic evaluations during developm ent or in older age related to exposures and outcom e s.

6. The underlying assum p tion that the internal dose of the active form of an agent to the target site is the relevant m easure of dose clearly underscores toxicokinetics as an
essential tool that m u st 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 assessm ent activities to address various issues, including but not lim ited to (a) design of studies, (b) delivery to the fetus/neonate, (c) dose scaling, (d) toxicokinetic and toxicodynam i c considerations, and (e) route extrapolation.

A white paper on pharm acokinetics com m i ssioned by the Technical Panel (Versar Inc., 2001b) is m eant to serve as a technical resource for the application of toxicokinetics to these and other issues addressed throughout this docum ent. Another white paper on aging (Versar Inc., 2001a) also addresses issues of changing pharm acokinetics during
this lif e stage.

7. Portal-of-entry effects (i.e., respirator y, gastrointestinal, derm al) are acknowledged as being im portant in the effects of chem icals, and they m a y preclude system ic toxicity as being sentinel. Chronic oral RfDs and i nhalation RfCs have been developed for a

3-28num ber of agents, but rarely have derm al RfDs been derived. In som e cases, oral RfDs and oral cancer potency factors have been used to assess system ic toxicity from derm al

exposures. However, the derm al route of exposure can result in different patterns of distribution, m e tabolism
, and excretion than those that occur from the oral route. Derm al
contact with a chem ical m a y also result in direct derm al toxicity, such as allergic contact
derm atitis, urticaria reactions, chem ical irritation, and skin cancer.

The dose-response relationship for the portal-of-entry effects in skin is likely to be independent of any associated system ic toxicity exhibited by a particular chem ical. Therefore, there is a long-term need for the developm ent of derm al RfDs that consider both the system ic toxicity effects and the porta l-of-entry effects of individual chem icals. In addition, there is a need for data on the derm al uptake of chem icals from soil, water, and air, including inform ation about specific chem ical form s and bioavailability from different soil types that contribute to varia tions in uptake. Different exposure duration RfDs, such as acute chem ical 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 m a kes the following recom m e ndations (in no particular order) regarding the developm ent of testing procedures and guidance for their use. In identifying the need for developm ent of specific protocols, the Technical Panel is not recom m e nding that these tests be used for every chem ical
or in all circum stances, 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. Inform ation on all aspects of a chem ical should be considered in the strategy for testing, including
chem ical-physical characteristics, intended use, and toxicokinetic and toxicodynam i c (m ode of action) data, to allow a m o re efficient and targeted testing approach. In addition, the strategy should consider life stages in evaluating exposures and

outcom e s, 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 inform ation that would be relevant to susceptible subpopulations, including life stages (i.e., inform the

3-29selection of the intraspecies UF). Such studies also could provide inform ation on species differences (i.e., inform the selection of the interspecies UF). Finally, such studies can provide inform ation to conduct route-to-route extrapolations and reduce
the num ber of route-specific tests required to derive a reference value.

• D evelop 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 m a y not be protective of m o re 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 exam ple, the evaluation of toxic effects that m a y occur in old age from prenatal or early postnat al exposure (including carcinogenesis) or

prem ature 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 som e developm ental toxicity studies, expose anim als up to the tim e of testing. Som e form of “stop exposure” studies would provide useful
inform ation 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. Im m unotoxicity and cardiovascular toxicity are presently looked at only in a cursory m a nner. Carcinogenicity is currently evaluated only after chronic exposure to adult anim als. There is a need to integrate functional m easurem ents into evaluations of these and other system s.

• 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 sim ilar effects on the skin is a glaring om ission.

3-303.3. 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 m a y 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 determ ination of an LD50 in adult test species. A gross necropsy is conducted on the anim als, and histologic evaluation of target organs m a y or m a y not be conducted. Therefore, very lim ited inform ation is obtained
from the current protocol that would be useful for determ ining an acute reference value. However, a num ber of alternative study designs are available that would provide inform ation 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

m i nim u m of three dose groups with 10 anim als/sex/group are used. The anim als are dosed once on day 1 and followed for 2 weeks. Clinical signs of toxicity are recorded daily, food

consum ption and body weights are recorded on days 1–4, 8, and 14. There is an interim sacrifice
of 5 anim als/sex/group at 3 days after dosing and a final sacrifice of the rem a ining anim als at 2 weeks after dosing. At both sacrifices, hem a tological and clinical chem istry analyses are conducted, as is a urinalysis. The anim als are necropsied, organ weights are recorded, and the

organs are exam ined histologically. Because the purpose of this study design is to provide hazard and dose-response
inform ation rather than determ ination of an LD50, the dose levels should be chosen accordingly. This study would initially be conducted on adult anim als. As inform ation is obtained from other toxicology and/or toxicokinetic studies, it m a y be necessary to conduct the study with anim als at different life stages and to include other endpoints.

3-31Acclimatization123456789101112131415StudydayAcclimatization123456789101112131415StudydayAcclimatization123456789101112131415StudydayAcclimatization123456789101112131415StudydayAcclimatization123456789101112131415StudydayAcclimatizationBodyweight,foodconsumptionBodyweight,Bodyweight,foodconsumptionfoodconsumptionStudyday123456789101112131415

3-32DosingGross necropsy,organ weight,histopathology,hematology,clinicalchemistryGross necropsy,organ weight,histopathology,hematology,clinical chemistryFigure 3-6.Alternative acute toxicity protocol.3.3.2. Alternative Chronic Toxicity Testing Protocols As stated, a review of currently availa ble EPA guideline toxicology studies (OPPTS 870 Series) dem onstrates that there is no single pr otocol 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 som e detail below and are illustrated in accom p anying figures.
The intent of this discussion is to dem onstrate the advantages (and disadvantages) of exploring nontraditional testing paradigm s; however, such discussion does not constitute a

recom m e ndation for im plem entation. For m a ny chem icals, the existence of adequate (by Agency standards) stand-alone studies would preclude the need for further testing, with or without expanded or com b ined 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 exam ple of a study design that would incorporate lifetim e (in utero through old age) exposure is the expanded chronic/carcinogenic ity study (shown in Figure 3-7), which could serve as a replacem ent for a standard guideline chronic/carcinogenicity study in rats. In this expanded study, fem a le rats are assigned to treatm e nt groups, m a ted, and treated with test substance throughout gestation and lactation. W h en pups are weaned on PND 21, they are assigned individual anim al num bers and m a intained within their established treatm e nt group. Prenatal and early postnatal exposure to the test substance in this study is sim ilar to that required for the in utero carcinogenicity study that is used to evaluate food additive chem icals for regulation by the Food and Drug Adm i nistrati on’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 num ber of anim als used in this expanded study is greater than for a standard guideline chronic/carcinogenicity study because of the additional interim sacrifice; for each annual

segm ent, the sacrifice of 25 rats/sex/group is required. To reduce this num ber, the study could
be conducted with fewer anim als per segm ent (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 f o r tum o r data or will elim inate exam ination of an im portant lif e phase.
Param e ters typical of a guideline chronic/carcinogenicity study are exam ined in this expanded study (e.g., m o rtality, clinical observa tions, body weight, food consum ption, clinical

3-33M a t e G e s t a t i o n L a c t a t i o n

W e a n a n d

a s s i g n F 1 p u p s

S a c r i f i c e P a n i m a l s1 yr2 yr3 yrInterimsacrifice1Interimsacrifice2Terminalsacrifice

I n i ti a t e t r e a t m e n t

Q u a r a n t i n eImmunotoxicityscreenClinical pathologyHistopathologyNeuropathologyImmunotoxicityscreenClinical pathologyHistopathologyNeuropathologyImmunotoxicityscreenClinicalpathologyHistopathologyNeuropathologyNeurobehaviorNeurobehaviorNeurobehaviorClinicalpathologyClinicalpathologyClinicalpathologyImmunotoxicityscreen

a t e s t a t i o a c t a t i o

I n a t

t r e a e n

u a r a n t i n ti e i m t

t n n

G LNeurobehavior1 yrNeurobehavior2 yrNeurobehavior3 yrClinicalClinicalClinical

W e a n a n dpathologyInterimsacrifice1pathologyInterimsacrifice2pathology

a s s i g n F 1 p u p s

S a c r i f i c e P a n i m a l sImmunotoxicityscreenImmunotoxicityscreenImmunotoxicityscreenClinical pathologyClinicalpathologyHistopathologyHistopathologyNeuropathologyNeuropathologyFigure 3-7.Expanded chronic/carcinogenicity study.

Q M eTerminalsacrificeImmunotoxicityscreenClinical pathologyHistopathologyNeuropathology

3-34 echem istry and hem a tology, ophthalm ology, gross pathology, and histopathology). In addition, neurological and im m unological evaluations are perform e d in the adult anim als at m u ltiple intervals into old age, which, along with the fact that the anim als are exposed to the chem ical during all life stages, contributes to the superiority of this study design. Although the tem poral linear nature of this study protocol m a kes it less com p licated to conduct in the laboratory, this attribute also results in the inability to easily assess som e other
im portant endpoints, such as prenatal devel opm ent, reproduction and endocrine function, and
DNT. Additionally, by 3 years of age, when this study would be term inated, survival in laboratory rats m a y be com p rom i sed; therefore, it m a y be necessary to consider using feed
restriction to m a xim i ze the num ber of anim als available for in vivo and post m o rtem assessm ent of aged anim als. In addition, housing from birth in specific-pathogen-free facilities m a y be necessary to m a intain sufficient viable anim als for such an extended period of tim e (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 com posed of at least four segm ents: a two-generation reproduction and fertility study, an expanded chronic/carcinogenicity study, a devel opm ental toxicity study, and a DNT study. Each of these is currently conducted as a separate study. An optional continuous-breeding study

segm ent could be added to the design. W h en conducted in the rat, the unified screening study assesses all life stages of the anim als and provides a m eans to evaluate prenatal developm ental toxicity, DNT, reproduction, and endocrine function, a ll within anim als that are derived from the

sam e 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 t ypical two-generation reproduction and fertility study, with 10 weeks of treatm e nt, m a ting, 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 anim als are sel ected and/or assigned to a different study segm ent, it is assum e d that the treatm e nt group rem a ins constant for each anim al.) The parental (P)
anim als from the first generation are not im m e diately term inated; rather, they are transferred to a prenatal developm ental study segm ent. After a short rest, they are m a ted. The P m a les can be

term inated at any tim e point; the P fem a les are continued through to caesarian section on approxim a tely GD 20. The resulting F1b fetuses are processed and exam ined for external, soft tissue, and skeletal

3-35Chronic/CarcinogenicityStudy Segment1 yr2 yr3 yrMateF1M &F -continuouscohabitationOptionalContinuous BreedingStudySegmentChronic/CarcinogenicityStudy Segment1 yr2 yr3 yrPrenatalDevelopmentalToxicityStudySegmentReproduction andFertilityStudySegmentInitiatetreatment10weeks10weeksMateMateMateGestationLactationGestationLactation W e a n &

A s s i g n F 2C-sectionHistopathologyF1b fetalevaluationsPND 60TermsacF2

H i s t o p a t h o l o g y F 1

N e u r o p a t h o l o g y

o f s e l e c t F 2(P Generation)(F1 Generation)PQuar.Neurobehavioral assessmentsInterimsac1Interimsac2Developmental NeurotoxicityStudySegmentTerminalsac

W ea n &

A s s i g n F 1 a(F2Generation)(F1Generation)F1GestationGestationGestationGestationGestationGestationLactation

R e m o v e F 2 p u p s R e m o v e F 2 p u p s R e m o v e F 2 p u p s Re m o v e F 2 p u p s

Cr o s s - o v e r m a t i n g o f F 1

c o n t r o l a n d t r e a t e d p a r e n t sPrenatalDevelopmentalToxicityStudySegmentReproduction andFertilityStudySegmentInitiatetreatment10weeks10weeksMateMateMateGestationLactationGestationLactation W e a n &

A s s i g n F 2C-sectionHistopathologyF1b fetalevaluationsPND 60TermsacF2

H i s t o p a t h o l o g y F 1

N e u r o p a t h o l o g y

o f s e l e c t F 2(P Generation)(F1 Generation)PQuar.Neurobehavioral assessmentsInterimsac1Interimsac2Developmental NeurotoxicityStudySegmentTerminalsac

W ea n &

A s s i g n F 1 a(F2Generation)(F1Generation)F1GestationGestationGestationGestationGestationGestationLactation

R e m o v e F 2 p u p s R e m o v e F 2 p u p s R e m o v e F 2 p u p s Re m o v e F 2 p u p s

Cr o s s - o v e r m a t i n g o f F 1

c o n t r o l a n d t r e a t e d p a r e n t s

3-36MateF1M &F -continuouscohabitationOptionalContinuous BreedingStudySegmentQuar. = QuarantineP = ParentalF1 and F2 = 1st and 2nd filial generationsFigure 3-8.Unified screening study.Study lines are not drawn to scale.abnorm a lities, as is typical in an OPPTS 870.3700 study. At necropsy, however, the Pgeneration anim als receive an extended postm ortem exam ination, according to the procedures for the two-generation reproduction and fertility study, that includes sperm m e asures for the

m a les and extensive histopathology of the reproductive and other organ system s for both sexes. The expanded chronic/carcinogenicity study segm ent, using F1 anim als, would continue as described above concurrently with all other segm ents of the unified screening study but continuing well past the tim e that the others have been term inated. The other F1 pups that are selected as second-generation parental anim als in the reproduction and fertility study segm ent are treated for 10 weeks and then undergo the standard reproductive functional assessm ents, as specified in the OPPTS 870.3800 guideline. Because a num ber of F2 pups from this generation will continue on into the DNT study segm ent, som e additional observations are required during the lactation segm ent of the second generation. Specifically, F2 pups are selected and assigned
f o r neurobehavioral assessm ents on PND 4 (at the tim e of litter standardization). Preweaning observations include weekly age-appropriate clinical/functional behavioral observations
conducted outside of the hom e cage and m o tor activity assessm ents on PNDs 13 and 17.
Additional assessm ents of physical, reflex, and sensory developm ent m a y also be conducted during this period.
At the tim e of weaning of the F2 pups on PND 21, those preselected for neurobehavioral

assessm ent continue into the DNT segm ent and other weanlings are sacrificed for postm ortem 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 m o rphom etric analysis). The DNT-segm e nt F2 anim als are evaluated as per OPPTS 870.6300, which includes m u ltiple assessm ents of clinical and functional observations, m o tor activity, auditory startle habituation, and learning and m e m o ry. They are m a intained until term ination (with postm ortem evaluations, including neuropathology following perfusion fixation) at approxim a tely PND 60.
Also at the tim e of weaning of the F2 pups, a decision could be m a de to either sacrifice the F1 parental anim als im m e diately (with the usual sperm m easures and postm ortem evaluations) or to m a intain them through a continuous-breeding reproduction study segm ent, sequentially m a ting the F1 adults for the production of five litters (the pups from these litters are

term inated in early lactation). This continuous-breeding study segm ent, which would extend the reproduction study for about 100 additional days, uses a standardized assessm ent protocol that has been well characterized in the peer-reviewed literature (Lam b, 1985; Lam b et al., 1985; Morrissey et al., 1989) but does not have a corresponding OPPTS guideline.

3-37As previously stated, in this unified st udy protocol, the anim als are both exposed and assessed during all life stages, and the evaluation of both structural and functional endpoints for

m

u ltiple organ system s are m a xim i zed in the overall design, for exam ple, by the inclusion of
im m unotoxicity and neurotoxicity endpoints. There is one notable exception to this statem ent in that reproductive senescence is not standardly exam ined. Nevertheless, if the two-generation reproduction study segm ent identifies problem s with fertility or cyclicity, this could be pursued

m o re rigorously by the addition of testing during the second or third year of the expanded chronic/carcinogenicity study, for exam ple, evaluating cyclicity in aged fem a le 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 m a ny fewer naive anim als for study initiation and it increases the efficient
utilization of anim als, particularly of the F2 offspring from the reproduction study. Although there are obvious benefits in using a unified screening study, there are also a

num ber of concerns or potential problem s involved with its conduct. Although it is assum e d that

treatm e nt levels and route of adm i nistration will rem a in constant across all study segm ents, this approach to dose-setting and route selection m a y not always be optim al for every phase. Generally, a tem poral nonlinear design of this nature is m o re difficult to m a nage 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 developm ental toxicity studies. The use of either strain for the unified study could com p rom i se the use of historical data for com p arison, for

exam ple, for the chronic/carcinogenicity study if the Sprague-Dawley is used and for the reproductive and developm ental toxicity studies if the F344 is used. As study com p lexity increases, so does the opportunity for error. In som e cases, a serious technical error in one study segm ent could com p rom i se subsequent study segm ents and
result in an extensive waste of anim als and resources. As with the expanded chronic/carcinogenicity study discussed a bove (section 3.3.2.1.), survival during the chronic/carcinogenicity study segm ent in this design m a y need to be enhanced via feed
restriction. Also, if the test substance interferes with reproduction or results in increased

m o rtality, the num ber of offspring that are available for assignm ent to subsequent study

segm ents (e.g., the selection of F2 anim als for the DNT phase) m a y be critically reduced. An
additional but sim ilar problem could arise when selecting F1 anim als for the expanded chronic/carcinogenicity study segm ent at the sam e tim e as for the second generation of the reproduction and fertility study segm ent, because a large num ber of offspring needs to be
available all at one tim e. Additionally, the offspring that are assigned to the

3-38chronic/carcinogenicity segm ent should be genetically diverse within each dose group and should originate from as m a ny litters as possible (i.e., not be siblings).
A num ber of possible solutions that could be used alone or in com b ination to increase the

num ber of F1 pups available for selection in other study phases include the following:

1. Reducing the num ber of anim als needed for the expanded chronic/carcinogenicity

segm ent by exam ining fewer anim als at each serial sacrifice or by abandoning the final year of evaluation, as described above.

2. Reducing the num ber of anim als assigned to the second generation of the reproduction and fertility study segm ent; however, this could com p rom i se the num ber of F1 offspring that would be available for the DNT study segm ent.

3. Standardizing litters to 10 rather than 8 pups per sex and assum i ng that no litter has less than 10 pups and that no pups die during lactation. Because som e sm all litters and neonatal pup deaths alm o st always occur, even in controls, it is wiser to design the study

m o re conservatively in order to avoid discovering that there are not enough F1 pups to assign to the later segm ent(s).

4. Assigning additional fem a les to the two-generation reproduction and fertility study

segm ent in order to produce extra F1 pups for selection. Although even a m odest
increase in the num ber in each group would increase the probability of producing a
sufficient num ber of F1 pups, a larger num ber of litters would generally be required in order to ensure genetic diversity am ong the weanlings that are assigned to the chronic/carcinogenicity study segm ent. This could be accom p lished by placing additional P-generation fem a les or breeding pairs on study, perhaps com b ined with 2:1

m a ting procedures, or by m a ting the m a les with the reproduction and fertility studysegm ent fem a les first and then with an extra set of fem a les. One adverse consequence of placing additional fem a les on study so that their litters can be used for selection of genetically diverse offspring for the chronic/carcinogenicity study segm ent is that this

m e thod results in a larger num ber 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 im m unotoxicity, specialized neurotoxicity tests, or adult onset disease or diseases of aging.

3-39Som e of the above options appear to be m o re advantageous and preferable than others; however, no recom m e ndation is proffered because the list is presented only to illustrate som e of
the m a ny possibilities that could be used in a custom ized study design. It should be noted that
sim p ly com b ining 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 num ber
of anim als that would be required to conduct the two studies individually. No additional anim als are required over the reproduction and fertility study al one, and there is greater efficiency in the use of the F2 offspring when the DNT study is conducted in that group.

3-404. FRAMEWORK FOR SETTING ACUTE, SHORT-TERM, LONGER-TERM, AND CHRONIC REFERENCE VALUES

As noted in Chapter 2, the Technical Panel is recom m e nding 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 assessm ents for health
effects known or assum e d to be produced through a nonlinear and/or threshold m ode of action. Although there has been a dichotom y between cancer and noncancer risk assessm ent in term s of the underlying assum p tion about the linearity or nonlinearity of the dose-response curve, there is

a m ove toward harm onization am ong approaches for all health effects (Butterworth and Bogdanffy, 1999; Bogdanffy et al., 2001). This includes recognition of the possibility that som e carcinogenic agents m a y work through nonlinear m echanism s (U.S. EPA, 1999a), whereas som e agents that produce effects other than cancer m a y work through linear m echanism s (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 m ode of action and

presum ed dose-response relationship. The approach described here is the default approach to be used when the assum p tion is a
nonlinear and/or threshold m ode of action, except for cases where other m e thods have been developed (e.g., in support of the NAAQS). This approach can and should be im proved upon or replaced when m o re specific data on toxicokinetics and m ode of action are available to allow the developm ent of a chem ical-specific or a biologically based dose-response m odel for prediction
of risks to hum ans and to susceptible individuals within the population. The acute, short-term
, longer-term
, and chronic reference values derived on the basis of the recom m e ndations 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 dosim etric adjustm e nt and application of UFs. A num ber
of recom m e ndations are m a de with regard to this process. In particular, the Technical Panel

recom m e nds incorporating the concept of life stage and expanding the endpoints evaluated as
well as consideration of duration and tim ing of exposure and latency to response in characterizing the extent of the database used for setting reference values. The Technical Panel

4-1strongly encourages the use of a narrative description of the database, including strengths and
lim itations, rather than a single confidence statem ent for support of a reference value. The adjustm e nts required for derivation of the hum an equivalent dose (HED) for oral and
derm al exposure and the hum an equivalent concentration (HEC) for inhalation exposure are
described and discussed. This is followed by recom m e ndations about the evaluation and

com p arison of data for the POD, based on an analysis of each potentially lim iting endpoint carried through the reference value derivation process, followed by selection of the appropriate health-protective reference value. Finally, the Technical Panel em phasizes that considerable use of scientific judgm ent is advisable and necessary in practically all phases of the process, especially in the application of
UFs. This review and its recom m e ndations build on the principles in the Agency’s handbook on risk characterization (U.S. EPA, 2000b), which ca lls for transparency, clarity, consistency, and
reasonableness in the risk assessm ent process.

4.1. DEFINITIONS OF EXPOSURE DURATIONS FOR USE IN SETTING
REFERENCE VALUES

The Technical Panel proposes the following de finitions of exposure duration as a first step in the developm ent of consistent approaches for the Agency. These definitions are based on exposure durations for hum ans; analogous exposure durations for rodents are indicated for the longer-term and chronic durations. The definitions are not intended to be rigid specifications,
but sim p ly general descriptions of the relevant exposure tim e period. Their application is m eant to be flexible, so that, for exam ple, if a 4-m onth anim al study is available, it m a y 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 com p atible with those definitions currently used by various program offices within the Agency. The definitions for various durations, as follow, were discu ssed at an EPA Risk Assessm ent Forum Colloquium (CDM Group Inc, 2000).
Acute: Exposure by the oral, derm al, or inhalation route for 24 hours or less.

4-2Short-term: Repeated exposure 4 by the oral, derm al, or inhalation route for m o re than 24 hours, up to 30 days. Longer-term: Repeated exposure by the oral, derm al, or inhalation route for m o re than 30 days, up to approxim a tely 10% of the life span in hum ans 5 (m ore than 30 days up to approxim a tely 90 days in typically used laboratory anim al species 6 ).
Chronic: Repeated exposure by the oral, derm al, or inhalation route for mo r e t h a n approxim a tely 10% of the life span in hum ans (m ore than approxim a tely 90 days to 2 years in typically used laboratory anim al 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 assessm ents in which the known or

assum e d exposure duration approxim a ted the exposure duration in the appropriate reference
value definition. The Panel recognizes that Program Offices m a y m a ke further adjustm e nts to the reference values depending on circum stances that are unique in their assessm ents. The Technical Panel recom m e nds 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 recom m e nded reference values for any specific chem ical. This analysis can becom e com p lex in a case where the toxicological endpoint m a y differ for the different durations of exposure.
The Technical Panel is aware that there will be data lim itations f o r an individual
chem ical that m a y preclude developm ent of all four reference values. For exam ple, currently, a chronic RfD or RfC would not ordinarily be consid ered for inclusion in the IRIS database unless

4 A repeated exposure m a y be either continuous, periodic, or interm ittent. 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 e xposure 6 hrs/day, 5 days/wk or oral exposure 5 days/wk. An interm ittent exposure is one in which there is no effect of one exposure on the ef

f ect of the next; this def i nition im plies suf f i cient tim e f o r the chem ical and its m e tabolites to clear the biological system before the subsequent exposure, that is, noncum ulative pharm acokinetics. A periodic exposure m a y or m a y not be interm ittent.

5 The lifespan value used depends on the situation under consideration. For exam ple, 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 anim al species refers to rats, m i ce, and rabbits, for exam ple.

4-3a subchronic or chronic study were available. Sim ilarly, 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 m a y wish to consider several options. One option would be to not develop a reference va lue 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 estim ate of a reference value that would be protective for a short-term exposure duration. For exam ple, the Office of W a ter (see Chapter 2) will use a longer-term health advisory for a child as a conservative estim ate for a 10-day exposure in the absence of data to derive a 10-day health advisory. Other program -specific options m i ght also be considered.
The Technical Panel is aware that tim e and resources need to be considered when
im plem enting its recom m e ndations. The IRIS program has begun to im plem ent a pilot program to test whether developm ent of the expanded array of reference values is practical and can be

accom p lished without unduly delaying the com p letion of an IRIS file. As a part of the pilot, the IRIS program will need to identify the m e thods 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 item s describe
the issues and the recom m e nded changes.

1. The parenthetical statem ent in
the current RfD and RfC
def i nitions—“with uncertainty spanning perhaps an order of

m a gnitude”—has been variously used by risk assessors and risk

m a nagers to m ean that the estim ate is at the upper end, the lower end,
or the m i ddle of the range of an B ox 4-1. Current definition s for the chronic oral RfD and inhalation RfC

RfD: an estim ate (with uncertainty spanning perhaps an order of m a gnitude) of a d a i l y oral exposure to the hum an population (including sensitive subgroups) that i s likely to be without appreciable ri sk of deleterious effects during a lifetim e

. It can be derived from a NOAEL, LOAEL, or B M D
, with UFs generally applied to reflect lim itations o f the data used. Generally used in EPA’s noncancer hea l t h

assessm ents.

RfC: an estim ate (with uncertainty spanning perhaps an order of m a gnitude) of a continuous inhalation exposure to the hum an population (i ncluding sensitive subgroups) that is likely to be without appreciable risk of deleterious effects during a lifetim e. It can be derived from a N OAEL, LOAEL, or BMD, wi t h UFs generally applied to reflect lim itations of the data u s ed. Generally used in EPA’s noncancer health assessm ents.

4-4order of m a gnitude. This statem ent has been rem oved from the proposed revision of the definition for reference value (Box 4-2), and it is recom m e nded that issues of uncertainty/variability be discussed qualitatively as part of the weight of evidence and
characterization of the database. Attem p ts to adapt such a qualitative derivation process
to form al quantitative procedures for prediction of accuracy presents m a jor difficulties. A particularly obvious difficulty is that the sam e definition and phrase were applied to
different reference values that m a y have varied m a rkedly in their underlying data and, thus, their potential for accuracy. For exam ple, the sam e “order-of-m a gnitude” range applied equally to a robust reference value with known exposures plus observable and quantifiable dose-response data derived from a segm ent of the hum an population and to a

m a rginal reference value based only on anim al data with m i nim a l supporting
inf o rm ation.

Box 4-2. Proposed revisions in the reference value definitions

Reference Value: an estim ate of an exposure, designated by duration a and route, to the hum an population (including susceptible subgroups b ) tha t is likely to be without an appreciable ri sk of adverse health effects over a lifetim e. It is derived from a BMDL, a NOAEL, a LOAEL, or another su itable point of departure, with uncertainty/variability factors c applied to reflect lim itations of the data used.

________________________________________

a The generalized durations are sim ilar 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 lifetim e), all considered to be continuous exposures throughout the duration specified.

b Susceptible subgroups m a y refer to life stages, e.g., ch ildren or the elderly, or to other segm ents of the

population, e.g., asthm a tics or the im m une-com prom ised , but they are likely to be som e what chem ical specific and they m a y 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
docum ent for any such prediction and recom m e nds that the database characterizations for reference values be approached in a com p rehensive way, as discussed in Section 4.3 to ensure that they are authoritative and as com p lete as possible in order to yield qualitative
inform ation about the range that could be predicted around the individual estim ates rather
than attem p ting quantitative evaluations of accuracy and ranges.

4-52. The term “deleterious” is considered am biguous by som e , so it has been replaced with
the term “adverse,” because the latter is m o re com m only understood in the context of data evaluation and selection of endpoints for setting reference values.

3. In the spirit of harm onization of risk assessm ent approaches for hum an health effects,
it has been recom m e nded 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 assessm ents for any type of health
effect known or assum e d to be produced through a nonlinear and/or threshold m ode of action (which m a y include U-shaped or other nonm onotonic dose-response curves as well as thresholds). In light of this recom m e ndation, the term “noncancer” has been rem oved
from the definition, denoting the m ove toward defining approaches for low-dose
estim ation or extrapolation based on m ode of action. It is recom m e nded that this issue be considered further in the deliberations by the Risk Assessm ent Forum ’ s Technical Panel
on a fram ework for harm onization of approaches for hum an health risk assessm ent.

To f u lf ill the need f o r consistency in the designation of various duration ref e rence values,
the Panel recom m e nds that the term inology for reference values be standardized. Rather than continuing to use RfD and RfC only to denote ch ronic oral and inhalation reference values, respectively, standardized term inology should be developed that denotes both duration and route of exposure. Although Technical Panel m e m b ers did not com e to agreem ent on the best way to do this (and we welcom e alternative suggestions), the term inology shown below is offered as an

exam ple of the way in which consistent labels could be developed and used. Either new
standard term inology, (e.g., reference value) could be used, or RfD and RfC could continue to be used, but they would always need to be accom p anied by the qualifying duration of exposure and, in the case of the RfD, by the route of exposure. Thus, the following alternatives for

term inology are offered:

Acute (Oral, Derm al) Reference Value or Dose, Acute (Inhalation) Reference
Value or Concentration: RfV AO , RfV AD , RfV AI ; Rf D AO , RfD AD , RfC AI or RfC A

Short-term (Oral, Derm al) Reference Value or Dose; Short-term (Inhalation) Reference
Value or Concentration: RfV SO , RfV SD , RfV SI ; Rf D SO , RfD SD , RfC SI or RfC S

4-6Longer-term (Oral, Derm al) Reference Value or Dose; Longer-term (Inhalation)
Reference Value or Concentration: RfV LO , RfV LD , RfV LI ; Rf D LO , RfD LD , RfC LI or RfC L

Chronic (Oral, Derm al) Reference Value or Dose; Chronic (Inhalation) Reference Value
or Concentration: RfV CO , RfV CD , RfV CI ; Rf D CO , RfD CD , RfC CI or RfC C

The Panel recom m e nds that endpoint- or life stage-specific reference values such as the
RfD DT (reference dose for developm ental toxicity), which were originally proposed in Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), not be derived. Rather, a sam p le 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 Rf D DT concept of a critical window of exposure for som e health effects is addressed in the adoption of the less-than-chronic reference values. This recom m e ndation does not preclude, however, using specific com m on endpoints in the assessm ent of cum u lative risk for m i xtures or chem icals that have a com m on m ode of action or for risk m a nagem e nt purposes.

4.3. CHARACTERIZ ATION 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 hum an and anim al data that are available in the open literature as well as data
subm itted to the Agency in response to various regulatory standards, data call-ins, or other requirem e nts and agreem ents.

4.3.1. Review of Studies
Data will be available from a wide variety of sources, including studies conducted according to EPA guidelines, studies conducte d by industry using Organization for Econom ic Cooperation and Developm ent or other protocol s, experim e ntal studies conducted by academ ic researchers, epidem iology studies, case reports or series, and controlled clinical studies in

4-7volunteers. 7 These studies will be of widely differing quality; EPA m u st evaluate each study to
determ ine 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
anim al and hum an studies.

All types of studies:

• W hat 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 m a ke
an evaluation?

• W ere the appropriate endpoints assessed in the study? 8 W e re the techniques used for
the assessm ent scientifically sound?

C W e re appropriate statistical techniques applied for each endpoint? W a s the power of the study adequate to detect effects?

C Did the study establish dose-response relationships? W a s a BMD lower confidence level (BMDL), LOAEL or NOAEL established?

• I s the shape of the dose-response curve consistent with the known toxicokinetics of
the test com pound?

7 Currently, OPP is reviewing its policy concerning use of hum an data from studies in which there is intentional pesticide exposure, and it has asked the National Academ y of Sciences for input on the acceptability of such studies and ethical criteria for their use under the Protection of Hum a n Subjects Rule (the “Com m on Rule”) (EPA, 2001c).

8 A chem ical m a y cause a variety of toxi c effects depending on the am ount, duration, tim ing, and pattern of exposure (i.e., continuous, periodic, or interm ittent). These effects m a y range from severe—such as death—to m o re sub tle biochem i cal, physiological, or pathological changes in one or m o re organ system s. In addition, the effects will vary depending on their latency following exposure and when the observations are m a de. Prim ary attention is given in risk assessm ent to those effects in the lower exposure range and/or the effects m o st biologically appropriate for a hum an health risk assessm ent.

4-8• D o effects fit with what is known about m ode 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 W h at 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:

• W hat were the data sources for exposure, health status, and risk factors (e.g., questionnaires, biological m easurem ents, exposure/work history record reviews, or exposure/disease registries) and what were their strengths and lim itations?

• W hat m e thods were used to control, m easure, or reduce various form s of error (e.g.,

m i sclassification or interviewer bias, confounding factors and potential effect

m odifiers) and their potential im pact on the findings? W h at is the validity (accuracy) and reliability (reproducibility) of the m e thods used to determ ine exposure and

outcom e ? W h at were the response rates?

• W hat m a jor dem ographic and other personal factors were exam ined (e.g., age, sex, ethnic group, socioeconom ic status, sm oking status, and occupational exposure)?
W h at other clim ate or life stage factors were im portant for the endpoints and exposures assessed?

• W ere the f i ndings exam ined f o r biologic plausibility, internal and external consistency of the findings, and the influence of lim itations of the design, data sources, and analytic m e thods?

4-9Animal studies:

C W a s the study sufficiently docum ented (e.g., conducted in accordance with good laboratory practices)?

C W e re appropriate analytical techniques used to m easure the stability, hom ogeneity, and actual level of the test substance in the study (in the water, feed, air, etc.)?

• W as an appropriate anim al species used? 9 W a s an appropriate num ber of anim als
used? W e re sex and age considered?

C W e re the dose levels appropriate? W h at was the basis for choosing the dose levels?

C W a s an appropriate m e thod used to assign the anim als to dose groups?

• W as an appropriate route and m a trix of exposure em ployed? 10

C W a s the duration of exposure adequate for the particular study design?

• W ere possible alterations in m e tabolism considered at the higher exposure levels?

9 The laboratory anim als used m o st often are the rat, m ouse, rabbit, guinea pig, ham s ter, dog, or m onkey. W h en reviewing these studies, the risk assessor m a kes judgm ents about the ability of the study to predict the potential for toxicity in hum ans and tries to select data from the species that is m o st relevant to hum ans using the m o st defensible biological rationale. W h en available, com p arative toxicokinetics can be used to support this decision. Absent a clearly

m o st-relevant species, the m o st sensitive m a m m a lian species is used, that is, the species that shows toxicity at the lowest exposure level.

10 The m o st appropriate route of exposure is the route for which an evaluation is to be

m a de. The toxicity of the chem ical m a y differ with route of exposure because of differences in

m echanism of action or toxicokinetics (absorption, distribution, m e tabolism
, and excretion). Developm ent of data to establish dosim etry for the purpose of route-to-route extrapolation is encouraged; however, route-to-route extrapolati on is inappropriate when based exclusively upon default assum p tions regarding exposure and toxicokinetics. Even within the sam e route of
exposure, responses m a y differ due to alterations in toxicokinetics, for exam ple, dietary or water exposure versus oral gavage.

4-10Professional judgm ent 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 m ode of ac tion. These judgm ents require the input of experts trained in toxicology, statistics, and epidem iology and, often, of specialists in the structure and function of the target organ system s. Both the biological and the statistical significance of the effects are considered when m a king these judgm ents. Biological significance
is the determ ination that the observed effect (a biochem i cal change, a functional im pairm e nt, or a pathological lesion) is likely to im pair the perf orm a nce 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 m ode 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 m a ny discrete or quantal endpoints (e.g., birth defects, tum o rs, or som e discrete pathological changes), this judgm ent is m o re 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 m easures (e.g., body weight, enzym e changes, physiological m easures), this tends to
be m o re difficult, because the am ount 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 relationshi p of changes in that endpoint to other effects of the agent.
Decisions about the am ount of change to consider adverse m u st always be m a de using professional judgm ent and m u st be viewed in light of all the data available on the endpoint of

concern. All toxicological data on a chem ical m u st be reviewed before deciding whether an effect is biologically significant and adverse. Us ing a default cutoff value to define adversity for
continuous m easures m a y result in an inappropriate interpretation of data and less than optim um evaluation of a chem ical’s effects.

4.3.2. Issues to be Considered in Characteriz i ng the Database for Risk Assessment
4.3.2.1. The Weight-of-Evidence Approach A weight-of-evidence approach such as th at provided in EPA’s RfC Methodology (U.S. EPA, 1994) or in EPA’s proposed guidelines for carcinogen risk assessm ent (U.S. EPA, 1999a) should be used in assessing the database for an agent. This approach requires a critical

4-11evaluation 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 m u ch

m o re weight than those of lower quality. W h en both epidem iological and experim e ntal data are available, sim ilarity of effects between hum ans and anim als is given m o re weight. If the

m echanism or m ode of action is well characterized, this inf o rm ation is used in the interpretation
of observed effects in either hum an or anim al studies. W e ight of evidence is not to be interpreted as sim p ly tallying the num ber of positive and negative studies, nor does it im ply an averaging of the doses or exposures identified in individual studies that m a y be suitable as PODs
for risk assessm ent. The study or studies used for the POD are identified by an inform ed and expert evaluation of all the available evidence.

4.3.2.2. Use of Human and Animal Data in Risk Assessment Adequate hum an data are the m o st relevant for assessing risks to hum ans. W h en sufficient hum an data are available to describe the exposure-response relationship for an adverse

outcom e (s) that is judged to be the m o st sensitive effect(s), reference values should be based on
hum an data. Much m o re data on a wide range of endpoints typically are required to establish confidence that there are no effects of exposure. If sufficient hum an data are not available to provide the basis for reference values, data from anim al studies m u st be em ployed. It is advantageous if som e hum an data are available to com p are with effects observed in anim als,
even if the hum an data are not adequate f o r quantitative analysis. Availability of data on ef f e cts in hum ans at least allows qualitative com p arison with ef f ects observed in anim als f o r determ ining whether toxicity occurs in the sam e organ system s and whether the nature of the
effects is sim ilar or different. If no hum an data are available, reliance m u st be exclusively on
anim al data. In that case, attention should be paid to whether data are available in m o re than one species and, if so, whether the sam e or sim ilar effects occur in different species and possible sources of any observed differences.
One of the m a jor def a ult assum p tions in EPA’s risk assessm ent guidelines is that anim al
data are relevant for hum ans (e.g., U.S. EPA, 1991, 1996, 1998c). Such defaults are intended to be used in the absence of experim e ntal data that can provide direct inform ation on the relevance
of anim al data. Several types of inform ation should be considered when determ ining the relevance or
nonrelevance of effects observed in anim al m odels for hum ans. This inform ation is used in a variety of ways, from determ ining the role of m e tabolism in toxicity (Is the parent chem ical or a

m e tabolite responsible for toxicity? ) , to assessing whether hom ologous activity would be

4-12expected across species (Do hum ans share the sensitivity of the anim al m odel, or is the response
due to som e species-specific idiosyncratic reaction? ), to determ ining whether or not a threshold is likely to exist for the response (Are repair m echanism s capable of m a intaining a hom eostatic process? ). All of this inform ation m u st be we ighed in light of the known heterogeneity of the
hum an population versus the relatively inbred status of laboratory anim als used in toxicity testing studies and housed under carefully controlled environm ental conditions. Table 4-1 presents several factors to consider when evaluating the weight of evidence
about the likelihood of the occurrence of effects in hum ans that is based on anim al data (in conjunction with hum an data, if available). The table is not necessarily intended to delineate all
factors that m a y need to be considered, but rather to provide a fram ework for evaluation and interpretation. It is im portant to evaluate the database in a holistic m a nner, determ ining strengths and weaknesses that are relevant to the overall assessm ent. Each chem ical and database presents a unique set of issues that m u st be evaluated critically and thoughtfully. The dose-response nature of the data is an im portant characteristic of the database or individual study. W h en data are dose related , that is, when the incidence and/or intensity of response changes in an orderly m a nner as a function of dose, the effect should be considered to be of greater im portance than when there is no apparent association between exposure and toxicity. Note, however, that the dose-response relationship need not be m onotonic. U-shaped (or inverted U-shaped) dose-response functions are not uncom m on in toxicology. For exam ple,
a chem ical m a y induce an enzym e at low doses and inhibit it at high doses. Sim ilarly, m a ny solvent-like chem icals (including alcohol) produce increased m o tor activity at lower doses and depressed activity at high doses.
Sim ilarly, com p arative toxicokinetic/metabolism data that suggest qualitative and quantitative com p arability to that in hum ans would support the relevancy of anim al data. Evidence suggesting a difference in toxicokinetics/m e tabolism would require additional exploration regarding whether the dif f e rence(s) results in a m a jor qualitative or quantitative
difference in internal dose in hum ans. The similarity of effects between species is also an im portant aspect in characterizing
the database. Sim ilar effects in m o re than one species indicate that the effect provides increased weight of evidence for the risk assessm ent process, even if such data are not available in hum ans. In contrast, response data that show inconsistency of effects am ong studies and/or species that cannot be explained by differences in toxicoki netics/m e tabolism or tim ing and/or m a gnitude of

exposure, m a y suggest that less em phasis be placed on the effect. “Sim ilarity” does not necessarily require identical effects between speci es. For exam ple, changes in m o tor activity in

4-13Table 4-1. Factors for evaluation of the w e ight 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 m onotonic)

Toxicokinetics/

m e tabolism Qualitative and quantitative

com p arability between hum ans and anim als

Sim ilarity of effects S im ilar effects in m o re than one anim al species or in anim als and hum ans

Mode of action D em onstration of hom ologous m ode of action in anim al m odel and hum ans

Tem poral relationship C onsistent tem poral relationship between exposure and effect No identified relationship between exposure and m a gnitude of effect

Qualitative and quantitative differences between hum ans and anim als

Inconsistency of effects am ong studies and/or species that cannot be explained by differences in tim ing and/or m a gnitude of exposure or toxicokinetics/m e tabolism
Evidence suggesting that the m ode of action is species specific and irrelevant to hum ans

Lack of tem porality between exposure and effect

anim als evaluated in the neurotoxicity screening test and cognitive ef f ects in hum ans would generally be considered sim ilar, because both are indicative of changes in nervous system
function.
Mode of action inform ation is also im portant in understanding whether a particular
effect m a y be im portant for hum ans. For exam ple, a transient reduction in anogenital distance in the postnatal anim al following perinatal exposure to an anti-androgen has increased weight if the
chem ical is also known to act as an anti-androgen in hum ans. Likewise, the interpretation of increased skeletal variants observed following exposure to m a ny chem icals would be enhanced by data indicating that the m echanistic pathways for these agents and the overall biological signif i cance def i ned were also a possibility in hum ans. Mode of action data are also im portant in
determ ining whether various chem icals work by com m on m odes or m echanism s of action, which
would then be considered in a cum u lative risk assessm ent.
Another criterion that is im portant 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 m ode of action of the agent. It m a y be the case, however, that higher doses produce a shorter latency to effect than do lower doses.

4-144.3.2.3. 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 m eans a differential (greater) response at the sam e internal dose in a particular segm ent 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 m a y predispose individuals to greater response to an exposure. Life stages m a y include the developing individual before and after birth up to m a turity (e.g., em bryo, fetus, young child, adolescent), adults, or aging
individuals. Other susceptible subpopulations m a y include people with specific genetic polym orphism s that render them m o re vulnerable to a specific agent or people with specific diseases or pre-existing conditions (e.g., asthm a tics). The term m a y also refer to gender differences, lifestyle choices, or nutritional state.
It is im portant to recognize that little basis currently exists for a priori identification of susceptible subpopulations for m a ny chem icals. W ithout other data to raise suspicions, only the evaluation of effects in various segm ents of the population such as those m e ntioned above can identify susceptible subpopulations for a particular chem ical and a particular set of exposure
conditions. In som e situations, differential exposure rather than differential susceptibility per se

m a y be the critical issue (e.g., hand-to-m outh activity in toddlers). Econom ic differences m a y 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 chem ical and providing som e 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 com p lem e ntary to, but not the sam e as, the other approaches. EPA’s developm ental toxicity (U.S. EPA, 1991) and reproductive toxicity (U.S. EPA, 1996) risk assessm ent guidelines describe an approach that characterizes the database as sufficient or insufficient to judge whether a chem ical does or does not pose a hazard within the context of dose, route, duration, and tim ing of exposure. The International Program m e on Chem ical Safety (IPCS) (IPCS, 1995) proposed an approach based on the quality of inform ation gathered in developm ental 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 recom m e ndations f o r characterizing risks to children’s health within the context of the FQPA by discussing issues that would increase or decrease the level of concern.

4-15The present report endorses and extends the recom m e ndations of the 10X Toxicology
W o rking Group’s report by incorporating the issues dealing with level of concern into a
fram ework for evaluating the evidence regarding the identification and characterization of susceptible subpopulations (see below). A workshop was held recently to discuss aspects of a
fram ework for children’s health risk assessm ent and to em phasize a broader perspective on the issues that should be considered in hazard characterization, dose-response assessm ent, exposure

assessm ent, and risk characterization for children as a susceptible subpopulation (ILSI RSI, 2001). In contrast with the attention paid to children and asthm a tics as potentially susceptible subpopulations in recent years, little attention has been focused on risk assessm ent for other potentially susceptible subgroups. As outlined in Chapter 3, there currently are no requirem e nts
in EPA anim al study protocols for exposure during old age or for outcom e evaluations near the end of the life span following earlier life stage exposures. Sim ilarly, healthy anim als that are

m o re genetically hom ogeneous than hum ans are used in standard toxicity testing protocols, and
inform ation on pre-existing conditions or genetic polym orphism s is largely unavailable from
anim al studies.
Hum a n studies also usually em ploy healthy nonelderly individuals, although som e studies in m o re susceptible populations have been conducted, such as studies of the effects of air pollutants in asthm a tics. Individuals who have id entified risk factors that are not the focus of a study are usually excluded from the study sam p le. It is im portant to consider such characteristics
of the database if hum an data are used as the basis for the risk assessm ent. As can be seen in Table 4-2, several issues m u st be considered in assessing the potential
for som e subpopulations, including different life stages, to have greater susceptibility than others
to a chem ical. These include the timing (life stage)-response relationship, indicating greater susceptibility to exposure at som e 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 le vels of exposure in different subpopulations.
Another im portant consideration is whether effects are observed at the sam e dose but
with a shorter latency in different subpopulations. Additionally, differences am ong groups in

term s of the seriousness and reversibility of effects m u st be considered. For exam ple, an agent

m a y produce relatively m ild and reversible neurological effects in adults but produce perm anent behavioral im pairm e nt following in utero exposure. It is also im portant to keep in m i nd that
ef

f ects that m a y initially appear to be reversible m a y re-appear later or be predictive of later
adverse outcom e s. This is probably best exem plified by certain outcom e s following a

4-16Table 4-2. Factors for evaluating evidence regarding identification and characteriz a tion of susceptible subpopulations a

Factor Increased w e ight Decreased w e ight

Tim i ng (life stage) - response relationship Effects occur at greater

m a gnitude at one or m o re life stage(s) No difference in effects at different life stage(s)

Type of effect Different types of effects in specific subpopulations Sam e effect(s) across all potential subpopulations
Dose-response relationship Effect occurs at lower exposures in one or m o re 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 ef f ects Effects different in seriousness or degree of reversibility in specif i c subpopulations and/or differences in later consequence of an initially reversible ef f ect No differences between subpopulations in seriousness and/or reversibility of ef f ects, or in later consequences of an initially reversible ef f e ct

a Subpopulations m a y be defined by gender, individuals at different life stages (fetus, child, adult, elderly), differences in genetic polym orphism s, and/or pre-exis ting diseases or conditions that m a y result in differential sensitivity to adverse effects from exposure to a specific toxic agent.

developm ental exposure; for exam ple, an initial depression in birth weight or weight gain or subtle developm ental retardation m a y be indicators of m o re serious abnorm a lities later in life.

4.3.3. Characteriz a tion of the Extent of the Database
The derivation of an RfD or an RfC is a m u ltifaceted process that involves the coordination of data gathering and evaluation, an alysis and judgm ent in varying proportions, and integration of all the inf o rm ation available. A vital part of the chronic RfD and RfC derivation process that relies heavily on judgm ent, for exam ple, is the current approach to characterizing
the database. For exam ple, the m i nim u m 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

4-17a high confidence chronic RfD or RfC is a chronic study in two species, a single two-generation reproductive toxicity study, and a developm ental toxicity study in two species by the appropriate route of exposure.
The Technical Panel is recom m e nding a som e what different approach. Instead of specifying particular studies, this approach em phasi zes the types of data needed (in term s of both
hum an and anim al data) for deriving reference values and recom m e nds the use of a narrative description of the extent of the database ra ther than a single confidence statem ent. The Technical Panel believes that this approach encourages the use of a wider range of inform ation in deriving reference values that take into consideration the issues of duration and route of exposure, the tim ing of exposures, the types and extent of endpoint assessm ent (i.e., structural and function), the susceptible subpopulations evaluated, and the potential for latent effects and/or reversibility of effects. In addition, this appr oach encourages the identification of data that
would be needed or useful for im proving the risk assessm ent for a particular chem ical or group
of chem icals. To characterize the database, the Technical Panel has developed a description of a
“m inim al” 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 judgm ent is necessary when evaluating the extent of the database for a particular chem ical. Defining the extent of the database requires an overall evaluation and judgm ent as to where in the m i nim a l–robust
continuum the available database should be ch aracterized. The Technical Panel purposely did not define additional categories between m i nim a l and robust (m oderate), and the Panel has serious concerns about developing such categories because of the tendency to try to characterize a database with single word descriptors. In stead, we strongly support a narrative description of the extent of the database, with em phasis on the strengths and lim itations of the data. It should
also be noted that a database that is less than m i nim a l should not be used to derive a reference
value. Rather than presenting separate “m inim al” and “robust” database descriptions for each type of reference value that m i ght 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, derm al, or inhalation routes of exposure). Additionally, it is expected
that the dif f e rent types of ref e rence values f o r a particular chem ical will be developed within the

sam e assessm ent. In this m a nner, the entire database for a chem ical m a y be relied upon in the developm ent of each of the different values (e.g., im portant and relevant insights m a y be gleaned

4-18from toxicity studies for exposure durations other than those directly corresponding to the type of reference value being developed).
A m i nim a l database as
defined above can be used to set
ref e rence values, but the lim itations of such a database should be clearly recognized and discussed in the narrative description. For exam ple,

a m i nim a l database m a y provide data on only one duration or route of exposure or it m a y 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 hum a n d a ta available, route-specific toxicity data are lim ited t o dose -response data applicable to the duration in question with assessm e n t o f endpoints other than

m o rtality. A study showing only effect levels for m o rtality or other
extrem ely severe toxicity would not be sufficient to set a reference value.

Robust Database: includes extensi v e hum an and/or anim al toxicology data that c over route-specific inform ation on m a ny health endpoin t s , durations of e xposure, tim ing of exposure, life stages and susceptible subpopulations. In the absence of com p lete hum an data, m echanistic and other data show the relevance of the anim al data for predicting hum an response. Specifically, the doseresponse data for the reference value in question includes endpointspecific data (e.g., developm ental toxicity, neurotoxicity) coupled with toxicokinetic inform a t i o n as needed for route-to-route extrapolation. The toxicity studies include the evaluation of a

v a riety of endpoints (e.g., hem a tological, clinical, histology o f 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 reversib i lity of effects and laten c y to response, taking into consideration the possibility that less-than-chronic exposure m a y l e a d to effects at

som e period o f t i m e after exposure. Biological and chem ical characteristics of the exposure and ou t c o m e s , as well as known lim its on reserve capacities and repair of dam a ge, form the basis for determ ining the appropriate length of follow-up.

database would address issues of potential toxicity in hum ans and anim als and include data on several durations and routes of exposure as well as a thorough assessm ent of a variety of health endpoints. It would also include sufficient da ta on toxicokinetics and m ode action to provide
extensive inform ation for extrapolation of effects to hum ans, including potentially susceptible subpopulations. A com p lete database on a si ngle health endpoint that does not contain
inform ation on other endpoints of possible relevancy would not necessarily constitute a robust database, nor would a database that provides co m p lete inform ation 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 m ean 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 judgem e nt will be required to determ ine which UFs are appropriate in each

case.

4-19A critical assessm ent of the extent and quality of the database will inf o rm 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 m o re inform ation from additional toxicology studi es, toxicokinetic studies, structure-activity relationships, and hum an data becom e s 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 hum an health risk assessm ent. As this additional inform ation
becom e s available, the use of UFs will likely decrease. The ultim ate objective is to account for
all hum an health endpoints resulting from exposures over all life stages from before conception to the elderly adult. The optim um assessm ent considers subtle ef f ects that im pact an individual’s quality of life as well as so-called “frank” effects (death and m a jor disease). The evaluation should

encom p ass im m e diate health outcom e s as well delayed responses to an exposure (i.e., latent responses), although m o st 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

assessm ent 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?

• A re 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 m a trix of exposure relevant to the specific reference value being
derived?

C Is the duration of exposure appropriate for the specific reference value being derived?

4-20C Is the anim al species and strain appropriate for extrapolation to hum ans?

C To what degree m a y the biological endpoints be extrapolated (qualitatively and quantitatively) to hum ans?

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 com pound?

C Are the m e tabolism and toxicokinetics in the anim al species sim ilar to those of
hum ans?

C Has the dose-response curve been replicated by or is it consistent with data from other laboratories and other test species?

• H ave the data for all relevant endpoints been adequately m odeled by the BMD or other appropriate quantitative analysis to determ ine the m o st sensitive endpoint(s)?

C How well is the toxicity characterized? Do the results of all the studies indicate the possibility of ef f ects on particular system s that have not yet been explored sufficiently or do they indicate that additional studies m a y 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 m u st be m a de and procedures applied before the final derivation of a
reference value. This section sum m a rizes the current procedures and points out assum p tions

m a de and areas for im provem e nt 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)

. Adjustm e nt of the study dose/exposure for duration is described in Section 4.4.2, and derivati on of a HED or HEC is discussed in Section
4.4.3.

4-21Other issues are discussed briefly in Section 4.4.4, such as varying levels of response at the BMDL, BMCL (lower confidence lim it 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 benchm ark 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 sum m a rizes 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 m o st sensitive species as the dose rate of an agent increases” (U.S. EPA, 2002c). The underlying assum p tion is that if the RfD or the RfC is derived to preven t the critical effect from occurring, then no other effects of concern will occur; in addition, this approach assum e s that the relationship of various health effects for a particular chem ical is m a intained across species. The Technical Panel is concerned that pres enting only a single critical effect and the critical study from which it was derived in the IRIS sum m a ry table that appears at the beginning
of each RfD or RfC file m a y not provide enough inform ation to the reader who is unfam iliar
with risk assessm ent and thus could be m i slead ing. Presenting a single endpoint as a POD for a system ic effect, for exam ple, cannot capture the nature of the dose-response curve for that particular endpoint, nor does it convey the possibility that other m o re serious endpoints m a y have a dose-response character m a rkedly different from the less serious endpoint. For exam ple, an agent m a y have a clear progression of responses with increasing dose that is seen as one type of effect at the lowest exposure level (e.g., prot einuria in the case of cadm i um ), but at a higher level it produces additional effects (proteinuria PLUS GFR decrem ents) and at the highest level

even m o re types of effects (proteinuria PLUS GFR decrem ents PLUS osteom alacia). Each of
these effects could have a m a rkedly different dose-response character. Focusing on a single critical effect also does not reflect the situation in which other types
of effects m a y be found at sim ilar levels of expos ure or the variety of health outcom e s that m a y result when an exposure significantly exceeds the RfD or the RfC. Most im portantly, in light of
the Technical Panel’s recom m e ndations for deriving an expanded num ber of reference values for different durations and routes of exposure, th e lim itations of focusing only on the critical effect

4-22becom e apparent because the m o st sensitive endpoi nt m a y be different for different durations or routes of exposure. Layered upon this com p lex consideration of dose-response is the further com p lication that all of the exposure levels producing these effects are or should be adjusted to a hum an equivalent exposure at the tim e of their com p arison. These adjustm e nts m a y profoundly affect
what is considered the m o st sensitive organ or system

. Ef f ects that occur at the sam e external inhaled concentration but in different organs in the sam e exposed anim als (e.g., effects in the liver and the nasal cavity) m a y have quite different HECs, based on the current RfC

m e thodology (U.S. EPA, 1994), because the underlying basis for the adjustm e nt used for system ic effects is m a rkedly different from that used for portal-of-entry effects between anim als
and hum ans. This adjustm e nt procedure is discussed further below but is noted here because of its interrelationship with identif ying what is to be considered a critical ef f ect. These aspects all support the case that a m o re com p rehensive approach to setting reference values requires a m o re extensive and system atic analysis of endpoints than has typically been conducted in the past. In the approach proposed here, the selection of the POD
would be sim ilar to the current critical effect approach (e.g., U.S. EPA, 1994) and would include the use of sound scientific judgm ent 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 sam p le reference values, with selection of the lim iting value(s) protective of all endpoints as the final step (the sam e approach would be used for deriving a
POD for low-dose m odeling, as discussed in the proposed cancer risk assessm ent guidelines [U.S. EPA, 1999a]).
For exam ple, the dose-response curves would be m odeled for several adverse endpoints and the corresponding BMDs and BMCs and their lower 95% confidence lim its (BMDLs/BMCLs) calculated (U.S. EPA, 2000c) or NOAELs determ ined if dose-response

m odeling is not possible. Next, duration adjust m e nt to the continuous exposure scenario would be perform e d for each endpoint, with further adjustm e nt to the corresponding HECs using the
RfC m e thodology (U.S. EPA, 1994) or adjusted BMDLs or NOAELs for oral or derm al 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 chem ical-specific data, such as known toxicokinetic differences between the laboratory anim al species tested and hum ans, and m ode of action inform ation would be applied to the adjusted values for each relevant endpoint. The sam p le reference values would

4-23then be com p ared across endpoints and organ system s to determ ine which are the m o st relevant for use in deriving the final reference value for each exposure duration that will be protective of
the hum an population (including susceptible subgroups).
The Technical Panel recom m e nds the use of a m o re visual and graphic exposureresponse array to depict the PODs for all releva nt endpoints for various routes and durations of

exposure, som e what 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 com p arison of relevant endpoints and values. (See exam ples 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 m a tch the intent of the reference value regarding species or duration. For exam ple, chronic RfD and RfC values are intended by definition to be for “a continuous exposure to the... hum an population.” Doses or exposures from studies in which anim als are exposed for less than a lifetim e or in which worker populations are exposed only during wo rking hours require adjustm e nt to continuous exposure in order to be concordant with the intended duration of the reference value (see
Rozm an and Doull, 2000; Rozm an 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 adjustm e nts is discussed, as is the applicability of these procedures to various routes of exposure. The Agency has invested considerable tim e and effort into exploring these aspects for the
inhalation route. A m a jor point that will becom e apparent in this discussion is that

m e thodologies for duration adjustm e nt via the inha lation route are currently in place as part of the existing m e thodology for the chronic RfC and as proposed for ARE derivations, whereas no

com p arable docum ents yet exist for the oral or derm al routes of exposure.

4.4.2.1. Duration Adjustment Procedures for Inhalation Exposures to Continuous-Exposure Scenarios Adjustm e nt 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 anim als and hum ans (U.S. EPA, 1994). Operationally, this is accom p lished by

4-24applying a C n x t product 11 for both the num ber of hours in a daily exposure period and the

num ber of days per week that the exposures are perform e d. In an inhalation study in which
anim als are exposed to 100 m g /m 3 for 6 hours, 5 days per week, the adjustm e nt to a continuous exposure concentration would consider both hours per day and days per week:

100 m g /m 3 × 6/24 hrs x 5/7 days/wk = 17.9 m g /m 3

,

with 17.9 m g /m 3 being the concentration adjusted for continuous exposure. Study designs that include exposures 7 days/wk, for exam ple, pren atal developm ental toxicity studies and DNT studies, do not require the 5/7 days/wk adjustm e nt. Exposures from hum an occupational studies are m o st often reported as 8-hr tim eweighted averages (TW A s) and are therefore al so discontinuous. Adjustm e nt of these exposures to derive a HEC is explained below in Section 4.4.3. These adjustm e nt procedures im ply that the C × t product and not C is associated with the endpoints observed; this m a y be restated as im plying that the area under the curve (AUC), C × t, rather than the peak concentration, C, is the dosim eter associated with toxicity. Although neither
of these dosim eters m a y be dem onstrable experim e ntally to be the appropriate m easure of dose, the Agency uses adjustm e nt to a continuous inhalation exposure based on the C × t relationship

as a m a tter of policy.
W h en applied to a discontinuous inhalation exposure regim e n from an experim e ntal study, adjustm e nt to a continuous exposure will always result in a lower value of C and m a intain

a m easure of total exposure, that is, C × t. Thus, application of this procedure provides an

autom a tic m a rgin of protectiveness for chem icals for which C alone m a y be appropriate, and it
ref l ects the m a xim u m dose f o r agents f o r which total or cum u lative dose is the appropriate

m easure. W h en 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 chem icals that support
the use of C x t or that offer alternative m odels for adjustm e nt of exposure duration.

4.4.2.2. Duration Adjustment for Inhalation Developmental Toxicity Studies—A Current Exception A notable exception to duration adjustm e nt of inhalation exposures is for inhalation developm ental toxicity studies in which this practice historically has not been done. The current

11 W h ere C n = C 1 , as described in Section 4.4.2.3.

4-25guidelines for developm ental toxicity risk assessm ent (U.S. EPA, 1991) recom m e nd against duration adjustm e nt (i.e., from a discontinuous to a continuous exposure) as a default procedure
unless toxicokinetic data are available to indicate an accum u lation with continuous exposure. This is contrary to the default approach used for other types of studies in which duration adjustm e nt is done without a requirem e nt for toxi cokinetic inform ation. In fact, for other types of studies, toxicokinetic inform ation is often used as the basis for m oving away from the default adjustm e nt. Furtherm ore, although the effects of som e agents that cause developm ental toxicity have
been shown to be m o re a function of peak con centration (Nau, 1991), the effects of other agents have been shown to be related to either AUC or C, depending on the tim ing of exposure and the developm ental tim ing of the organ system affected (Terry et al., 1994). In addition, recent studies have shown that the developm ental effects of certain agents that have a short half-life, such as all-trans-retinoic acid (Tzim a s et al., 1997) and ethylene oxide (W eller et al.,1999), or a very discrete exposure period, for exam ple, hypertherm ia (Kim m e l et al., 2002), are a function of
AUC.
On the basis of this inform ation and the rationale used for duration adjustm e nt for other health effects (i.e., that exposure adjustm e nt based on C × t tends to be m o re health protective),
the Technical Panel recom m e nds that duration adjustm e nt procedures to continuous exposures based on C × t be used as a default procedure for inhalation developm ental toxicity studies as it
is for other health effects from inhalation expos ure. The Technical Panel also urges continued developm ent of data, m odeling, and im proved procedures for dose-duration adjustm e nts related to developm ental toxicity.

4.4.2.3. Duration Adjustment for Acute Reference Values—Discontinuous Scenarios of
24 Hours or Less As discussed above, the m a gnitude of response to a toxic chem ical exposure usually depends on both the concentration and the duration of the exposure, such that the com b ination of
these com ponents, C × t, determ ines the response and, by logical extension, the internal dose of a
chem ical at the target tissue. In deriving acute, short-term , or longer-term reference values, there

m a y be a need to specifically adjust or present these values under alternative C × t com b inations.
For exam ple, an acute reference value m a y 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 m e thodology for developm ent of AREs (U.S. EPA, 1998a). This section presents the adjustm e nt procedures recom m e nded in the draft ARE m e thodology.

4-26Because of the recognized lim itations of the C x t m odel, a m odification has been developed such that C n × T = k, with n being em pirically derived. the values of the “n” exponent are shown in Figure 4-1. the

Figure 4-1. Concentration-by-duration plot show ing the effect of the exponent in the C n x T = k on extrapolation across time.

Source: Adapted from ten Berge et al., 1986.

current version of the Agency’s ARE m e thodology, 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 num ber of chem icals 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”

4-27Note that for any degree of downward slope with increasing duration (lines m a rked 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 longe r duration. Several possible approaches for extrapolation in this situation could be envisioned. One approach would be to assum e a value of 1 for “n,” such that C n × 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 m o st chem icals so far exam ined has shown an appreciable downward slope (e.g., 0.8 < n < 3.5 [ten Berge et al., 1986]). The optim al approach for extrapolating from one dose-duration response situation to another is the use of a physiologically based pharm acokinetic m odel (PBPK) m odel. The principle of using PBPK m odels 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 inform ation using PBPK m odels requires a chem ical database that is rich in toxicity data; theref ore, this approach is not applicable to m o st chem icals f o r which
toxicokinetic data are scarce or nonexistent. In the absence of such a database to support the developm ent of a PBPK m odel, the approach recom m e nded by the draft ARE m e thodology is the use of chem ical-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 adjustm e nt approach (i.e., averaging to continuous exposure), when applied to m u ltiple 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 chem ical-specific data to inform duration adjustm e nt, the response has

m o st often been related to the sim p le C × t product. This is also the default in the draft ARE

m e thodology for adjustm e nt to longer durations. For adjustm e nt to shorter durations, the ARE

m e thodology conservatively recom m e nds that there be no change in concentration. Further investigation would increase confidence in the basic assum p tions m a de for the
latter two m e thods of duration adjustm e nt, including the applicability of the C x t relationship over spans of exposure from m onths 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.

4-284.4.3. Derivation of a HEC or a HED
Anim al data often form the basis for dose-response assessm ent. By definition, the IRIS
risk values are for hum ans, thereby m a king anim al-to-hum an extrapolation requisite. The specific point of this extrapolation is to estim ate from anim al exposure inform ation the hum an exposure scenario that would result in the sam e response. The sim p lest m a nner in which this

m a y be done is application of an anim al-to-hum an UF (discussed further below), typically with a
value of 10; in application this m eans that hum ans are assum e d to be m o re sensitive to ef f e cts than are anim als by a factor of 10.
Much of the RfC m e thodology (U.S. EPA, 1994) focused on im proving the science underlying the anim al-to-hum an UF, segregating it into toxicokinetic and toxicodynam i c

com ponents and providing generalized procedures to derive dosim etric adjustm e nt factors (DAF). Application of DAFs to the anim al air borne exposure values yields estim ates of the
concentration that would result in the sam e concentration to hum ans, that is, the HEC. Application of a DAF in the calculation of a HEC is considered to address the toxicokinetic aspects of the anim al-to-hum an UF (i.e., to estim ate from anim al exposure inform ation the
hum an exposure scenario that would result in the sam e dose to a given target tissue). Current Agency practice is to accom m odate uncertainty about the rem a ining toxicodynam i c com ponent through application of a partial anim al-to-hum an UF (10 0.5 , which is typically rounded to 3). The theoretical basis for deriving DAFs used in calculating HECs, along
with recom m e ndations for im provem e nt of this process, is discussed in this section. Exposures from hum an occupational studies ar e m o st often reported as 8-hr TW As for exposures during work days (5 days/wk). As with discontinuous exposures of anim al studies (e.g., 6 hrs/day, 5 days/wk), exposures from occ upational studies are also adjusted to derive
continuous HECs relevant to the hum an populati on (U.S. EPA, 1994). As described below for
anim al data, the optim al approach is to use a biologically m o tivated m a them atical, or PBPK,

m odel. An occupational exposure can be extrapolated in the sam e fashion as interm ittent exposure regim e ns from experim e ntal laborator y anim als, using particle deposition or PBPK

m odels with hum an exertion (work) ventilation rates and exposure durations appropriate to the occupational setting.
In the event that a PBPK m odel or required physicochem i cal and physiological

param e ters are not available, the default approach for hum an exposure scenarios is to adjust by
the def a ult occupational ventilation rate and f o r the interm ittent work week schedule. The ventilation rate adjustm e nt is based on the assum e d am ount of air used by a worker during the work period, that is, half of the daily ventilatory capacity of an adult m a le hum an is assigned (10

4-29m 3 of 20 m 3 total) to the 8-hour occupational exposure (i.e., instead of 1/3 or 8/24 hrs) (ICRP, 1994). By basing this adjustm e nt on a functioning physiological param e ter, that is, a fractional ventilatory capacity based on the assum p tion that activity levels are higher in this setting than in others, such as at rest or asleep, this adjustm e nt m a y be considered to have a toxicokinetic basis.
The 8-hour TW A concentrations are m u ltiplied by this factor, 10/20 m 3 , and the product is considered to be an average continuous airborne concentration. In parallel with the anim al studies, an adjustm e nt for days per week (usually 5/7 days/wk) is also m a de, if applicable. This adjusted airborne concentration is considered to be a
HEC. This default calculation, as with those desc ribed below for extrapolation from anim al data, was developed for the general hum an population. It m a y be appropriate to further evaluate this approach or to develop an alternate default approach to ensure adequate consideration of
intrahum an variation. Currently, no procedures parallel to the inhalation RfC m e thodology exist for deriving
either oral or derm al hum an equivalents from an im al data. Default factors (usually of 10) are routinely applied to address the issue of anim al-to-hum an extrapolation. Thus, no parallel to the HEC, that is, a HED, is derived nor are other adjustm e nts applied to the anim al oral or derm al
dose.
This section recom m e nds that dose adjustm e nts sim ilar to those by which HECs are
estim ated be explored in deriving HEDs for oral and derm al exposures. This would be

accom p lished in a m a nner parallel to the HEC derivation, by instituting and applying a DAF to
anim al oral or derm al exposures. Specific r ecom m e ndations are also presented and discussed concerning the basis for deriving DAFs for HED calculation. These recom m e ndations, along with current procedures f o r estim ating hum an equivalent values, are illustrated in Figure 4-2. This figure also dem onstrates how calculati on of the HEC through application of a DAF is considered to address the toxicokinetic but not the toxicodynam i c com ponent of the anim al-tohum an extrapolation. Procedures outlined in this figure for deriving a HEC m a y be applied to any anim al 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 chem ical-specific PBPK

m odel param e terized for the species and regions (e.g., respiratory tract) involved in the toxicity, as shown on the left-hand side in Figure 4-2. W h en sufficiently param e terized, a PBPK m odel is capable of calculating internal doses to a target organ from any exposure scenario in an anim al

4-30Oral/Dermal/InhalationExposureChemicalSpecific PBPKModelHEC/HEDInhalation ExposureOral/Dermal ExposureExposure conditions,Exposure conditions,e.g., mg/m3, hr/d, d/wke.g., mg/kg-day, d/wkAdjust toContinuousExposure24 hr/d, 7 d/wkAdjusttoContinuousExposure(7 d/wk)Application ofDAF(for toxicokinetics)Application ofDAF(for toxicokinetics)HECCurrentHEDProposed

4-31DAF = DosimetricAdjustment FactorFigure 4-2.Current and proposed generalized proceduresfor deriving HECs or HEDs from animal exposures.and then estim ating what hum an exposure would result in this sam e internal dose, that is, the
HED or the HEC. A form al DAF is not calculated in this process; rather, the m odel itself serves
as a DAF in estim ating HECs or HEDs. However, constructing a PBPK m odel is an
inform ation-intensive process that requires m u ch chem ical-specific data, including route-specific
data. Such sophisticated data and m odels are available usually for only a subset of chem icals
that have extensive databases. It should be noted that even these sophisticated m odels are often param e terized on the
basis of adult m e m b ers of the species. Many of the param e ters critical to PBPK m odel solutions are sensitive to life stages, such as lung function/developm ent in hum ans (Pinkerton and Joad, 2000), for which no or few data are available. Thus, these m odels are available but often cannot specifically address species differences at life stages other than m a ture adults (and then usually

m a les). The Technical Panel encourages research and data gathering to support the construction
of PBPK m odels, it endorses attem p ts to produce PBPK m odels that are sensitive to life stages, and it supports fully attem p ts to produce tem p late m odels for suites of related chem icals, 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 com p lexity 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 anatom ic 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 rem o tely. These factors are all em ployed in determ ining the appropriate DAF. For HECs, DAFs are applied to the “duration-adjusted” concentration to which the anim als were exposed (e.g., to a weekly average). The generalized DAF procedures m a y also em ploy chem icalspecific param e ters, such as m a ss transport coefficients, when available. In lieu of such data, however, default procedures that yield generalized adjustm e nts are recom m e nded. Although these generalized procedures were developed from the existing scientific understanding of the relevant processes, they have not been com p rehensively evaluated (e.g., using data from hum ans and anim als). They are explained fully in the RfC m e thodology (U.S. EPA, 1994).
For exam ple, the m a nner 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 hum ans and rats and applying it to the external exposure concentration for rats. The current default values used for both the hum an and the rat

4-32extrathoracic surface area are single estim ates from the literature and are apparently estim ated
f r om adult specim e ns. The ventilation m easure f o r hum ans is set at a def a ult value of 20 m 3 , and
the ventilation m easure for rats is based on an algorithm of body weight (from U.S. EPA, 1988).
A m a jor assum p tion m a de in this particular adjustm e nt is that the distribution of a gas in the region of interest is uniform , although it is known to be highly nonuniform (Kim bell et al., 1993, 1997). Data are not available to address this sim p lified assum p tion directly. Use of the

m e thod, for exam ple on effects in the extrathoracic region, results in a DAF of about 0.2, such
that the resultant HECs are about 20% of the anim al-duration-adjusted concentration. Although
inform ation is not yet available to address this assum p tion, indications are that resolution with
actual data m a y produce DAFs that are m u ch closer to unity, that is, that are near the anim aladjusted concentration.
In com p arison 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 rem o te to the site of absorption in the pulm onary region) is usually accom p lished by creating a ratio of the blood:gas partition coefficient for the laboratory anim al species to the hum an value. The ratio is used as the DAF and applied to the experim e ntal exposure concentration. In lieu of data on the values for blood:gas partition coefficients for the chem ical or when the data indicate the ratio to be >1, the
default assum p tion is that the ratio of anim al coefficient to hum an coefficient is 1, and therefore
the DAF would be 1. However, available data on partition coef f i cients f o r a num ber of

com pounds indicate that the anim al/hum an 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 recom m e nds further investigation into using data-derived values in constructing the anim al/hum an ratios—even when m u ch greater than 1—in place of the default.
The default dosim etric adjustm e nt procedure for particulate substances is an em pirical

m odel that estim ates regional deposition only, although it is recognized that with the developm ent of the relevant data, clearance and the retained dose m a y be used as a DAF (U.S. EPA, 1994). The DAF for particles is m o re specifically term ed the regional deposited dose ratio
and is derived from a norm a lizing factor (surface area being the recom m e nded factor for all three regions of the respiratory tract), the ratio of anim al-to-hum an m i nute volum es (where the hum an default value is the traditional adult value of 13.8 L vs. the adult value for the relevant anim al),
and the ratio of anim al-to-hum an regional fractional deposition. Physiological param e ters used
in estim ating the regional deposition include body weight, m i nute volum e, and surface area for the three areas of the respiratory tract. Defaults for the hum an values are based on adult data

4-33(e.g., 70 kg body weight, 13.8 L m i nute ventilatory volum e, etc); the anim al values are also traditionally based on adult data. To evaluate protectiveness of these default calculations for different life stages, it m a y be appropriate to perform ratio calculations using data for other life stages. As a general recom m e ndation, the Technical Panel encourages further consideration of the existing anim al-to-hum an extrapolation procedures described in current m e thodologies (e.g.,
the chronic RfC m e thodology [U.S. EPA, 1994]) and the developm ent of procedures for inhalation adjustm e nt to incorporate the m o st current scientific thought and data to address, as needed, issues of variability due to lif e stage and other intrinsic f actors. This consideration
would include exam ining the extent to which calculating a HEC (or any recom m e nded HED) addresses cross-species toxicokinetics as well as identif ication and parallel investigation into issues of toxicodynam i cs.

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 adu lts for inhaled toxic agents (including both gases and particulates) for reasons relating to either t oxicokinetics or toxicodynam i cs. It is clear for any of a variety of reasons related to toxicokinetics that an adult and a child breathing the sam e concentration of an agent such as a reactive gas m a y 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 sam e concentration of a reactive gas, for

exam ple, would be to com p are the am ount of gas breathed in (which would be directly proportional to the ventilatory volum e) with the overall surface area in the respiratory tract on which the gas m a y im pinge. The current Agency default assum p tion used in deriving HECs for particles and reactive gases that elicit respirator y effects is that the surface area of the total respiratory tract of an adult m a le, estim ated at 54.3 m 2 , is exposed to a total daily air intake of 20

m 3 , a volum e for an adult m a le derived from a com b ination 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 hum an respiratory system passes through several distinct stages of m a turation and growth that involve branching m o rphogenesis and cellular differentiation during the first several years of life and into adolescence (Pinkerton and Joad, 2000). The proportion of surface area to ventilation volum e m a y be m a rkedly different during these developm ental 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.

4-34The Technical Panel recom m e nds that issues involving dose to the young from inhalation exposures be pursued both theoretically and experim e ntally 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 estim ate of the toxicokinetic aspect of dose only, and toxicodynam i c 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 BW 3/4

as a Cross-Species DAF As indicated above, the Agency currently does not provide a procedure for calculating a
HED for oral or derm al exposure scenarios that would parallel calculation of the inhalation HEC. Instead, assum p tions are m a de regarding the com p arability of ingested or applied dose, based on

a m g /kg body-weight basis, and there is no adjustm e nt for portal-of-entry alterations to internal dose or on portal-of-entry versus system ic effects. The Technical Panel recognizes the work of an interagency workgroup to develop and propose dosim etric adjustm e nt procedures for both
derm al and oral routes of exposure in order to address those aspects of cross-species dosim etric adjustm e nt that are m i ssing in Figure 4-2. Som e of these proposals have already appeared in
abstract form (Jarabek, 2000; Hanna and Jara bek, 2000; Hubal et al., 2000; Rigas et al., 2000). Figure 4-2 dem onstrates that dosim etric adjustm e nt procedures for estim ating hum an equivalents from anim al values are not cons istent 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 em ploying it to estim ate a HED. For exam ple, in the absence of m o re sophisticated physiologically based m odels, the Agency has endorsed scaling of doses for carcinogens between species according to body m a ss raised to the 3/4 power (BW 3/4 ) (U.S. EPA, 1992). This procedure presum es that equal doses in these units (i.e., in m g /kg 3/4 /day) when
adm i nistered daily over a lif etim e, will result in equal lif etim e cancer risks across m a m m a lian species. This sam e relationship (i.e., BW 3/4 ) has been affirm ed to apply across entire phyla, including plants (Gillooly et al., 2001), for general m e tabolic rates. The basis for the less-than-full-power relationship for general m e tabolic processes (i.e.,
< BW 1 ) is thought to be related to species differences in exchange surfaces and distribution
networks that constrain concentration and flux of m e tabolic reactants (W est et al., 1997; Enquist et al., 1998). Thus, when this procedure is applied to anim al data, the resulting scaled hum an dose m a y be viewed as a valid cross-species rela tionship not only of cancer potency but also for general m e tabolic processes and, by extension, for other phenom ena involving the fundam e ntal
determ inants of concentration and flux, the sam e ones that drive basic toxicokinetics.

4-35This brief analysis of the BW 3/4 cross-species relationship and toxicokinetic processes and the Agency’s endorsem e nt of this procedure for carcinogenic agents m a kes this process a possible candidate for estim ating cross-species toxicokinetic relationships in the absence of adequate toxicokinetic inform ation. That is, BW 3/4 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 toxicokine tic aspects of cross-species extrapolation and does not address toxicodynam i c differences that m a y exist between species. As with the HEC, consideration of toxicodynam i cs is proposed to be through application of a portion of the anim alto-hum an extrapolation (10 0.5 , which is typically rounded to 3). Table 4-3 shows the general

m a gnitude of the DAFs that would be applied to various species to obtain the HED along with the default UF of 3 to cover toxicodynam i c differences.

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

Species Weight (kg) DAF a

7

4

3

Rabbit 2.5 2

Hum a n 70 1 Mouse 0.03

Rat 0.25
Guinea pig 0.5

a Derived on the basis of BW 3/4 relationship. All variables in BW 3/4 relationship containing tim e will scale BW -1/ 4

, such that anim al BW -1/ 4 /hum an BW -1/ 4 = DAF.

The Technical Panel encourages consideration of cross-species extrapolation procedures
for oral and derm al reference values, including evaluation of the m o st current scientific thought and data to address, as needed, issues of variability due to lif e stage and other intrinsic f actors. This consideration would include exam ination of the extent to which calculation of a HED addresses cross-species toxicokinetics and identification and parallel investigation into issues of toxicodynam i cs.

4-364.4.4. 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 assessm ent. 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 exam ple, the use of a quantitative dose-response m odeling 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 lim it of sensitivity for m o st long-term bioassays is in the range of 10%, as
determ ined from both the typical num ber of anim als used in bioassays (~50/group) and a low spontaneous background rate (e.g., 0.1%) for a given effect (Hasem an, 1984; Hasem a n et al., 1989). For other types of studies, however, the lim it of sensitivity m a y be lower or higher than
10%. For exam ple, in an analysis of a large num ber of standard prenatal developm ental toxicity studies with an average sam p le size of 15–20 litters , the lim it of sensitivity averaged 5% for the proportion of pups affected per litter, whereas when the quantal endpoint (i.e., the num ber of
litters af f ected) was analyzed in dam s f r om the sam e studies, the lim it of sensitivity averaged 30% (Allen et al., 1994). For data from som e hum an studies (e.g., high-quality, large epidem iology studies), the lim it of sensitivity m a y be in the range of 1 to 5%. In the BMD guidance docum ent (U.S. EPA, 2000c), the BMDL or BMCL is

recom m e nded for the POD in order to ensure that a m a jority 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% m a y not be appropriate to use in deriving an exposure to the hum an 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 ex trapolate to a lower dose/exposure level considered
to reflect a m o re appropriate level of risk (e.g., <10%).
Sim ilarly, the NOAEL is not necessarily a no-effect level, and it depends on the study design, including sam p le size, background rate, and response variability, which can be used to
determ ine the lim it of detection for a particular study. Thus, a NOAEL m a y be equivalent to no response or it m a y actually represent a substantial response rate. Previously, there has been no

attem p t to apply a factor to the NOAEL on the basis of power calculations, sam p le size, or response variability for deriving a POD, although professional judgm ent is recom m e nded in deciding whether the study is acceptable for use in deriving a POD.

4-37Adjustm e nt 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

agreem ent about any recom m e ndation for change to the current m e thodology, and it

recom m e nds that they be considered further by the Agency. The Technical Panel also

recom m e nds 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 t o true heterogeneity or diversity. For

exam ple, am ong a population that drinks water from the sam e source and with the sam e contam inant concentration, the risks from consum ing the water m a y vary. This m a y be d u e to differences in exposure (i.e., different people drinking different am ounts of water and having different body weights, different exposure frequencie s, an d different exposure d

u r at i o n s ) as w e l l as d i f f e r e n ces i n r e sponse ( e .g ., g e n e t i c differences in resistance to a chem ical dose). Those inherent differences are referred to as variability. Differences am ong individuals in a population are referred to as inter-individual variabi l ity, while differences for one individual over tim e is referred to as intra-individual variability.

Uncertainty occurs because of a lack of knowledge. It is not the sam e as variability. For exam ple, a risk assessor m a y be very certain that different people drink different am ounts of
water but m a y be uncertain about how m u ch variability there is in water inta kes within the population. Uncertainty can often be reduced by collecting m o re and bette r data, while variability is an inherent property of the popul a tion being evaluated. Variability can be better characterized with m o re data, but it cannot be reduced or elim inated. Efforts to clearly distinguish between variability and uncertainty are im portant for both risk assessm ent and risk characterization.

Source: U.S. EPA, 1997b.

Reference values are derived in a way that attem p ts to account for both the uncertainty and the variability in the data available (see Box 4-4). The existing def i nition of UF in the IRIS glossary m i xes 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 experim e ntal data. UFs are intended to account for (1) the variation in sensitivity am ong the m e m b ers of the hum an population (i.e., interhum an or intraspecies variability); (2) the uncertainty in extrapolating anim al data to hum ans (i.e., interspecies variability); (3) the uncertainty in extrapolating f r om data obtained in a study with less-than-lifetim e exposure to lifetim e 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
anim al data when the database is incom p lete.

4-38Following the logic above, the LOAEL-to- NOAEL extrapolation, the subchronic-to

chronic extrapolation, and the database deficiency factors are UFs. The variation in susceptibility am ong m e m b ers of the hum an population is a variability factor. W h en a default
f actor is used f o r intrahum an variability, however, this f actor also contains som e degree of uncertainty, because the range of uncertainty is not really known, although it is presum ed to be

no m o re than 10-fold. Rather than adding a ne w definition of variability factor, we propose to

m odify 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 experim e ntal data. The factors are intended to account for (1) the variation in sensitivity am ong the m e m b ers of the hum an population (i.e., inter-individual variability); (2) the uncertainty in extrapolating anim al
data to hum ans (i.e., interspecies uncertainty); (3) the uncertainty in extrapolating from data obtained in a study with less-than-lifetim e exposure to lifetim e 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 incom p lete.

In setting pesticide tolerances, the FQPA directs EPA to use an additional 10-fold m a rgin of safety to protect infants and children, taking into account the potential for pre- and postnatal toxicity and the com p leteness 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 docum ents (U.S. EPA, 1999b, c, 2002b). The Agency has concluded that in m a ny cases, concerns regarding pre- and postnatal toxicity can be addressed by calculating an RfD or an MOE using pre- or postnatal developm ental 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 developm ent of an RfD/RfC, OPP assessm en ts of pesticide risk to children also consider applying part or all of the FQPA factor in cer tain situations to account for areas of residual

4-39uncertainty 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
f o r an observed susceptibility. This risk m a nagem e nt approach is consistent with procedures used in the past for m a naging potential risks, although the FQPA has brought a significant new
focus on im proving the process of risk assessm ent relative to children’s health risks from
environm ental exposures. In considering the robustness of the RfC/RfD m e thodology 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 exam ple, the
database UF m a y 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 W o rking 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 recom m e nded in this review, will be adequate
in m o st cases to cover concerns and uncertainties regarding the potential for pre- and postnatal toxicity and the com p leteness of the toxicology database. In other words, an additional UF is not
needed in the RfC/RfD m e thodology 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 m a y be decreased or increased
from the default value of 10). Guidance is needed on the use of developm ental toxicity data in all reference values, including the appropriate application of UFs, because of the assum p tion that a single exposure during developm ent m a y produce an effect (U.S. EPA, 1991) and the concom itant recognition
that m u ltiple exposures m a y result in effects at lower doses in m a ny cases or cause tolerance in
other cases. These issues are chem ical specific, and scientific judgem e nt about when and how to apply UFs m u st include consideration of toxicokinetics/m e tabolism as well as the m ode 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 judgm en t. It is im perative that the IRIS docum entation contain a justification for the individual UFs select ed for a particular agent. The default factors typically used cover a single order of m a gnitude (i.e., 10 1 ). By convention, in the Agency, a value of 3 is used in place of one-half power (i.e., 10 0.5 ) when appropriate. The Technical Panel

recom m e nds that these half-power values be factored as whole num bers when they occur singly

4-40but as powers or logs when they occur in tandem

. A com posite UF of 3 and 10 would be expressed as 30 (3 ×10 1 ), whereas a com posite UF of 3 and 3 would be expressed as 10 (10 0.5

×10 0.5 = 10 1 ). It should be noted, in addition, that rigi d application of log or 1⁄2 log units for UFs could lead to an illogical set of reference values; therefore, the Technical Panel em phasizes that application of scientific judgm ent is critical to the overall process.
It is im perative that the IRIS docum entation contain a justification for the individual
factors selected for each chem ical or assessm ent and for each duration reference value. Although default factors of 10 are recom m e nded, with 3 used in place of half-power values (i.e.,
10 0.5 ) when occurring singly, the exact value of the UF chosen should depend on the quality of the studies available, the extent of the data base, and scientific judgm ent. Sound scientific judgm ent 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 m a xim u m uncertainty exists in all five areas, it is unlikely that the database is sufficient to derive a reference value. Uncertainty in four areas

m a y also indicate that the database is insufficient to derive a reference value. In the case of the RfC, the m a xim u m UF would be 3000, whereas the m a xim u m would be 10,000 for the RfD.
This is because the derivation of RfCs and RfDs have evolved som e what differently. The RfC

m e thodology (U.S. EPA, 1994) recom m e nds dividing the interspecies UF in half, one-half (10 0.5 ) each for toxicokinetic and toxicodynam i c considerations, and it includes a DAF to account for toxicokinetic differences in calculating the HEC, thus reducing the interspecies UF to 3 for toxicodynam i c issues. RfDs, however, do not in corporate a DAF for deriving a HED, and the interspecies UF of 10 is typically applied.
The Technical Panel recom m e nds lim iting the total UF applied for any particular
chem ical to no m o re than 3000 and avoiding the derivation of a reference value that involves application of the full 10-fold UF in four or m o re areas of extrapolation. This m a xim u m of 3000 applies only to the UFs discussed in the following sections and does not include the various adjustm e nt factors that have been discusse d previously (Sections 4.4.2. and 4.4.3.). Sim ilar
concerns would need to be considered for the less-than-lifetim e reference values, taking into account those UFs that are appropriate for each duration reference value.

4-414.4.5.2. Interspecies UF The interspecies UF is applied to account for the extrapolation of laboratory anim al data
to hum ans, and it generally is presum ed to include both toxicokinetic and toxicodynam i c 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., toxicodynam i cs) between the laboratory anim al species used for testing and hum ans. Seldom are there data available to inform toxicodynam i c differences. One-half the default 10- fold interspecies UF (i.e., 10 0.5 ) is assum e d to account for such differences, but m o re specific data should be used when available (see discussion of chem ical-specific adjustm e nt 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 m o re or equally as susceptible to the pollutant as are hum ans, and in the absence of any other specific toxicokinetic or toxicodynam i c 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
hum an population (interhum an 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 hum an population that are m o st sensitive to the health hazards of the chem ical 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 reaffirm s the im portance of this UF, recom m e nding 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 m o st susceptible subpopulation(s). Various authors who have evaluated the intraspecies UF using data from anim al or
hum an studies (as sum m a rized by Dourson et al. [ 1996]) have concluded that the 10-fold default factor appears to be protective when starting from a m e dian response—by inference a NOAEL

assum e d to be from an average group of hum ans. Renwick and Lazarus (1998) considered data on toxicokinetics and toxicodynam i cs to support the idea that the 10-fold intraspecies factor can be divided into two factors to account for kinetics and dynam i cs. W h en they evaluated the

com posite 10-fold factor to account for variability in both kinetics and dynam i cs, they concluded
that a 10-fold factor would cover the vast m a jority (>99%) of the population. These evaluations, however, did not specif i cally consider children as part of the range of hum an variability when evaluating the adequacy of the intraspecies UF.

4-42In papers that have evaluated this factor for the general population as well as for specific subpopulations, including children (Renwick a nd Lazarus,1998; Renwick, 1998) and the elderly (Abdel-Mageed et al., 2001), the 10-fold intraspecies factor appears to be sufficient in m o st cases, and chem ical-specific factors often indicat e a requirem e nt for less than a l0-fold factor. Renwick (1998) indicated that the 10-fold factor is m o re likely to be sufficient if developm ental toxicity data are available on the specific agent. Calabrese (2001) reviewed the data available on

a num ber of chem ical classes and concluded that the young are often m o re susceptible than
adults but that there is a not-inf requent occurrence of greater susceptibility in adults. The

som e tim es greater sensitivity am ong the elderly than am ong m a ture adults appears to be related prim arily to reduced renal clearance (Abdel-Mageed et al., 2001; Skowronski and AbdelRahm an, 2001). The Technical Panel urges continued research and evaluation of the sim ilarities 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 docum ented by Dourson et al. (1996). These include 2/46 RfCs and 13/346 RfDs (overall f r equency 3.6%). In those cases where developm ental ef f ects were the m o st sensitive endpoint (0 RfCs, 6 RfDs), reduction of the intraspecies UF from 10 to 3 was based on data
derived either from hum an data showing which age groups or tim e periods were m o st susceptible (e.g., m e thyl m e rcury exposure to the developing fetus) or from an anim al study with support
from strong hum an or other data (e.g., Aroclo r 1016 in utero exposure in m onkeys, strontium

induced 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 som e 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 chem ical’s m ode of action) so th at 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

recom m e ndation of the Toxicology W o rking Group of the 10X Task Force (U.S. EPA, 1999b) that reduction of the intraspecies UF from a defau lt 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

4-43m u ch the intraspecies UF m a y be reduced m u st be linked to how com p letely the susceptible subpopulation has been identified and their sensitivity described (vs. assum e d). At the other

extrem e, a 10-fold factor m a y som e tim es be too sm all because of factors that can influence large
dif f e rences in susceptibility, such as genetic polym orphism s. The Technical Panel urges the developm ent of data to support the selection of th e 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 m a y be altered, depending on the m a gnitude and nature of the response at the LOAEL. It is im portant to consider the slope of the dose-response curve in the range of the LOAEL in m a king the determ ination to reduce the size ofthe LOAEL-toNOAEL UF. Several papers have described the m a gnitude of the difference between the dose at
the LOAEL and at the NOAEL. For exam ple, Le wis et al. (1990) and Faustm an et al. (1994)
showed that the ratio of the LOAEL-to-NOAEL in m a ny cases was approxim a tely threefold, but
in a few cases the difference was as m u ch as 10-fold. In general, the ratio of the doses at the LOAEL and the NOAEL is likely to vary considerably am ong studies and m a y not be inform ative. 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 som e significant response is observed, rather than selecting doses that will give a m a xim u m NOAEL and a m i nim u m 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 incom p lete characteriza tion of the chem ical’s toxicity. In addition to identifying toxicity inform ation that is lacking, review of existing data m a y also suggest that a
lower reference value m i ght 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

m a gnitude, the assessor should consider both the data lacking and the data available for particular organ system s as well as life stages.
In m a ny respects, the additional 10-fold factor for infants recom m e nded by the National Research Council (NRC, 1993) and by Schilter et al. (1996) and called for in the 1996 FQPA is

4-44sim ilar to the database UF. If the Rf D/Rf C is ba sed on anim al data, a factor of 3 is often applied if either a prenatal toxicity study or a two-ge neration reproduction study is m i ssing, or a factor of
10 m a y be applied if both are m i ssing (Dourson et al., 1996). Dourson et al. (1992) exam ined the use of the database UF by analyzing ratios of NOAELs for chronic dog, rat, and m ouse studies and reproductive and developm ental toxicity studies in rats. They concluded that reproductive and developm ental toxicity studies provide useful inform ation for establishing the lowest NOAEL, and if one or m o re bioassays are m i ssing, 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 developm ental toxicity and signal the need for developm ental data on specific organ system s (e.g., detailed nervous system , im m une system , carcinogenesis, or endocri ne system ), then the database factor should
take into account whether or not these data are available and used in the assessm ent and their potential to affect the POD for the partic ular duration RfD or RfC under developm ent. If the RfD/RfC is based on hum an data, a sim ilar assessm ent regarding the com p leteness of the database is necessary. Inform ation on life stages and organ system s m a y com e from either
anim al or hum an studies. If data on specific life stages or organ system s are unavailable or
lim ited data suggest that availability of m o re extensive data m i ght decrease the POD, this should be taken into account in assigning a database UF. For exam ple, depending on the database and
what is known about the chem ical, the lack of a two-generation anim al reproductive toxicity study m i ght be considered a deficiency even if the reference value is based on hum an data. In any case, the size of the database f actor to be applied will depend on other inf o rm ation in the
database and on how m u ch im pact the m i ssing data m a y have on determ ining the toxicity of a
chem ical and, consequently, the POD.

4.4.5.6. Subchronic-to-Chronic-Duration UF As indicated earlier, a duration adjustm e nt 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 subchr onic study on the assum p tion that effects
from a given com pound 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 adjustm e nt of the exposures from interm ittent 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. Som e work has been published on this aspect of extrapolation (Lewis et al., 1990; Pieters et al., 1998). Guidan ce for replacem ent of the default factor of 10 by

4-45CSAFs m a y be forthcom ing. It would be appropria te to incorporate such data into applicable

assessm ents. 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 m a y 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 m a y 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 com m e nts located
about the MF are in the RfC m e thodology (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 judgm ent and assessm ent. Som e exam ple applications are also given, such as accounting for sm all sam p le size or for poor exposure characterization in the principal study. The definition in the IRIS glossary gives sim ilar exam ples. The description of the database UF shows substantial sim ilarity to that of the MF. Text
on the database UF indicates that this factor attem p ts to recognize that without a com p rehensive array of endpoints there is uncertainty as to whether all possible toxicologic endpoints at the various life stages are adequately addressed. W ithout this inform ation, uncertainty rem a ins as to
whether the critical ef f ect chosen f o r Rf D or Rf C derivation is either the m o st sensitive or the

m o st appropriate. There are only seven cases in IRIS for which an MF has been applied: RfDs
for chrom i um III, chrom i um VI, nitrite, 1,1-biphenyl, and m a nganese and RfCs for m e thyl 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 developm ents in the IRIS process include the obligation for risk characterization
within the assessm ents. A central aspect of ri sk characterization includes discussing confidence and uncertainties in the quality of data used and the “clarity, transparency, consistency and
reasonableness” of the assessm ent (U.S. EPA, 2000b). W ithin the risk characterization, the assessor has a pathway provided to discuss and analyze all aspects of uncertainty about the database, including the adequacy or lim itations of the database, directly in the assessm ent.

4-46The Panel considers the purpose of the MF to be sufficiently subsum ed in the general
database UF. The Panel also notes that the risk characterization section of assessm ents m a y be used to provide a full and com p lete characterization of all uncertainty, including any residual uncertainty that m a y not be addressed by the other UFs. In view of these factors, the Panel

recom m e nds 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 chem ical-specific data perm it the replacem ent of com ponents of interspecies or in ter-individual variation with data-derived values
in the context of the traditional default fram ework as developed by Renwick (1993) and revised by IPCS (1994). The following is a brief discussi on of available m e thodologies that prom ote the
use of CSAFs in risk assessm ent. Renwick (1993) described the use of toxicokinetic and toxicodynam i c data as a m eans of replacing the traditional 10-f o ld saf e ty f actors f o r hum an sensitivity and experim e ntal anim al-tohum an extrapolation in developing acceptable daily intakes. His data-derived approach assigns default values for both toxicokinetic and toxicodynam i c differences within each traditional 10- fold safety factor. Specifically, Renwick propos ed dividing both the interspecies and the interindividual UFs into a factor of 2.5 for toxicodyna m i cs and a factor of 4.0 for toxicokinetics. IPCS (1994) has adopted the data-derived approach initially proposed by Renwick (1993), with a slight m odification in the UF for inter-indivi dual variation (3.16 for toxicodynam i cs and 3.16 for toxicokinetics). IPCS has used this approach in several of its recent risk assessm ents (e.g., IPCS, 1998), and EPA is proposing a sim ilar approach for boron (U.S. EPA, 2001b). IPCS has developed a draft guidance docum ent (IPCS, 2001) to assist risk assessors in the use of experim e ntal data in deriving CSAFs for interspecies differences and hum an variability in dose/concentration response assessm ent. CSAFs have been adopted because they
better describe the nature of the refinem e nt to the usual default approach. For several years, EPA used a m o re qualitative approach to m odif y the usual 10-f o ld default values (Dourson et al., 1996). Recently, it has used a data-derived approach as one of the

m e thods to derive a UF for boron (U.S. EPA, 2001b). EPA has not yet established guidance for the use of chem ical-specific data for deriving UFs, but the division of UFs into toxicodynam i c and toxicokinetic com ponents is in the RfC

m e thodology (U.S. EPA, 1994). EPA’s assessm ents of data assum e a division of both

4-47interspecies and intraspecies UFs into toxicokinetic and toxicodynam i c com ponents that have assigned default values of 3.16 (10 0.5 ) each. The Agency will develop its own guidance f o r the
use of CSAFs in risk assessm ent, based on som e of the available m e thodologies (e.g., IPCS).
The Technical Panel would like to caution the user that for m a ny substances there are relatively few data available to serve as an adequate basis to replace defaults for interspecies
dif f e rences and hum an variability with m o re inf o rm ative CSAFs. Currently, relevant data f o r consideration are often restricted to the com ponent of uncertainty related to interspecies
differences in toxicokinetics. Although there are fewer relevant data with which to address the
other four com ponents nam e ly—interspecies (ani m a l-to-hum an) differences in toxicodynam i cs, intraspecies (hum an) variability in toxicokinetics, intraspecies (hum an) variability in toxicodynam i cs, and adequacy of the database—it is anticipated that availability of such
inf o rm ation will be needed to apply CSAFs. Specif i cally, the data-derived CSAF approach f o r any single substance is necessarily determ ined principally by the availability of relevant data. The extent of data available is, in turn, of ten a function of the econom ic im portance of the substance, and this is frequently related to the extent of potential hum an 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 chem ical-specific or biologically based dose-response m odel is probabilistic analysis. W h en the available data are suf f i cient to m eaningf ully characterize the
distributions of interest, a probabilistic approach would provide results as a distribution rather than as a single m easure for the dose/concentration-response. For exam ple, distributions could be used for inputs into a toxicokinetic m odel to derive a distribution of internal dose m e trics. Also, the approaches described in the draft IPCS guidance docum ent (IPCS, 2001) are am enable to probabilistic analysis. Probabilistic analysis f o r hum an health assessm ents generally has been conf ined to the exposure variables. In deriving hum an health toxicity reference values, inter-individual variability in toxicokinetics and toxicodynam i cs is usually represented with a UF because data are insufficient to support a m o re quantitative representation of these sources of inter-individual variability. Several studies have been published addressing the use of probabilistic data for
health assessm ents (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

recom m e nds that the Agency f u rther evaluate approaches such as probabilistic analysis f o r characterizing variability and uncertainty in toxicity ref e rence values.

4-484.4.7. Summary of Key Points from the Case Studies Two case studies were developed to illustrate m a ny of the recom m e ndations in this report. The studies are for two hypothetical chem icals: 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 expos ure (acute, short-term , longer-term
, and chronic). Very little is known about the m ode of action for Inhalate except for the tum o rigenic effects in liver, which are thought to be produced as a result of prolonged cytotoxicity caused by oxidative

m e tabolism

. Thus, a nonlinear m ode of action is assum e d 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 inhalati on reference values, including carcinogenic effects

assum e d to have a nonlinear dose-response. Luteinate belongs to a class of chem icals known to work through a neuroendocrine m ode
of action. In order to ascertain its potency and confirm a sim ilar m ode of action, a num ber of
short-term studies were conducted, followed by testing in m o re 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 exam ple of the usefulness of m ode-of-action inform ation in establishing the shortand long-term effects of Luteinate on relevant targ et organ system s at different life stages. Such
inform ation enables the developm ent of a targeted robust data set for use in establishing reference values for various durations of exposure.
A detailed sum m a ry of the case studies is provided in Appendix B. Several key points
are described here that dem onstrate the use of the proposed fram ework outlined in this chapter. First, the data are reviewed and characterized on the basis of the hazard and dose-response
inform ation, including consideration of the weight-of-evidence factors discussed in Section 4.3.2, above. A narrative statem ent is used to describe the extent of the database for each
chem ical as well as the gaps in inform ation that would m a ke the database m o re robust.
Dosim e tric adjustm e nts were m a de to derive HECs in the case of Inhalate. For Luteinate, adjustm e nts for oral exposure were m a de on a BW 1 basis and do not incorporate the BW 3/4 scaling factor or other DAF, as further work is needed on the harm onization of approaches for deriving of oral and derm al 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.

4-49Then, the reference values are derived by considering all of the relevant data for each duration reference value, weighing the evidence in the database, developing sam p le 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 m u ltiple-duration reference values are not without precedent. Several offices within EPA, as well as ATSDR and
the AEGL com m ittee, derive m u ltiple duration values f o r various purposes (see review in Chapter 2). The derivation of sam p le reference values in selecting the final reference value also
is not a new idea. For exam ple, EPA’s assessm ent for m e thylm e rcury included the derivation of

sam p le RfDs from prospective longitudinal st udies of the effects of in utero exposure to

m e thylm e rcury (Table 4-4) in deriving a chronic RfD (U.S. EPA, 2001d).
Sam p le RfDs were derived from a num be r 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 m e thylm e rcury exposure and deficits in neuropsychological function were reported. The sam p le RfDs converged on 0.1 : g/kg/day, providing strong support for the appropriateness of this value. This RfD is not a developm ental RfD per se, and its use is not restricted to pregnancy or developm enta l periods. The RfD, derived from an overall evaluation of the database, is applicable to lifetim e daily exposure for all populations, including sensitive subgroups. In a recently released health assessm ent docum ent on 1,3-butadiene (U.S. EPA, 2002d),

sam p le RfCs were derived for determ ining the chronic RfC.

4-50Table 4-4. BMDLs, ingested dose, a nd RfDs for various endpoints from the Faroe Islands, New Z e aland, and the NRC integrative analysis

Test b

BNT Faroes

W hole cohort PCB adjusted d Lowest PCB tertile CPT Faroes

W hole cohort PCB adjusted Lowest PCB tertile CVLT Faroes

W hole cohort PCB adjusted Lowest PCB tertile Finger Tap Faroes

W hole cohort PCB adjusted Lowest PCB tertile Geom etric m ean

W hole cohort PCB adjusted Lowest PCB tertile Median values

Faroes New Zealand Sm oothed values

BNT Faroes CPT Faroes CVLT Faroes Finger Tap Faroes MCCPP New Zealand MCMT New Zealand Integrative

All endpoints BMDL a (ppb mercury cord blood)

5 8 71 40

4 6 49 28

103 78 52

7 9 66 24

6 8 65 34

48 2 4

48 48 60 52 2 8 3 2

3 2 Ingested dose b ( : g/k g /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 RfD c ( : g/k g /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 BMDL 05 s from NRC (2000), Tables 7-4, 7-5, 7-6. Hair m e rcury was converted to blood m e rcury using a 250:1 ratio and an assum p tion of equivalent m a ternal and cord levels.

b Calculated using a one-com partm e nt m odel.

c Calculated using an UF of 10.

d There was significant co-exposure to PCBs in the Faro e Islands study, with PCB cord tissue concentrations available for about half of the whole cohort. Anal yses were perform ed 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 Nam i ng Test; CPT = Continuous Perfo rm ance Test; CVLT = California Verbal Learning Test; MCCPP = McCarthy Perceived Perform ance; MCMT = McCarthy Motor Test.

4-515. RECOMMENDATIONS

A num ber of recom m e ndations have been m a de in other parts of this report. This chapter

sum m a rizes those recom m e ndations, based on the Technical Panel’s review of the RfD and RfC

process. The Technical Panel assum e s that it will be possible to im plem ent som e of the

recom m e ndations 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 m ode of action inform ation early in the process, as well as when to im plem ent new testing guidelines in the process of developing a data package on a particular chem ical. OPPTS, together with scientists in other parts of the Agency, will consider the recom m e ndations to develop additional or alternative testing guidelines as part of the Harm onized Health Effects Test Guidelines (870 Series). As part of its deliberations, the Technical Panel considered the recom m e ndations of the Toxicology W o rking Group of the 10X Task Force (U.S. EPA, 1999b, and Appendix A). The
Technical Panel endorses those recom m e ndations and extends and expands them to deal with a broader view of life stages, tim ing and duration of exposure, and evaluation of endpoints, both
structural and functional. The recom m e ndations are presented here in the order of the chapters in which they appear. Further discussion of the specific recom m e ndations can be found in the earlier chapters.

Chapter 2
The Technical Panel concurred with the recom m e ndation 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, derm al, and inhalation routes and that they be included in the IRIS database for use by EPA program s, where a pplicable. 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 recom m e ndation in Chapter 2 concerning the derivation of lessthan-lifetim e reference values. This review was undertaken to determ ine what inform ation is currently gathered with regard to life stage assessm ent, endpoint assessm ent, route and duration of exposure, and latency to response. The intent of this review is not to suggest that additional

5-1testing be conducted for each and every chem ical in order to fill in the inform ation gaps identified for those organ system s evaluated. Nor is it suggested that the alternative testing protocols discussed in this chapter be conducted for every chem ical or becom e part of current toxicology testing requirem e nts or that these alternative protocols are the only options available. Rather, it is the goal of this docum ent to provide a basis for the developm ent of innovative alternative testing approaches and the use of such data in risk assessm ent. The recom m e ndations
include:

• D evelop a strategy for alternative approaches to toxicity testing, with guidance on how and when to use existing and newly recom m e nded guidelines.

• D evelop guidelines or guideline study protocols that will provide m o re system atic
inform ation on toxicokinetics and toxicodynam i cs (i.e., m echanism or m ode of action), including at different life stages.

• D evelop protocols for acute and short-term studies that provide m o re

com p rehensive data for setting reference values.

• M odify existing guideline study protocols to provide m o re com p rehensive coverage of life stages for both exposure and outcom e s.

• C ollect m o re inform ation from less-than-lifetim e exposures to evaluate latency to
ef

f ect and reversibility of ef f ect.

• D evelop guidelines or guideline study protocols to assess im m unotoxicity, carcinogenicity, and cardiovascular toxicity at different life stages.

• E xplore the f easibility of setting derm al ref e rence values f o r direct toxicity at the portal of entry, including sensitization.

Chapter 4
The Technical Panel discussed a num ber of m odifications to the existing fram ework 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

5-2illustrate m a ny of these concepts are sum m a rized in Chapter 4 and discussed in detail in Appendix B. The recom m e ndations for im provem e nt and expansion of the existing approaches
are aim e d at taking a broader approach to the characterization of the entire database and what
im pact that will have on the dose-response assessm ent and risk characterization of a chem ical.
Included are recom m e ndations for setting several less-than-lifetim e reference values, broader characterization of the database instead of using a checklist of a m i nim u m set of studies for setting a reference value, using an exposure-response array and carrying appropriate and relevant endpoints through the derivation of sam p le 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 RfD DT ) but using a process that facilitates the evaluation of risk to particular subgroups for specific program office needs, including cum u lative risk assessm ent. The specific recom m e ndations follow:

1. Include the acute, short-term , longer-term
, and chronic reference values derived on the
basis of the recom m e ndations in this report in IRIS af ter 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
aim e d at clarifying that the approach to reference values discussed here is intended for
risk assessm ents for any type of health effect known or assum e d to be produced through a
nonlinear and/or threshold m ode of action (which m a y include U-shaped or other

nonm onotonic dose-response curves as well as thresholds). Thus, the term “noncancer”
has been rem oved from the definition in the spirit of overall harm onization of risk

assessm ent approaches for hum an health effects because it has been recom m e nded 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 m ove toward defining approaches for low-dose estim ation or extrapolation based on m ode of
action.

5-3The term “deleterious” has been replaced with the term “adverse,” because the latter is

m o re com m only used and understood in data evaluation and selection of endpoints for setting reference values. The parenthetical statem ent in the current RfD and RfC definitions, “with uncertainty spanning perhaps an order of m a gnitude,” has been

rem oved from the proposed revision of the definition for reference value, and it is

recom m e nded 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

recom m e nds that the term inology for reference values be standardized; this standardized

term inology should reflect both duration and r oute of exposure. Consistent term inology

recom m e ndations for reference values are proposed in this report, but additional suggestions are welcom e.

5. The Technical Panel recom m e nds that endpoint-specific reference values per se not be developed, including the RfD DT , which was originally proposed in Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991). Rather, a sam p le 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 recom m e nded; 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 W o rking 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, m e dium , or low should be used in describing the extent of the database. The extrem es for the extent of the database (i.e., m i nim a l or robust) are defined in Chapter 4. The narrative approach is
intended to em phasize the types of data available (both hum an and anim al) as well as
research needed to f ill the data gaps that could im prove the derivation of ref e rence

5-4values, and it should encourage the use of a wider range of inform ation in deriving reference values, taking into consideration the life stages evaluated; the issues of tim ing, duration, and route of exposure; the types and extent of endpoint assessm ent (i.e.,
structure and f unction); and the potential f o r latent ef f e cts and/or reversibility of

responses.

8. Duration adjustm e nt procedures to continuous exposures for inhalation developm ental toxicity studies should be done in the sam e way as for other health endpoints.

9. Additional consideration of the HEC and HED derivation m e thodology 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 determ ine the range of num erical 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 sam p le reference value derivation, with selection of
the lim iting value(s) as the final step, rather than on a single “critical study” and “critical
effect.”

12. It is im perative that the IRIS docum entation contain a justification for the individual
factors selected for each chem ical or assessm ent, because rigid application of UFs could lead to an illogical set of reference values. Although default factors of 10 are

recom m e nded, with 3 used in place of half-power values (i.e., 10 0.5 ) 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 judgm ent. Sound scientific judgm ent 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).

5-513. The Technical Panel recom m e nds that if there is uncertainty in m o re 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 determ ine whether the derivation of a reference value is appropriate. In addition, the
Technical Panel recom m e nds lim iting the total UF applied to a chronic reference value for any particular chem ical to 3000. This m a xim u m of 3000 applies only to the UFs and does not include the various adjustm e nt factors discussed in Chapter 4.

14. The intraspecies UF is applied to account for variations in susceptibility within the
hum an population (interhum an 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 hum an population that are m o st sensitive to the
health hazards of the chem ical 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 reaffirm s the im portance of this UF, recom m e nding 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 m o st susceptible subpopulation(s).
At the other extrem e, a 10-fold factor m a y som e tim es be too sm all because of
f actors that can inf l uence large dif f e rences in susceptibility, such as genetic polym orphism s. The Technical Panel urges the developm ent 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 sim ilarities 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 assum e d to overlap to som e extent, the Technical Panel agrees with the 10X Task Force Toxicology W o rking Group (U.S. EPA, 1999b) that the current interspecies, intraspecies, and database deficiency UFs, if appropriately applied using the approaches recom m e nded in this review, will be

5-6adequate in m o st 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 m a nagem e nt (e.g., by rete ntion of all or part of the FQPA safety factor for pesticides).

17. The Panel considers the purpose of the MF to be sufficiently subsum ed in the general
database UF. Therefore, the Panel recom m e nds 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 toxicodynam i c and toxicokinetic

com ponents is in the RfC m e thodology (U.S. EPA, 1994) and m a y apply to the intraspecies UF as well. The Agency is encouraged to develop its own guidance, based

on som e of the available m e thodologies (e.g., IPCS).

The following issues were discussed by the Technical Panel but were considered m o re appropriate for discussion and recom m e ndation by other panels/com m ittees:

1. There have been inconsistencies in the use of BMD m odeling approaches to deriving RfDs and RfCs currently in IRIS. The Technical Panel was unable to fully evaluate these issues or to reach agreem ent about any recom m e ndation for change to current

m e thodology and recom m e nds that they be considered further by the Agency. The
Technical Panel also recom m e nds 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 recom m e nds harm onization of the approaches for HEC and HED derivation for all types of health effects. Developm ent of the appropriate adjustm e nt procedure is referred to the Harm onization Fram ework Technical Panel.

3. The Technical Panel recom m e nds that the Agency further evaluate approaches such as probabilistic analysis f o r characterizing variability and uncertainty in toxicity ref e rence values.

5-74. The Technical Panel recom m e nds further evaluation of appropriate adjustm e nt of doses for duration of exposure. The m e thod de rived from ten Berge et al. (1986) is raised as a possibility for acute exposures on the basis of its recom m e ndation in the ARE

m e thodology. Duration adjustm e nt 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.

5-8APPENDIX A: ISSUES RAISED BY THE 10X TASK FORCE

A num ber of issues were raised by the 10X Task Force 1 in its discussions of the requirem e nts for protecting children’s health and application of an additional 10X safety factor,

as m a ndated 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

com m unity 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 m a y be adequate in the m a jority of cases for protecting children’s health. However, when data specific to children’s health are

m i ssing or inadequate for a particular agent, application of the database m odifying factor in addition to the intraspecies variability factor m a y be sufficient to account for the possibility that children m a y be signif i cantly m o re sensitive than adults. This issue needs
further exam ination.

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 W o rking Group of the 10X Task Force and include factors such as (a) hum an data on pre- and postnatal toxicity; (b) pre- and postnatal toxi city in anim al studies, including effects of a
different or sim ilar type as those in adults; (c) dose-response nature of the experim e ntal
anim al data, including the dose-related incidence of response, relative potency of response, slope of the dose-response curve when the m a rgin of exposure is sm all, and how well the NOAEL or BMD is defined; and (d) relevance of the experim e ntal anim al
data to hum ans, including toxicokinetics, sim ilarity of the biological response, and knowledge of the m echanism of action. For each of these areas, criteria are given for
estim ating a level of concern for children’s health as high, m oderate, or low. The level of

concern m a y be taken into account in the uncertainty and m odifying factors applied to the RfD, although there is currently no form al process for doing so.

1 See 10X Task Force docum ents: 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-13. As indicated in the toxicology docum ent 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

assum e d that m o st endpoints of developm ental toxicity can be caused by a single

exposure. If, however, developm ental effects are m o re 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 developm ental toxicity involve repeated exposures, data are not often available on which endpoints m a y be induced by acute, subacute, subchronic, or chronic dosing regim e ns and, therefore, on which should be used in setting various
duration reference values. Further consideration of the appropriate application of developm ental toxicity endpoints to various duration reference values is recom m e nded. As part of this recom m e ndation, an in-depth review of the HED docum ent on Hazard Identification—Toxicology Endpoint Sel ection 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
Rf Ds are also set f o r pesticides, and interm ediate ref e rence values are set f o r residential exposures as well as for drinking water. Data on developm ental toxicity will often be a greater factor in calculating the acute and interm ediate reference values, and exposures to
children are m o re often of this type as well. Consideration should be given to setting
interm ediate reference values for environm ental 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 developm ental toxicity using, for exam ple, a 6-hr/day exposure regim e n, are adjusted to a continuous (24 hr/day) exposure for calculating
RfDs/RfCs. The developm ental toxicity risk assessm ent guidelines (U.S. EPA, 1991)

recom m e nded against m a king this adjustm e nt because it was assum e d that there was a threshold above which exposure would have to occur before an effect would result. This

recom m e ndation needs to be reconsidered, along with the adjustm e nt of NOAELS/BMDs in general.

A-2Several im provem e nts in testing approaches were also proposed for consideration in the 10X Task Force report as a way to im prove the assessm ent 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 assessm ent purposes. The im provem e nts to be considered
include

• pharm acokinetics that include data from different developm ental stages, perhaps done in a tiered approach as suggested in Kim m e l and Francis (1990);

• d irect dosing of neonates, especially when early exposure is of concern, because
this is the tim e when dif f e rences in m e tabolic capability are greatest;

• p erinatal carcinogenesis studies and appropriate triggers for when they should be required;

• d evelopm ental im m unotoxicity testing and appropriate triggers; and

• a dvanced DNT testing, in particular, cognitive testing that is m o re sim ilar to that
used in hum ans.

An additional issue was how to m a ke exposure assessm ents com p atible with the doseresponse assessm ent. For exam ple, how should the appropriate durations of exposure be
determ ined for toxic endpoints of concern? Should standard exposure durations be used?

A-3APPENDIX B: CASE STUDIES—EVALUATING AND SELECTING
HEALTH ENDPOINTS FOR DERIVING REFERENCE VALUES

Two case studies were developed by the Technical Panel to illustrate m a ny of the points discussed in this report. The first case study is of a hypothetical volatile chem ical for which
lim ited data are available and little inform ation is known about the m ode of action except that there is support for a nonlinear m ode of action for cancer. The second case study is of a hypothetical endocrine disruptor for which the m ode of
action is known or assum e d from other chem icals in the sam e class. This case study is used to illustrate in part how the inf o rm ation on m ode of action can inf o rm a m o re f o cused collection of data as well as the interpretation of the data and its use in risk assessm ent. 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 m odeled. However, the Technical Panel strongly encourages the use of dose-response m odeling and calculation of BMDLs or BMCLs for selection of the POD to be used as the basis for deriving reference values.

B-1CASE STUDY 1: INHALATE

Inhalate, a synthetic halogenated aliphatic alkene, is a nonflam m a ble volatile liquid at

room tem p erature. The chem ical enters the air through its industrial and com m e rcial use, prim arily as a solvent. It is also found in surface and ground water and soil upon disposal. The

m o st im portant route of hum an exposure is inhalation of the chem ical in the am bient and indoor air, although there is a lower possibility of ingestion through contam inated drinking water. Because of its high volatility, derm al exposure to the chem ical is expected to be m i nim a l. This case study illustrates the use of single or m u ltiple health endpoints for deriving
reference values for different durations of exposure following inhalation exposure. It also
illustrates the harm onized approach for all effects (including cancer) that are known or assum e d to be produced through a nonlinear or threshold m ode of action. For the purpose of illustration, results of key studies are sum m a rized 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 chem ical 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 inf o rm ation on the absorption and distribution of Inhalate in hum ans and laboratory anim als following oral, inhalation, or derm al exposure. However, sim ilar effects are seen by oral and inhalation exposures, suggesting that Inhalate or its m e tabolites reach their target sites after absorption from either exposure route. Available in vitro m e tabolic studies indicate that Inhalate is extensively m e tabolized in target tissues including the liver and kidney
of rats and m i ce. Lim ited in vitro studies with hum an tissues show a sim ilar pattern of

m e tabolism

. As discussed below, m u ch of Inhalate-induced toxicity appears to be due to its

m e tabolites. These m e tabolites have been detected in the urine of rats and m i ce f o llowing inhalation and oral exposure to the parent chem ical.

Postulated Mode of Action
No inform ation is available on m ode of action except for the carcinogenicity of Inhalate. The carcinogenic effects of Inhalate in rodent liver are attributed to oxidative m e tabolism

m e diated cytotoxicity in the target organ. The oxidative m e tabolismproduces highly tissuereactive m e tabolites that lead to tissue injury and cell death. The persistent cell prolif eration

B-2B-3Table B-1. Summary results of major inhalation exposure studies on InhalateSpecies SexExposure durationand frequencyConcentrations(mg/m3)LOAEL/NOAEL(mg/m3)HECa(mg/m3) ResponsesHuman M/F 2 hrs 4, 40, 400 40/4 40/4,2-hrHeadache, dizziness, incoordination,drowsiness, anesthesia at 2000 mg/m3Accidental exposure NA NA - Narcosis, proteinuria, hematuria6 hrs/day for 7 days(clinical exposure)b10, 20, 100, 150 20/10 4/2 Headache, dizziness, incoordination,drowsinessOccupational(>15 yrs)TWA of 56 20/NA 20/NA Dizziness, forgetfulness; changes inserum liver enzymes; increased urinarylevels of lysozymes, beta-glucuronidaseF Occupational NA NA - Menstrual disorders; spontaneousabortion; cardiac anomalies in childrenof workersRat/mouseM/F 4 hrs 0, 13, 24, 50 13/NA 13/NA,4-hrDose-related hyperactivity, ataxia,hypoactivity, narcosis6 hrs/day, 5 days/wkfor 2 wks0, 50, 100, 200 50/NA 9/NA Dose-related hyperactivity, ataxia,hypoactivity, narcosisRat M/F 6 hrs/day, 5 days/wkfor 13 wks0, 30 30/NAc– Changes in fatty acid composition of thebrainsTable B-1. Summary results of major inhalation exposure studies on Inhalate (continued)Species SexExposure durationand frequencyConcentrations(mg/m3)LOAEL/NOAEL(mg/m3)HECa(mg/m3) Responses

B-4Rat(cont)M/F 6 hrs/day, 5days/wkfor 13 wks0, 22, 39, 88 39/22 7/4 Dose-related hyperactivity, ataxia; liverhypertrophy, vacuolization ofhepatocytes, necrosis; cytomegaly,toxic nephrosis of tubular epithelialcellsMouse M/F 6 hrs/day, 5 days/wkfor 13 wks0, 28, 50, 100 50/28 9/5 Dose-related hyperactivity, ataxia; liverhypertrophy, vacuolization ofhepatocytes, necrosis; cytomegaly,toxic nephrosis of tubular epithelialcellsRat M/F 6 hrs/day, 5 days/wkfor 104 wks0, 11, 22, 44 22/11 4/2 Clinical signs of neurotoxicity; dose-related liver hypertrophy, vacuolizationof hepatocytes, necrosis, hepatocellularcarcinoma; cytomegaly, toxic nephrosisof tubular epithelial cellsMouse M/F 6 hrs/day, 5 days/wkfor 78 wkse0, 11, 28, 56 28/11 5/2Rat F GDs 70–13,6 hrs/day0, 10, 90 90/10 22.5/2.5 Decreased motor activity in pups;ataxia in damsat high doseM/F Two-generationreproductive study,6hrs/day, 5 days/wk0, 28, 100 100/28 18/5 Reduced litter size, reduced survival ofoffspring, sedation at high doseaThese values are approximate HECs derived in accordance with theRfC methodology (U.S. EPA, 1994) and Chapter 4 (this report) forcategory 3 Gases using pharmacokinetic data for Inhalate.bData included here for comparison; data from intentional humanexposures are not currently used by EPA in risk assessment.cSpecial study for neurotoxicity, no HEC calculated.dConsidered to be a lifetime exposureHEC = human equivalent conentrationNA = not availableTWA = time-weighted averageGD = gestation daypresum ably would lead to higher probabilities of cell m u tation and subsequent cancer. Liver

tum o rs were produced only at dose levels that resulted in repeated or sustained cytotoxicity and regenerative cell proliferation. This postulated m ode of action is further supported by the observation in specialized studies that neither the cytotoxicity nor cell proliferation occurred in
the CYP2E1 null m ouse or in the wild type treated with a P450 inhibitor at the sam e exposure. The weight of the evidence indicates that a m u tagenic m ode of action via DNA reactivity is not a significant com ponent of Inhalate-induced liver tum o rs in rats and m i ce. There are no data indicating that the m ode of action observed in rodents is not also likely to apply to hum ans.

Nervous System Inhalate has been found to elicit dose-dependent clinical signs of CNS effects in adult
hum ans following acute inhalation exposure and acci dental ingestion. CNS sym p tom s 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 m i ce exposed to Inhalate by inhalation following different duration of expos ures. There are lim ited data indicating that prenatal exposure to Inhalate adversely affects the developing nervous system in rats and m i ce (see Growth and Developm ent below). There are no data on the ability of Inhalate to af f ect the nervous system at other life stages (e.g., duri ng the fetal period, infancy, childhood, or old age).
The m echanism of action for the CNS effects has not been clearly established, but it is believed to be related to effects of the parent com pound on lipid and fatty acid com position of the

me mb r a ne s

.

Inhalation Exposure Several reports available in the open literature indicate dose-dependent clinical signs of CNS sym p tom s in adults exposed acutely and subacutely via inhalation to Inhalate. Males and
fe m a les exposed acutely to high concentrations (40– 4 00 m g / m 3 for 2 hours) showed dosedependent effects, including headache, dizziness, incoordination, drowsiness, and anesthesia. No effect was reported following acute exposure to 4 m g / m 3

. Si m ilar effects were observed in
adult hum an volunteers at lower concentrations (10, 20, 100, 150 m g /m 3 ) for 6 hours per day for up to 7 days, with a NOAEL of 10 m g/m 3

. An acute accidental exposure of a sm all group of workers to an unknown (presum a bly 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 presum ed that these workers were exposed to a

B-5daily TW A exposure of 56 m g /m 3

. Subjective neurological sym p tom s , including dizziness and forgetfulness, were consistently reported across studies. No other inform ation 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

m i ce following acute (13, 24, 50 m g /m 3 for 4 hours) and short-term inhalation exposure (50,100,
200 m g /m 3 6 hrs/day for 2 weeks) to Inhalate at high concentration. Sim ilar effects were observed in rats exposed at 39 and 88 m g /m 3 and in m i ce at 50 and 100 m g /m 3 for 13 weeks.
The subchronic NOAELs for rats and m i ce were 22 and 28 m g /m 3 , respectively. Chronic exposure to Inhalate at lower concentrations resulted in less serious clinical signs of CNS effects in rats (22 or 44 m g /m 3 ) and m i ce (28 or 56 m g /m 3 ). The chronic NOAELs
for rats and m i ce in these studies were both 11 m g /m 3

. It should be noted that neurological endpoints exam ined in these anim al studies are lim ited to clinical signs and histopathology. In a special study, changes in fatty acid com position of the brain were observed in rats exposed at 30

mg / m 3 (the only tested concentration) for 90 days. Pregnant rats were exposed by inhalation to Inhalate at 0, 10, or 90 m g /m 3 for 6 hrs/day on days 7–13 of gestation. Decreased m o tor activity was observed in 21- or 60-day-old pups
from dam s exposed to 90 m g /m 3

. A NOAEL of 10 m g /m 3 for developm ental neurotoxicity was identified in this study.

Oral Exposure Acute neurological effects in adult hum ans after ingestion of Inhalate are sim ilar to those seen after inhalation. Accidental exposure to approxim a tely 6–8 m L (or about 100 m g /kg/day)
resulted in narcotic effects. Neural tube def ects and eye anom alies were reported in studies of offspring of residents exposed to drinking water contam inated with Inhalate and other solvents. Exposure levels were not determ ined in this study. Single oral gavage adm i nistration of Inhalate to adult rats (1000 m g /kg) caused ataxia. Ataxia was also observed in pregnant rats treated by gavage at 900 m g /kg on gestation days (GDs) 6–19. No CNS effects were reported in a chr onic oral gavage study in rats and m i ce at 50, 100, or 300 m g /kg/day. However, neurological endpoi nts exam ined in these studies were lim ited to clinical signs and histopathology. In a study that investigated the effect of Inhalate on the developing nervous system , m a le

m ouse pups were treated by gavage at 50 or 300 m g /kg/day for 7 days (postnatal days [PNDs]

B-610–17). Hyperactivity was reported in anim als during adulthood at the high dose. No studies with exposure throughout developm ent (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 show n to induce dose-dependent liver toxicity in adult
rats and m i ce following subchronic and chronic e xposure by inhalation and oral gavage. Liver

tum o rs were also observed in chronic studies of rats and m i ce. Available data support the
conclusion that liver tum o rs were produced only at dose levels that resulted in repeated or sustained cytotoxicity and regenerative cell prolif eration. Inhalate carcinogenic effects in rodent
liver are attributed to oxidative m e tabolism

- m e diated cytotoxicity in the target organ. The
oxidative m e tabolism produces highly tissue-reactive m e tabolites that lead to tissue injury and
cell death. The persistent cell prolif eration presum ably would lead to higher probabilities of cell

m

u tation and subsequent cancer. This postulated m ode of action is further supported by the observation in specialized studies that neither the cytotoxicity nor cell proliferation occurred in the CYP2E1 null m ouse or in the wild type treated with a P450 inhibitor at the sam e exposure. The weight of the evidence
indicates that a m u tagenic m ode of action vi a DNA reactivity is not a significant com ponent of
Inhalate-induced liver tum o rs in rats and m i ce. No data exist indicating that the m ode of action observed in rodents is not also likely to apply to hum ans. There are no data that provide any insights into possible dif f e rential sensitivity across lif e stages.

Inhalation Exposure One study reported changes in serum levels of liver enzym e s in workers exposed to the
chem ical at a daily TW A exposure concentration of about 56 m g /m 3 over an 8-hour work shift. These workers, however, did not exhibit any clinical sym p tom s of liver dysfunction. Dose-related liver effects (liver hypertrophy, vacuolization of hepatocytes, necrosis) have
been observed in m i ce following subchronic exposure (13 weeks) to Inhalate at 50 or 100 m g /m 3

, with a NOAEL of 28 m g /m 3

. Dose-related liver toxicity and hepatocellular carcinom as were also found in m i ce following chronic exposure at 28 and 56 m g /m 3

. The NOAEL for liver toxicity in m i ce in this chronic study was 11 m g /m 3

. Rats showed sim ilar liver responses but at higher exposure concentrations following subchronic exposure (39 or 88 m g /m 3 ), with a NOAEL of 22 m g /m 3

. Liver toxicity and hepatocellular carcinom as were also observed at 22 or 44 m g /m 3 in a chronic study in rats. The

B-7NOAEL f o r liver ef f ects in rats was 11 m g /m 3

. It should be noted that liver effects exam ined in
these subchronic and chronic studies were lim ited to clinical chem istry, m o rphology, and histopathology.

Oral Exposure
Liver effects were observed in m i ce and rats treated subchronically (100, 300, 500

m g /kg/day) or chronically (50, 100, 300 m g /kg/day) with Inhalate via oral gavage and were
sim ilar to those seen after inhalation exposure. Mice showed m o re severe effects than did rats. Dose-related hepatocellular carcinom as were also found in treated m i ce in a chronic study.

Kidney
Available hum an and anim al studies indicate that Inhalate also has the potential to cause renal toxicity in adults. The m echanism s for the developm ent of kidney effects in hum ans and
anim als are not known. No data were available to evaluate the effects of Inhalate at life stages other than in adults (i.e., during developm ent or old age).

Inhalation Exposure Sym p tom s of renal dysfunction (proteinuria, hem a turia) have been associated with
accidental hum an exposure to anesthetic concentrations of Inhalate. Subtle or no renal effects were reported in workers exposed chronically. Increased urinary levels of lysozym e and beta

glucuronidase suggestive of m ild renal tubular dam a ge have been observed in workers exposed for an average of 15 years to a daily TW A concentration of 56 m g /m 3

. Dose-related renal toxicity (cytom egaly, toxic nephrosis of tubular epithelial cells in the inner renal cortex) was induced in rats (39, 88 m g /m 3 ) and m i ce (50, 100 m g /m 3 ) exposed to
Inhalate for 13 weeks. Subchronic NOAELs for renal effects in rats and m i ce were 22 m g /m 3
and 28 m g /m 3 , respectively. Sim ilar renal effects were observed in a chronic study in rats (22,
44 m g /m 3 ) and m i ce (28, 56 m g /m 3 ). Chronic NOAELs for renal effects in rats and m i ce were both 11 m g /m 3

.

Oral Exposure Dose-related toxic nephropathy characterized by degenerative changes in the proxim a l convoluted tubules and necrosis of the tubular epithelium were found in rats and m i ce treated with Inhalate via oral gavage for 90 days at 100, 300, or 500 m g /kg/day and for 2 years at 50,

B-8100, or 300 m g /kg/day. Subchronic and chronic NOAELs for renal effects in rats and m i ce were at 100 and 50 m g /kg/day, respectively.

Grow th and Development
Available studies in hum ans and anim als indicate that Inhalate has the potential to cause developm ental effects by inhalation and oral inge stion. Lim itations of hum an studies could not resolve whether the observed developm ental effects were causally related to the chem ical or
were a result of chance or bias. However, the epidem iologic findings are supported by anim al studies with exposure to Inhalate by inhalation a nd oral gavage that show that the developing nervous system is the m o st sensitive target in rats and m i ce.

Inhalation Exposure Epidem iologic studies of wom e n occupationally exposed to Inhalate and other related solvents have reported elevated risk of spontaneous abortion and cardiac anom alies in their offspring. Due to lim itations 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 m g /m 3 for 6 hrs/day on days 7–13 of gestation. Decreased m o tor activity was observed in 21- and 60-day-old pups
from dam s exposed to 90 m g /m 3

. A NOAEL of 10 m g /m 3 for developm ental effects was identified in this study. No studies included exposure throughout gestation and lactation to
determ ine effects at other developm ental 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 / m 3 , a concentration that also resulted in sedation and renal effects. No effects were reported
at 28 m g /m 3

.

Oral Exposure Neural tube defects and eye anom alies have been reported in studies of residents exposed to drinking water contam inated with Inhalate and other solvents. Exposure levels were not
determ ined in this study.
An increased incidence of m i cro/anophthalm ia were observed in the offspring of rats treated with Inhalate by gavage at 900 m g /kg/da y on GDs 6–19. In a study that investigated the effect of Inhalate on the developing nervous system , m a le m ouse pups were treated by gavage
with Inhalate at 50 or 300 m g /kg/day for 7 days (age 10–17 days). Hyperactivity was reported in
anim als during adulthood at the high dose. No e ffects were found at the low dose. No studies

B-9included exposure throughout gestation and lactation to determ ine effects at other developm ental stages.

Reproductive System
Available studies in hum ans and anim als suggest that Inhalate m a y have the potential to cause reproductive effects. The underlying m echanism of action for potential reproductive
effects is not known. Inhalation Exposure There is suggestive evidence of spontaneous abortion and m e nstrual disorders am ong

wom e n occupationally exposed to Inhalate. However, no definitive conclusions can be m a de
because of the lim itations associated with these studies. Reduced litter size and reduced survival of offspring were reported in rats exposed to
Inhalate at 100 m g /m 3 in a two-generation reproduction inhalation study; this concentration also
resulted in sedation and renal effects. No effects were identified at 28 m g /m 3

. The protocol used, however, was not the m o st recent one, in which reproductive developm ent (e.g., tim ing of puberty or anogenital distance) and adult reproductive function (sem en quality, estrous cyclicity) are evaluated, nor were organ weights m easured. No effects on the reproductive system were noted in any other studies.

Oral Exposure
No inform ation is available on the potential reproductive effects of Inhalate in anim als via oral exposure.

SELECTION OF HEALTH ENDPOINTS AND DERIVATION OF REFERENCE
VALUES Narrative Description of the Extent of the Database
No inform ation is available on possible m odes of action except for liver carcinogenic
effects in rats and m i ce. In this case, the m ode of action is attributed to oxidative m e tabolism

m e diated cytotoxicity and persistent cell proliferation in the liver. Persistent regenerative cell prolif eration presum ably would lead to higher probabilities of cell m u tation and subsequent

cancer. No data exist to indicate that the m ode of action observed in rodents is not also likely to apply to hum ans. Pharm acokinetic data indicate that Inhalate is extensively m e tabolized and that

m

u ch of its toxicity is due to the m e tabolites.

B-10The database for inhalation exposure is lim ited but adequate for deriving reference
values. Som e hum an data on acute, short-term , and longer-term exposures are available, although the range of endpoints evaluated and the dose-response inform ation for different durations of exposure are lim ited. The anim al data include acute, short-term , longer-term
, and chronic studies with exposures beginning in young adult anim als. The acute and short-term data
are lim ited to clinical signs of m o rbidity and m o rtality, whereas the longer-term and chronic
studies include som e histopathology as well. A developm ental neurotoxicity (DNT) study was conducted in rats with prenatal exposure lim ited to GDs 7–13 (as opposed to m o re extensive exposure throughout a m a jor part of CNS developm ent, e.g., GD 6 to PND 11 or 20 in the standard DNT study testing protocol). No other studies of prenatal or postnatal developm ental toxicity were done except for evaluations of survival and growth in a two-ge neration reproduction study in rats. However, the protocol used was one in which reproductive developm ent (e.g., tim ing of puberty or anogenital distance) and adult reproductive function (sem en quality, estrous cyclicity) were not evaluated, nor were organ weights m easured. No studies we re conducted that considered issues related to the toxicity of the agent in old age, either from earlier exposures or from exposures in aged
anim als. The database for oral exposure is m u ch m o re lim ited than the database for inhalation exposure, with acute accidental ingestion data in hum ans at a single, high-dose level resulting in narcosis and chronic drinking water exposure (no dose inform ation) associated with an increase
in birth defects. The anim al data are likewise very lim ited, with a single-dose acute toxicity study in rats in which clinical signs of m o rbidity and m o rtality were evaluated and subchronic (90-day) and chronic toxicity data in rats and m i ce indicating effects sim ilar to those seen with inhalation exposure. Prenatal developm ental toxicity data were available in rats following exposure on GDs 6–19, and an evaluation of adult neurotoxicity was conducted in m i ce following developm ental exposure on PNDs 10–17. No other developm ental toxicity data, and
no inform ation 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 anim als.

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 hum an equivalent concentrations (HECs) based on the dosim etric adjustm e nts discussed in Chapter 4 of

B-1105101520253035402 hrs-Human 4 hrs-Rat 4 hrs-Mouse GD 7 - 13-RatTwogenerationRatNOAELLOAELTiming 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-toxicityHEC HEC05101520253035402 hrs-Human 4 hrs-Rat 4 hrs-Mouse GD 7 - 13-RatTwogenerationRatNOAELLOAELTiming and duration of exposure–Species

Dose (mg/m 3 ) N eurot oxicity Neurotoxicity Neurotoxicity Developmental Neurotoxicity Repro- Neuro-toxicityHEC HEC

B-12Figure B-1. Exposure-response array of data considered for the Inhalate acute reference value.B-1305101520257d-Human GD 7 - 13-Rat2 wks-Rat 2 wks-Mouse TwogenerationRatNOAELLOAELTiming and duration of exposure–Species

Neurotoxicity Developmental Neurotoxicity Neurotoxicity Neurotoxicity Repro- Neuro-toxicity

Dose (mg/m 3 )HECHEC05101520257d-Human GD 7 - 13-Rat2 wks-Rat 2 wks-Mouse TwogenerationRatNOAELLOAELTiming and duration of exposure–Species

Neurotoxicity Developmental Neurotoxicity Neurotoxicity Neurotoxicity Repro- Neuro-toxicity

Dose (mg/m 3 )HECHECFigure B-2. Exposure-response array of data considered for the Inhalate short-term reference value.0510152025GD7 -13-RatTwo-generationRat13wks-Rat13wks-MouseTiming and durationof exposure–Species

Dos e (m g/ m 3 )NOAELLOAELHECHECNOAELLOAEL

D e v e lopmental N eurotoxi cit y Repro-, Ne uro-t o xi ci t y Ne u r o-, L i v e r, Ki dn e y T o x i c i t y Ne u r o-, L i v e r, Ki dn e y T o x i c i t yHECHEC2520

Dos e (m g/ m 3 )1510

B-1450GD7 -13-RatTwo-generationRat13wks-Rat13wks-MouseTiming and durationof exposure–SpeciesFigure B-3.Exposure-response array of data considered for the Inhalate

longer-term reference value.0510152025GD7 -13-RatTwo-generationRat104 wks-Rat78 wks-MouseTiming and durationof exposure–Species

Dos e (m g/ m 3 )NOAELLOAELHECHECNOAELLOAEL Neuro-, L i v e r, Ki dne y T o xi ci t y Ne u r o-, L i v e r, Ki d n e y To xi c i t y

Dev e lopme n t a l N eurot oxi c i t y Repro-, N e uro-toxicit yHECHEC2520

Dos e (m g/ m 3 )1510

B-1550GD7 -13-RatTwo-generationRat104 wks-Rat78 wks-MouseTiming and durationof exposure–SpeciesFigure B-4.Exposure-response array of data considered for the Inhalate

chronic reference value.this report, including dosim etric adjustm e nt of the developm ental toxicity data in the sam e

m a nner as for other types of toxicity data, as recom m e nded in U.S. EPA (1994) and Chapter 4.
No toxicokinetic m odel is available for Inhalate, so adjustm e nts to the applied concentrations for dose adjustm e nts 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 docum ent. As the effects observed are system ic, with no indica tions of portal-of-entry effects, the specific default procedures are based on the vapors of I nhalate being a category 3 gas. The principal

param e ter for interspecies extrapolation of category 3 gases, the blood:gas (air) partition coefficient (H b/g ), is unknown for both hum ans and anim als and assum e d to be 1. Therefore, the
dosim etric adjustm e nt factor (DAF) (see Chapter 4, Section 4.4.3) applied to the duration adjusted concentrations is 1, such that the duration adjusted values then becom e the HECs. Duration adjustm e nt was accom p lished by factoring the exposure concentration (in

mg / m 3 ) by 6/24 (for hours of exposure) and, where applicable, by 5/7 for the num ber of days per week exposed. As noted in Chapter 4, Section 4.4.2.1, duration adjustm e nts were not m a de to acute (i.e., less than 24-hr) single exposures.

Uncertainty Factors (UFs) An interspecies UF (A in Table B-2) of 10 1/2 was applied in all cases to anim al studies
because the dosim etric adjustm e nt procedures applied to Inhalate are considered to address the toxicokinetics portion of this UF, leaving the rest of the UF to cover interspecies toxicodynam i cs (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 estim ation of within-hum an variability and the m o st sensitive lif e 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 hum an 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 we re 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 developm ental 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-chronic

duration UF should be explored, as there m a y 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-16B-17 Table B-2. Derivation of reference values for Inhalate—inhalation exposure

Reference

value duration Exposure duration HEC (mg/m 3 ) Species Type of effect a Uncertainty factors b 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 13L d 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 9L d 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 20L d 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 1 e 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 20L d 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 1 e 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 dayB-18 A database UF of 10 1/2 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., feta l 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 LOAEL HEC of 40 mg/m 3 and a NOAEL HEC of 4 mg/m 3 in a 2- hour exposure. Animal studies also show that Inhalate has the potential to cause neurotoxicity in adults
with a LOAEL HEC of 13 mg/m 3 in a 4-hr exposure, and developmental neurotoxicity and other reproductive effects with LOAEL HEC s of 22.5 mg/m 3 and 18 mg/m 3 and NOAEL HEC s of 2.5 mg/m 3 and 5 mg/m 3 , respectively.
Default UFs of 10 1/2 (animal-to-human extrapolation), 10 (inter-individual differences), and

10 1/2 (database deficiencies: no adequate prenatal developmental toxicity studies in two species, no adequate developmental neurotoxicity study) were applied to all the NOAEL HEC s to derive sample reference values. In addition, a UF of 10 (LOAEL to NOAEL) was applied to the 4-hr rat and mouse

LOAEL HEC s. 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/m 3 (Table B2).

Short-term Inhalation Exposure The reference value for short-term inhalation exposure is based on the human data

NOAEL HEC s of 2 mg/m 3 as well as the animal developmental neurotoxicity NOAEL of 2.5 mg/m 3 andB-19 reproductive toxicity NOAEL of 5 mg/m 3 (LOAEL HEC s of 4 mg/m 3 , 22.5 mg/m 3 , and 18 mg/m 3

, respectively). In addition, the 14 day exposures of rats and mice resulted in a LOAEL HEC of 9 mg/m 3

. For the human NOAEL HEC of 2 mg/m 3 , applying a 10-fold default UF for intraspecies uncertainty and variability and a 10 1/2 -fold UF for database deficiencies results in a sample reference value for shortterm inhalation exposure of 0.07 mg/m 3 . Sample reference values based on animal data include default
factors of 10 1/2 (interspecies), 10 (intraspecies) and 10 1/2 (database deficiencies) applied to the

NOAEL HEC s for developmental neurotoxicity and reproductive toxicity (2.5 and 5 mg/m 3 , respectively) and result in sample reference values of 0.03 and 0.05 mg/m 3 . 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/m 3 . 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/m 3 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/m 3 ) 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 LOAEL HEC s range from 7 mg/m 3 to 22.5 mg/m 3 and the

NOAEL HEC s range from 2.5 mg/m 3 to 5 mg/m 3 . 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 10 1/2 (interspecies), 10 (intraspecies), and 10 1/2 (database deficiencies) were applied to

NOAEL HEC s 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/m 3 would result. Without this additional factor, the sample reference value from the developmental toxicity study was still the lowest value (0.03 mg/m 3 ), 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/m 3 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 LOAEL HEC s range from 4 mg/m 3 to18 mg/m 3

and the NOAEL HEC s range from 2 mg/m 3 to 5 mg/m 3 . UFs of 10 1/2 (interspecies), 10 (intraspecies),
and 10 1/2 (database deficiencies) applied to the chronic exposure NOAEL HEC s for neurotoxicity and liver, kidney, and reproductive toxicity data result in sample reference values of 0.02–0.05 mg/m 3 (Table B-2). Applying these UFs to the NOAEL HEC of 2.5 mg/m 3 for developmental toxicity yields a sample reference value of 0.03 mg/m 3 , 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 NOAEL HEC , 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/m 3 , the value chosen for the
chronic inhalation reference value. With regard to the liver effects of Inhalate, the NOAEL HEC s 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. A lthough 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/m 3 , 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. Environm ental fate studies have shown that it will persist in soils and will therefore likely to m ove into ground
and surface water. The general population m a y be exposed to Luteinate through consum ption of food and drinking water. Luteinate belongs to a class of pesticides for which the neuroendocrine m ode of action is
known. It was designed to be less potent than other m e m b ers of the class. In order to ascertain its potency, a num ber of short-term studies were first conducted; they confirm e d a sim ilar m ode
of

action and the f act that Luteinate was less potent. Following this, Luteinate was tested in

m o re traditional toxicology studies to establish its long-term effects and dose-response relationships. This case study provides an exam ple of the usefulness of m ode of action inform ation in establishing the short- and long-term effects of Luteinate on relevant target organ system s at different life stages. Such inform ation enables the developm ent of a targeted robust data set for use in establishing reference values for various durations of exposure.

Postulated Mode of Action
Other m e m b ers of this class of pesticides have been shown to act on the hypothalam i cpituitary-ovarian axis. These pesticides affect the hypothalam u s, leading to a decreased secretion of hypothalam i c norepinephrine (NE). Decreased NE levels result in decreased release of gonadotropin releasing horm one (GnRH) fr om the hypothalam u s. GnRH is the horm one responsible for inducing the pituitary gland to release luteinizing horm one (LH). Thus, the decrease in GnRH leads to a suppression of the pituitary LH release. These com pounds also
decrease the neurotransm itter dopam i ne, which in turn leads to a decrease in pituitary prolactin. Decreased LH and prolactin levels have the potential to im pact several organ system s at different life stages. In hum ans, there are robust pulses of the LH surge in the fetus and prior to
birth. The LH pulsatility continues with dim i nishing am plitude during the early m onths of postnatal life, and then LH secretion becom e s barely detectable during m u ch of the first decade of postnatal life. Around the age of 10, there is the reem ergence or re-awaking of LH pulses while sleeping. The natural progression from prepubertal to postpubertal status is dependent on the

norm a l function of the hypothalam i c-pituitary-gonadal axis. Likewise, m a ny of the sam e hypothalam i c m echanism s that control pituitary function and the pituitary horm ones them selves

B-21(especially LH and prolactin) play a key role in pubertal developm ent. In the adult, ovulation is dependent on sufficient LH levels. Therefore, this class of pesticides im pacts critical reproductive processes, including puberty, ovarian cyclicity, pregnancy, and lactation (m ilk quality/production). Given the role of NE and dopam i ne in the developm ent of the CNS, this class of pesticides m a y also affect the deve loping CNS; however, relevant toxicology studies have not yet been conducted. In addition, suppression of prolactin during the early postnatal period in rodents m a y lead to prostate inflam m a tion in m a le offspring. This class of pesticides has also been shown to lead to m a m m a ry tum o rs in SpragueDawley (SD) rats but not in other strains of rats or m i ce. Mam m a ry tum o rs result from the prolonged decrease in serum LH, which leads to a cessation of ovulation and eventually causes
the ovarian f o llicles to continue to secret estradiol. In concert with prolactin, estrogen acts on
the m a m m a ry gland and leads to the form ation of m a m m a ry tum o rs. However, the induction of
the tum o rs has been shown to be due to unique features of the reproductive aging process in Sprague-Dawley rats; because hum ans do not age in the sam e m a nner, this m ode of action is unlikely to be operative in hum ans.

SUMMARY OF HEALTH EFFECTS INFORMATION Studies with other pesticides in this chem ical class have found negligible differences in response (with the exception of the induction of m a m m a ry tum o rs in SD rats) am ong various
strains of rats and between rats and m i ce. 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 sum m a rized below in the context of the specific effects associated with either decreas es in LH or prolactin. In addition, the effect levels for specific toxicological endpoints are sum m a rized in Table B-3. It is im portant to note that as the spectrum of effects are m echanistically related to decreases in serum LH or prolactin, there is great sim ilarity in the doses that affect LH and prolactin levels and those that cause the
related ef f ects.

Absorption, Distribution, Metabolism, and Elimination Studies in adult rats have shown that Luteinate is rapidly absorbed following oral

exposure. It is not m e tabolized and is elim inated in the urine. No other inform ation is available. The toxicokinetic profiles of other pesticides in this chem ical class have not been extensively

exam ined either.

B-22Table B-3. Summary of endpoints and effect levels for rat studies of Luteinate

Response Exposure period Dose levels (mg/k g/day) NOAEL/LOAEL (mg/k g/day)

Decreased LH Pregnant fem a les: GDs 1–8 50, 100, 200 100/200

PNDs 22–41 25, 50, 100 50/100

Adults: 3 days 28 days 6 m onths 50, 100, 200 40, 80, 160 4, 8, 16 100/200 40/80 4/8
Two-generation reproduction study: 10 weeks - F0 13 weeks - F1 a 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 m onths 40, 80, 160 4, 8, 16 40/80 4/8
Two-generation reproduction study: 10 weeks - F0 13 weeks - F1 a 5, 10, 25, 50 5, 10, 25, 50 5/10 5/10

Altered
pregnancy

m a intenance Pregnant fem a les: 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 - F1 a 5, 10, 25, 50 5, 10, 25, 50 5/10 5/10
Delayed parturition Pregnant fem a les: GDs 6–10 100, 200, 400 100/200
Two-generation reproduction study: 14 weeks - F0 17 weeks - F1 a 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 - F1 a 5, 10, 25, 50 25/50
Delayed preputial separation PND 23–53 25, 50, 100 50/100

Two-generation reproduction study: 6–7 weeks - F1 a 5, 10, 25, 50 25/50

B-23Table B-3. Summary of endpoints and effect levels for rat studies of Luteinate (continued)

Response Exposure period Dose levels (mg/k g/day) NOAEL/LOAEL (mg/k g/day)

Attenuation of prolactin release Lactating fem a les: PNDs 1–4 25, 50, 100 25/50

Adults: 3 days 28 days 6 m onths 50, 100, 200 40, 80, 160 4, 8, 16 100/200 40/80 4/8
Two-generation reproduction study: 10 weeks - F0 13 weeks - F1 a 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 m onths 40, 80, 160 4, 8, 16 >200/NA b >160/NA b

Two-generation reproduction study: 16 weeks - F0 16 weeks - F1 a 5, 10, 25, 50 5, 10, 25, 50 >50/NA b 5/10

Reduced weight of

sem i nal vesicles and ventral prostate PNDs 23–53 25, 50, 100 50/100

Adults: 28 days 6 m onths 40, 80, 160 4, 8, 16 >200/NA b >160/NA b

Two-generation reproduction study: 16 weeks - F1 a 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/NA b

a F1 exposures are indicated for the duration of postnatal exposure, but it is assum e d that 3 weeks of prenatal exposure also occurred.

b W h en no effect on a particular endpoint wa s noted in a study, the NOAEL is indicated

as > the highest dose, and the LOAEL as NA (not applicable).

B-24Decreased 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 m a le and fem a le rats exposed to levels of 0, 50, 100, and 200 m g /kg/day, there were significant decreases in serum
LH at 200 m g /kg/day. Serum LH was significantly reduced at doses of 80 m g /kg/day and above in a 28-day study and at doses of 8 m g /kg/day a nd above in a 6-m onth study of SD rats. Levels in the latter study were sim ilar 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

m g /kg/day and higher following 10 weeks of exposure.
Serum LH was also m easured in pregnant dam s exposed to doses of 0, 50, 100, and 200

m g /kg/day on GDs 1–8 and was significantly redu ced at 200 m g /kg/day. Exposure of weanling SD rats on PNDs 22–41 to doses of 0, 25, 50, and 100 m g /kg/day of Luteinate resulted in a significant decrease in serum LH at 100 m g /kg/day. In a two-generation reproductive toxicity study, serum LH was m easured in the F1 generation prior to m a ting (13 weeks postnatal exposure) and was significantly reduced at doses of 10 m g /kg/day and higher.

Disruption of Estrous Cyclicity
In the adult fem a le, ovulation is dependent on sufficient levels of LH. Because exposure to Luteinate suppresses LH, it would be anticipated to also disrupt the norm a l estrous cycle. Estrous cyclicity was abnorm a l in SD rats exposed to 80 m g /kg/day for 28 days and 8 m g /kg/day
for 6 m onths. In the two-generation reproductive toxicity study, estrous cyclicity was abnorm a l
in the F0 fem a les exposed to 10 m g /kg/day for 10 weeks and in the F1 fem a les exposed to 10

m g /kg/day for 13 weeks postnatally (as well as prenatally). In addition, exposure of weanlings
to 100 m g /kg/day on PNDs 22–41 resulted in abnorm a l estrous cycles.

Pregnancy
Altered LH levels also im pact the ability of the f e m a le to m a intain pregnancy as well as
the tim ing of parturition. Thus, in a variety of exposure scenarios, an increase in pre- and postim plantation loss was observed. Im plantation was affected following exposure to 200

m g /kg/day on GDs 1–8 or 1–20 and after exposure to 400 m g /kg/day on GDs 6–10. Parturition was delayed following exposure to 200 m g /kg/day and above on GDs 6–10. In the twogeneration reproductive toxicity study, there was an increase in pre- and postim plantation loss and a delay in parturition in the F0 and F1 fem a les at doses of 10 m g /kg/day and higher.

B-25Sexual Maturation
At the tim e of puberty, the CNS and pituitary respond to increased concentrations of estradiol in a positive feedback fashion culm inating in the first LH surge. Thus, exposure to Luteinate would likely im pact sexual m a turation. Vaginal opening was delayed in fem a le SD rats following exposure to 100 m g /kg/day on PNDs 22–41 and preputial separation was delayed
in m a les exposed to 100 m g /kg/day on PNDs 23–53. In the two-generation reproductive toxicity study, vaginal opening was delayed in the F1 fem a les exposed to 50 m g /kg/day and preputial separation was delayed in the F1 m a les exposed to 50 m g /kg/day.

Decreased Prolactin and Related Effects Prolactin levels were significantly decreased in adult SD rats following exposure to 200

m g /kg/day of Luteinate for 3 days, to 80 m g /kg/day for 28 days, and to 8 m g /kg/day for 6

m onths. In a study in which lactating dam s were exposed during PNDs 1–4, prolactin levels
were reduced at doses of 50 m g /kg/day. In the two-generation reproductive toxicity study, prolactin levels were reduced in the F0 and F1 anim als at doses of 10 m g /kg/day.

Prostatitis As a consequence of the reduced prolactin levels, an increased incidence of prostatitis
was observed in m a les following m a ternal expos ure to 50 m g /kg/day during lactation days 1–4,
and in the F1 m a les exposed to 10 m g /kg/day and higher. Prostatitis was not observed in m a les exposed during adulthood only (i.e., F0 m a les, m a les in 28-day and 6-m onth studies).

Organ W e ights
In m a les exposed to doses of 100 m g /kg/day Luteinate during PNDs 23–53, there was a decrease in absolute and relative weights of the sem i nal vesicles and ventral prostate. This was
also observed in the F1 m a les exposed to doses of 10 m g /kg/day or greater at the tim e of

term inal sacrifice following m a ting. Histopathological exam ination revealed no lesions in either study. No organ weight changes or histopathologi cal lesions were noted in any of the other
studies.

Effects Unrelated to LH or Prolactin A prenatal developm ental toxicity study was conducted in which pregnant SD rats were exposed to doses of 0, 50, 100, or 200 m g /kg/day Luteinate on GDs 1–20. In addition to the
increase in im plantation loss noted above, there was an increase in delayed ossification at 200

B-26m g /kg/day but no effect on fetal body weight. It is unlikely that this effect is related to LH
levels. However, the m ode 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 m ode of action was known for other pesticides in this chem ical class, it was possible to study known targets during relevant life stages. Luteinate was shown to interfere with the pituitary-hypothalam i c axis, resulting in a decrease in serum LH and prolactin. The decrease was shown to be dependent on dose and duration of exposure. Norm al LH levels are known to be required for ovulation, m a intenance of pregnancy, tim ing of parturition, and sexual

m a turation. Sim ilarly, decreases in prolactin can lead to prostatitis and effects on the m a le reproductive organs. Each of these events were exam ined in several short-term studies as well as in traditional prenatal developm ental toxicity studies and a two-generation reproductive toxicity study. An acute toxicity study of Luteinate was not conducted. However, inform ation was
available on serum LH and prolactin levels from other short-term exposures that was inform ative for acute exposures. The database as a whole dem onstrates 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 m g /kg/day; longer exposures to weanlings on PNDs 22–41 or to adults for 28 days required a dose of 100 or 80

m g /kg/day, respectively. The ef f ective dose of 10 m g /kg/day was still lower f o llowing 10 to 13 weeks exposure. Therefore, it is unlikely that se rum LH levels and related effects would occur at
doses less than 200 m g /kg/day following an acute exposure, but higher doses m a y 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 m g /kg/day and following exposure to lactating dam s at 50 m g /kg/day. A 28-day exposure to adult rats resulted in decreased prolactin levels at 80 m g /kg/day, whereas 10 m g /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

m g /kg/day following an acute exposure.

B-27Knowledge about the effect of Luteinate on serum LH and prolactin levels from these other studies was considered in conjunction with the prenatal developm ental toxicity study for deriving the acute reference value. It is assum e d that effects resulting from developm ental exposures (both prenatal and postnatal) m a y be the result of a single exposure. Therefore, the fetal and offspring effects resulting from exposur es during gestation and postnatally to the tim e of sexual m a turation were also considered for the acute reference value. Data fromthe twogeneration study were not considered because there were always shorter-term studies showing effects at higher doses. Taken as a whole, the l ack of an acute toxicity study was not considered
to be a m a jor data gap for Luteinate. A chronic study of Luteinate was not conducted. However, serum LH was decreased at
10 m g /kg/day in the F0 and F1 anim als in the two-generation reproductive toxicity study and at 8

m g /kg/day in the 6-m onth study. Because there is essentially no change in the effective dose levels following 13 weeks of exposure and 6 m onths 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 m a jor 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
chem ical class that lifetim e exposure results in m a m m a ry tum o rs in SD rats, but this m ode of action is unlikely to be operative in hum ans due to differences in the aging process. Thus, this was not considered to be a data gap for Luteinate. A developm ental neurotoxicity study was not conducted. Given that Luteinate interferes with two neuroreceptors, norepinephrine and dopam i ne, the potential for developm ental 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 e xposures are shown in Figures B-5 through B-8, respectively. Although data on delayed ossificati on 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 com posite of the NOAELs for each endpoint

B-28with each exposure duration. In addition, the NOAELs for each relevant endpoint, the uncertainty factors (UFs), and the sam p le 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 toxicodynam i c differences between rodents and
hum ans. An intraspecies UF (H in Table B-4) of 10 was applied in all cases because the data did
not allow the estim ation of hum an 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-to

chronic UF (S in Table B-4) was not applied because there were data available for each exposure
duration. A database UF of 10 1/2 was applied in all cases because there are concerns for the potential developm ental neurotoxicity of Luteinate and there were no available data.
A database UF of 10 1/2 (rather than a UF of 10) was applied on basis of the observation that for other pesticides in this chem ical class, the doses that result in a decrease in norephinephrine and dopam i ne are very sim ilar to the doses that lead to decreases in serum LH. Therefore, it is likely that any developm ental ne urotoxicity effects would be observed at sim ilar
doses. Because the effects of Luteinate on serum LH levels have been well characterized, additional developm ental neurotoxicity inform ation is unlikely to dram atically affect the
reference values. However, a UF of 10 1/2 was retained to reflect underlying uncertainties of this data gap.

Acute Exposure For the derivation of the acute reference value, a standard prenatal developm ental toxicity study provided inform ation on the effect s of Luteinate on fetal ossification in the absence of an effect on fetal weight. There was no inform ation 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 m g /kg/day following an acute exposure, and it could be higher. Sim ilarly, it is unlikely that the NOAEL for serum prolactin levels and related effects would be less than 25

m g /kg/day following an acute exposure. The effects on sexual m a turation occurred at a dose of
100 m g /kg/day (NOAEL of 50 m g /kg/day) and eff ects on ossification occurred at a dose of 200

m g /kg/day (NOAEL of 100 m g /kg/day). Thus, derivation of the acute reference value based on these endpoints would also be protective of effects on serum LH and related eff ects, but m a y not cover effects on prolactin and

B-29Timingand Duration of Exposure020406080100120140160180200PNDs1-4PNDs1-4PNDs22-41PNDs23-53GD1-20

Dose (m g/ k g/day )NOAELLOAEL catio nNOAELLOAEL

D e cr eas e d P ro lactin Prostatitis D e la y e d O s s i f i catio n

Del a y e d V a g i na l Ope n in g D e la y e d Preput ia l S e para t i on200180160

B-30 Dose (m g/ k g/day )140120100806040200PNDs1-4PNDs1-4PNDs22-41PNDs23-53GD1-20Timingand Duration of ExposureFigure B-5.Exposure-response array of data considered for the Luteinate

acute reference value.0501001502002503003504003days28daysPNDs22 -4128 daysPNDs22 -41PNDs21 -414 -5weeks(F1)PNDs23 -536 -7weeks(F1)28 daysPNDs23 -53Timing and Duration ofExposure

Dose (mg/k g /d ay )NOAELLOAEL

D ecrea s e d L H D i s r up ted Es tru s Cy clici t y

D ecrea s e d L H D i s r upted Es trus C y cli c it y Pre g na nc y Loss Pre g na nc y Los s

D e la y e d P a rturi tion D e l a y e d Va g i na l Ope n i n g D e l a y e d P r ep utial s e p a rati on

D e la y e d V a g i nal O p ening D e la y e d P r eputial S e parat i on D e cr eas ed P r o l acti n D ecr eas ed P r ol actin P o rs t a titis Org a n W ei g hts

D e la y e d O s s i f i cation

D ecr ea se d L H400350
Dose (mg/k g /d ay )300250200150100500

B-313days28daysPNDs22 -4128 daysPNDs22 -41GD1 -8,GD1 -20GD6 -10GD6 -10PNDs21 -414 -5weeks(F1)PNDs23 -536 -7weeks(F1)PNDs1 -428 daysPNDsGD1 -20PNDs1 -423 -53Timing and Duration ofExposureFigure B-6.Exposure-response array of data considered for the Luteinate

short-term reference value.010203040506070809010010 -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)16 weeks(F1)Timingand Duration ofExposure

Dos e ( m g/ k g /day )NOAELLOAEL

D ecreas ed L H D i srupt ed Est r us C y cl ici t y Pr e g na n c y Loss D e la y e d Pa rt uri t i on P r os tatitis

D e creas ed Pr olactin Org a n W e i g ht s10090

Dos e ( m g/ k g /day )807060504030

B-322010010 -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)16 weeks(F1)Timingand Duration ofExposureFigure B-7.Exposure-response array of data considered for the Luteinate

longer-term reference value.01020304050607080901006months10-13weeks(F0,F1)6 months10 -13weeks(F0,F1)11 -16 weeks(F0,F1)14-17 weeks(F0,F1)10-13 weeks(F0,F1)16 weeks(F1)Timing and DurationofExposure

Dose ( m g/ k g /day )1009080NOAELLOAEL

D ecreas ed L H Dis r up ted Es tru s C y clicit y Preg nanc y Loss Dela y e d Pa rturition P r o s tatitis

D e cr eas e d L H D i srupted Estrus C y cl ici t y D ecreased Prolacti n

Dose ( m g/ k g /day )7060504030

B-33201006months10-13weeks(F0,F1)6 months10 -13weeks(F0,F1)11 -16 weeks(F0,F1)14-17 weeks(F0,F1)10-13 weeks(F0,F1)16 weeks(F1)Timing and DurationofExposureFigure B-8.Exposure-response array of data considered for the Luteinate

chronic reference value.0501001502002500510

D o s e ( m g/ k g / day ) A l t e r e d p r e g n a n c y

m a i n t e n a n c e

L H r e d u c t i o n

E s t r o u s d i s r u p t i o n D e l a y e d p a r t u r i t i o n P r o l a c t i n r e l e a s e a t t e n u a t i o n R e d u c e d s e m i n a l

v e s i c l e w e i g h t

R e d u c e d f e t a l o s s i f i c a t i o n

D e l a y e d p r e p u t i a l s e p a r a t i o n

D e l a y e d v a g i n a l o p e n i n g P r o s t a t i t i s

l t e r e p r e g n a n c

a i n t e n a n

e d u c t i o s t r o u d i s u p t i o e l a y e p a r t u r i t i o r o l a c t r e l e a s a t e n u a t i o e u e i a

v e s i c l e i g h

e u e e a o s s i f i a t i

e l a y e r p t i a s e p a t i

a y v g i a o e n i r o s a t i t i43219876GDs1-8GDs6-10GDs1-20PNDs 1-4PNDs 22-41PNDs 23-533D28D6moF0-10wkF1-13wkF1-5wkF1-7wkF1-16wk250200

D o s e ( m g/ k g / day )150100

B-345000123456789

y n

n n
c o

n g

n n s n l

a o
r

d a n l p n m e s

P t w e

R d c d R d c d t

r e c

H L r e t

s d

d p e u l

d f t l

E A m D l e e D n i

D PFigure B-9. NOAELs for Luteinate with timing/duration of exposure.10Table B-4. Summary of reference values for Luteinate

Reference

value duration Exposure duration NOAEL (mg/k g/day) Type of effect Uncertainty factors a Reference value (mg/k g/day)

A H L S D T otal Sample Final

Acute P NDs 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 1 0 1 0 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-35Table B-4. Summary of reference values for Luteinate (continued)

Reference

value duration Exposure duration NOAEL (mg/k g/day) Type of effect Uncertainty factors a Reference value (mg/k g/day)

A H L S D T otal 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 W t 10 10 1 1 300

GDs 1–20 100 Fetal ossification 10 10 1 1 300

Longerterm 10–13 weeks 5 L H 1 0 0 1

3 300

10–13 weeks 5 E strus 1 0 1 0 1 1 300

11–16 weeks 5 P regnancy Maintenance 10 10 1 1 300

14–17 weeks 5 P arturition 1 0 1 0 1 1 300

4–7 weeks 25 Sexual Maturation 10 10 1 1 300

10–13 weeks 5 P rolactin 10 10 1 1 300

16 weeks 5 Prostatitis 10 10 1 1 300

16 weeks 5 Organ W t 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-36Table B-4. Summary of reference values for Luteinate (continued)

Reference

value duration Exposure duration NOAEL (mg/k g/day) Type of effect Uncertainty factors a Reference value (mg/k g/day)

A H L S D T otal Sample Final

Chronic 6 m onths 4 L H 1 0 1 0 1 1

3 300 0.01

0.01 6 m onths 4 E strus 1 0 1 0 1 1 300 0.01

11–16 weeks 5 P regnancy Maintenance 10 10 1 1 300 0.02

14–17 weeks 5 P arturition 1 0 1 0 1 1 300 0.02

10–13 weeks 5 P rolactin 10 10 1 1 300 0.02

16 weeks 5 Prostatitis 10 10 1 1 300 0.02

16 weeks 5 Organ W t 10 10 1 1 300 0.02

GDs 1–20 100 Fetal ossification 10 10 1 1 300 0.33

a A = anim al-to-hum an (interspecies); H = inte r-individual (intraspecies); L = LOAEL-to-NOAEL; S = subchronic-to-chronic duration; D = database deficiency

related effects. For this reason, the NOAEL of 25 m g /kg/day for prolactin and related effects following a 4-day developm ental (neonatal) exposure was used as the basis for the acute reference value, with the assum p tion that a single exposure during a critical period of developm ent 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), m i ght 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 tim e in the early postnatal period. Effects on prolactin (and therefore dopam i ne) also indicate a concern for developm ental neurotoxicity (Figure B-5). Becau se there is a strong relationship between the data gaps of the acute toxicity study and the developm ental neurotoxicity study, a database UF of
10 1/2 was applied. In addition, UFs of 10 for interspecies and intraspecies uncertainty/variability were applied. The resulting reference value was 0.08 m g /kg/day.

B-37Short-term Exposure A variety of endpoints were exam ined for calculating the short-term reference value.
Inform ation was available on serum LH from the 3-day and 28-day adult studies as well as the study in which pregnant dam s were exposed on GDs 1–8. Inform ation on estrous cyclicity was
available from the 28-day study in adult rats and in the study in which fem a le weanlings were exposed on PNDs 22–41. Inform ation on pregnancy m a intenance was available from studies in which pregnant dam s were exposed on GDs 1–8, 6–10, and 1–20, and inform ation on parturition was available for exposures on GDs 6–10.
Inform ation on sexual m a turation in fem a les was available from the study in which
fem a le weanlings were exposed on PNDs 22–41, and inform ation on sexual m a turation in m a les
was available from the study in which weanling m a le rats were exposed on PNDs 23–53. In addition, inform ation on sexual m a turation from the two-generation reproductive toxicity study
was also considered relevant. The latter study also provided inform ation on the effects of
Luteinate on the sem i nal vesicles and ventral prostate. Inform ation on prolactin was available
from the 28-day study in adults and the study in which lactating dam s were exposed on PNDs 1–4; inform ation 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 10 1/2 was applied in the derivation of the final reference value for the lack of a developm ental neurotoxicity study. The resulting reference values ranged from 0.08 to 0.67 m g /kg/day, and the final reference value
was 0.08 m g /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 inform ation following longer exposures for serum LH, estrous cyclicity,

pregnancy m a intenance, 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

sam p le reference values were 0.33 m g /kg/day, based on delayed ossification and 0.08

m g /kg/day, based on sexual m a turation; all the other endpoints yielded a value of 0.02

m g /kg/day. The latter was chosen as the final value for the longer-term reference value.

B-38Chronic Exposure
Inform ation from all of the exposure scenarios described above was considered in deriving the chronic reference value. In addition, inform ation on serum LH and estrous cyclicity
was available from the 6-m onth study in adult rats. For endpoints where there was a clear exposure-dose-effect relationship, only inform ati on 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 m g /kg/day in the 6-m onth study and at 10 m g /kg/day in the two-generation reproductive toxicity study; the NOAELs were 4 and 5 m g /kg/day, respectively. Estrous cyclicity was affected at these sam e dose levels in the two studies. The NOAEL for all other endpoints in the two-generation reproductive toxicity study was also 5 m g /kg/day.
Given the sim ilarity 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 inform ation 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 da tabase deficiency for the purposes of deriving a
chronic ref e rence value. UFs of 10 f o r 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 m g /kg/day, and 0.01 m g /kg/day was chosen as the final value.

B-39GLOSSARY

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 m u ltiple 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 biochem i cal change, functional im pairm e nt, or pathologic lesion that affects the perform a nce of the whole organism or reduces an organism ' s ability to respond to an additional environm ental challenge.

Benchmark Dose (BMD) or Concentration (BMC) : A statistical lower conf idence lim it on the dose that produces a predeterm i ned change in response rate of an adverse effect (called the benchm ark response or BMR) com p ared 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 benchm ark 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 lim it of responses typically observed in well-conducted anim al experim e nts.

Bioassay : An assay for determ ining the potency (or concentration) of a substance that causes a biological change in experim e ntal anim als.

Bioavailability : The degree to which a substance becom e s available to the target tissue after adm i nistration or exposure.

G-1Biologically Based Dose Response (BBDR) model : A predictive tool used to estim ate potential hum an health risks by describing and quantifying the key steps in the cellular, tissue, and organism al responses as a result of chem ical 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 chem ical' s concentration between blood and air when at equilibrium

. Chronic Exposure : Multiple exposures occurring over an extended period of tim e or a significant fraction of the anim al' s or the indi vidual' s lifetim e. [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 m easure the (toxic) effects of chronic exposure to a chem ical.

Critical Effect : The first adverse effect, or its known precursor, that occurs to the m o st sensitive species as the dose rate of an agent increases.

Critical Study : The study that contributes m o st significantly to the qualitative and quantitative assessm ent of risk; also called Principal Study.

Developmental Toxicity : Adverse effects on the developing organism that m a y result from exposure prior to conception (either parent), during prenatal developm ent, or postnatally until the tim e of sexual m a turation. The m a jor m a nifestations of developm ental toxicity include death of the developing organism , structural abnor m a lity, 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-2the delivered or biologically effective dose for that organ or cell. [New definition proposed to be added to IRIS.]

Dose-Response Assessment : A determ ination of the relationship between the m a gnitude of an adm i nistered, applied, or internal dose a nd a specific biological response. Response can be expressed as m easured 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 dem onstrating speci fic, 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 m easurable biological event or chem ical concentration (e.g.,

m e tabolite concentration in a target tissue) used as an index of an effect of a chem ical exposure.

Epidemiology : The study of disease patterns in hum an 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 m a de between a chem ical, physical, or biological agent and the outer boundary of an organism

. Exposure is quantified as the am ount of an agent available at the exchange boundaries of the organism (e.g., skin, lungs, gut).

Exposure Assessment : An identification and evaluation of the hum an population exposed to a toxic agent, describing its com position and size, as well as the type, m a gnitude, frequency, route, and duration of exposure.

Exposure Pathw a y : The physical course an environm ental agent takes from the source to the individual exposed.

G-3Extrapolation, Low Dose : An estim ate of the response at a point below the range of the experim e ntal data, generally through the use of a m a them atical m odel.

Haz a rd : A potential source of harm

.

Haz a rd Assessment : The process of determ ining 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 hum ans.

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. [ N ew def i nition proposed to be added to IRIS]

Human Equivalent Concentration (HEC) : The hum an concentration (for inhalation exposure) of an agent that is believed to induce the sam e m a gnitude of toxic effect as the experim e ntal anim al species concentration. This adjustm e nt m a y incorporate toxicokinetic inform ation on the particular agent, if available, or use a default procedure.

Human Equivalent Dose (HED) : The hum an dose (for other than the inhalation route s of exposure) of an agent that is believed to induce the sam e m a gnitude of toxic effect as the experim e ntal anim al species dose. This adjustm e nt m a y incorporate toxicokinetic inform ation on the particular agent, if available, or use a default procedure, such as assum i ng that daily oral doses experienced for a lifetim e are proportional to body weight raised to the 0.75 power.

Incidence : The num ber of new cases of a disease that develop within a specified population over a specified period of tim e.

Incidence Rate : The ratio of new cases within a population to the total population at risk given a specified period of tim e.

Latency Period : The tim e between exposure to an agent and m a nifestation 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 am ount 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-4approximately 90 days in typically used laboratory animal species)

. [Proposed new definition to be used relative to the Longer-term Reference Value. Sim ilar to the current definition for Subchronic Exposure. Because subchronic exposure studies will continue to be used in risk

assessm ent, the latter termshould be retained as well but replaced with the definition for Longerterm Exposure.]

Low e st-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 m e thodology, U.S. EPA, 1994]

Margin of Exposure (MOE) : The LED10 or other point of departure divided by the actual or projected environm ental exposure of interest.

Mechanism of Action : The com p lete sequence of biological events that m u st occur to produce the toxic effect.

Mode of Action (MOA) : A less-detailed description of the m echanism of action in which som e but not all of the sequence of biologi cal events leading to a toxic effect is known.

Modifying Factor (MF) : A factor used in derivation of a reference dose or reference concentration. The m a gnitude of the MF reflect s the scientific uncertainties of the study and database not explicitly treated with standard uncertainty factors (e.g., the com p leteness 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 recom m e nds 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; som e effects m a y 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 m e thodology, U.S. EPA, 1994.]

G-5Nonlinear Dose Response : A pattern of frequency or severity of biological response that does not vary proportionately with the am ount of dose of an agent. W h en m ode of action inform ation indicates that responses m a y not follow a linear pattern below the dose range of the observed data, non-linear m e thods for determ ining risk at low dose m a y 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

com p artm ental m odel used to characterize pharm acokinetic behavior of a chem ical. Available data on blood flow rates, and m e tabolic and other processes which the chem ical undergoes within each com p artm ent are used to construct a m a ss-balance fram ework for the PBPK m odel. [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 m a rks the beginning of a low-dose extrapolation. This point is m o st often the upper bound on an observed incidence or on an estim ated incidence from a dose-response m odel. [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 m easure expressed as parts per billion. Equivalent to 1 x 10 -9

.
Ppm : A unit of m easure expressed as parts per m illion. Equivalent to 1 x 10 -6

.

Prevalence : The proportion of disease cases that exist within a population at a specific point in tim e relative to the num ber of individuals within that population at the sam e point in tim e.

Reference Concentration (RfC) : An estim ate (with uncertainty spanning perhaps an order of m a gnitude) of a continuous inhalati on exposure to the hum an population (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a

G-6lifetim e. It can be derived from a NOAEL, LOAEL, or benchm ark concentration, with uncertainty factors generally applied to reflect lim itations of the data used. Generally used in EPA' s noncancer health assessm ents. [Current IRIS definition.]

Reference Dose (RfD) : An estim ate (with uncertainty spanning perhaps an order of

m a gnitude) of a daily oral exposure to the hum an population (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a lifetim e. It can be derived from a NOAEL, LOAEL, or benchm ark dose, with uncertainty factors generally applied to reflect lim itations of the data used. Generally used in EPA' s noncancer health assessm ents. [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 s ubscript would be used with the RfV to denote route and duration, e.g., RfV AO 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 f o r ventilatory volum es and the surf ace area of the respiratory region affected (m g/m i n-sq. cm ). For extrarespiratory effects of particles, the deposited dose in the total respiratory system is adjusted for ventilatory volum es and body weight (m g/m i n-kg).

Regional Deposited Dose Ratio (RDDR) : The ratio of the regional deposited dose calculated for a given exposure in the anim al species of interest to the regional deposited dose of the sam e exposure in a hum an. This ratio is used to adjust the exposure-effect level for interspecies dosim etric differences to derive a hum an equivalent concentration for particles.

Regional Gas Dose : The gas dose calculated for the region of interest as related to the observed ef f ect f o r respiratory ef f ects. The deposited dose is adjusted f o r ventilatory volum es and the surface area of the respiratory region affected (m g/m i n-sq.cm )

. Regional Gas Dose Ratio (RGDR) : The ratio of the regional gas dose calculated for a given exposure in the anim al species of interest to the regional gas dose of the sam e exposure in hum ans. This ratio is used to adjust the exposure effect level for interspecies dosim etric differences to derive a hum an equivalent con centration for gases with respiratory effects.

Risk (in the context of human health) : The probability of injury, disease, or death f r om exposure to a chem ical agent or a m i xture of chem icals. In quantitative term s, risk is expressed in values ranging f r om zero (representing the certainty that harm will not occur) to one

G-7(representing the certainty that harm will occur). The f o llowing are exam ples 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. Sim ilarly, 1.3 E-3 or 1.3 x 10-3 = a ri sk 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 ri sk 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 determ ination of potential adverse health effects from exposure to chem icals, including both quantitative and qualitative expressions of risk. The process of risk assessm ent involves four m a jor steps: hazard identification, dose-response assessm ent, exposure assessm ent, 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 tim e, 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 [ s ic] 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 tim e or less. Study design considerations

m a y 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-8Subchronic Exposure : Exposure to a substance spanning approxim a tely 10% of the lifetim e of an organism

. [See note for Longer-term Exposure.]

Subchronic Study : A toxicity study designed to m easure effects from subchronic exposure to a chem ical.

Supporting Studies : Studies that contain inform ation 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 fr om its entry point, at which point effects are produced. Not all chem icals that produce system ic effects cause the sam e 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) m o st adversely affected by exposure to a chem ical 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 chem ical substance elicits a deleterious or adverse effect upon the biological system of an organism exposed to the substance over a designated tim e 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-9Toxicodynamics : 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 harm ful interactions between chem icals and biological system s. [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 chem ical substance or agent which m a y cause an adverse effect or effects to biological system s. [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 experi m e ntal data. UFs are intended to account for (1) variation in sensitivity am ong the m e m b ers of the hum an population, i.e., interhum an or intraspecies variability; (2) the uncertainty in extrapolating anim al data to hum ans, i.e., interspecies variability; (3) the uncertainty in extrapolating from data obtained in a study with less-than-lifetim e exposure to lifetim e 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 f r om anim al data when the data base is incom p lete. [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) uncertain ty in extrapolating animal data to humans (i.e.,

G-10interspecies 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-11REFERENCES

Abdel-Mageed, MA; Suh, DH; Abdel-Rahm an, MS. (2001) Intra-species extrapolation in risk assessm ent of different classes of antim icrobials. Hum a n Ecol Risk Assess 7:15–36.

Allen, BC; Kavlock, RJ; Kim m e l, CA; et al. (1994) Dose-response assessm ent for developm ental toxicity: II. Com p arison of generic benchm ark dose estim ates with NOAELs. Fundam Appl Toxicol 23:487–495.

Andersen, ME; Gargas, ML; Clewell, HJ, III; et al. ( 1987) Quantitative evaluation of the m e tabolic interactions between trichloroethylene and 1,1-dichloroethylene in vivo using gas uptake m e thods. Toxicol Appl Pharm acol 89:149–57.

ATSDR (Agency for Toxic Substances and Disease Registry). (1996) Minim a l risk levels for priority substances and guidance for derivation. Federal Register 61:25873–25881.

Baird, SJS; Cohen, JT; Graham , JD; Shlyakhter, AI; Evan s, JS. (1996) Noncancer risk assessm ent: a probabilistic alternative to current practice. Hum a n Ecol Risk Assess 2:79-102.

Barnes, DG; Dourson, M. (1988) Reference dose (RfD): description and use in health risk assessm ents. Regul Toxicol Pharm acol 8:471-486.

Barton, HA; Deisinger, PJ; English, JC; et al. (2000) Fam ily approach for estim ating reference concentrations/doses for series of related organic chem icals. Toxicol Sci 54(1):251–61.

Bogdanffy, MS; Daston, G; Faustm an, EM; et al. (2001) Harm onization of cancer and non-cancer risk assessm ent: proceedings of a consensus-building workshop. Toxicol Sci 61:18–31.

Brand, KP; Rhom berg, L; Evans, JS. (1999) Estim a ting noncancer uncertainty factors: are ratios NOAELs inform ative? Risk Anal 19:295–308.

Butterworth, BE; Bogdanffy, MS. (1999) A com p rehensive approach for integration of toxicity and cancer risk

assessm ents. Regul Toxicol Pham acol 29:23-36.

Calabrese, EJ. (2001) Assessing the default assum p tion that children are always at risk. Hum a n Ecol Risk Assess 7:37–59.

CDM Group, Inc. (2000) Colloquium on setting acute and inte rm ediate reference values, sum m a ry report, Septem ber 22. Contract num ber 68-D6-0060. Chevy Chase, MD.

Creason, J; Neas, L; W a lsh, D; et al . (2001) Particulate m a tter and heart rate variability am ong elderly retirees: the Baltim ore 1998 PM study. J Expo Anal Environ Epidem iol 11(2):116–122.

Dourson, ML; Knauf, LA; Swartout, JC. (1992) On refe rence dose (RfD) and its underlying toxicity data base. Toxicol Ind Health 8:171–189.

Dourson, ML; Felter, SP; Robinson, D. (1996) Evolution of science-based uncertainty factors in noncancer risk

assessm ent. Regul Toxicol Pharm acol 24:108–120.

Enquist, BJ; Brown, JH; W e st, GB. (1998) Allom e tric s caling of plant energetics and population density. Nature 95(6698):163–165.

R-1Evans, JS; Rhom berg, LR; W illiam s PL; W ilson, AM; Bair d, SJS. (2001) Reproductive and developm ental risks from ethylene oxide: a probabilistic characterization of possible regulatory thresholds. Risk Anal 21:697-717.

Faustm an, EM; Allen, BC; Kavlock, RJ; et al. (1994) Dose-response assessm ent for developm ental toxicity: I. Characterization of data base and determ in ation of NOAELs. Fundam Appl Toxicol 23:478–486.

Gad, SC; Chengelis, CP. (1998) Acute toxicology testing. 2 nd ed. San Diego: Academ ic Press.

Gargas, ML; Burgess, RJ; Voisard, DE; et al. (1989) Partition coefficien ts of low-m o lecular-weight volatile chem icals in various liquids and ti ssues. Toxicol Appl Pharm acol 98:87–99.

Gaylor, DW ; Kodell, RL. (2000) Percentiles of the produc t of uncertainty factors for establishing probabilistic reference doses. Risk Anal 20:245–250.

Gillooly, JF; Brown, JH; W e st, GB; et al. (2001) Effects of size and tem p erature on m e tabolic rate. Science 293:2248–2251.

Hanna, LM; Jarabek AM. (2000) Proposed inhalation rout e m odel suites: m ode-of-action-m o tivated dosim etry project. Presented at: Society of Risk Analys is annual m eeting; Dec. 3–6, 2000; Arlington, VA.

Hasem a n, JK. (1984) Statistical issues in the design, anal ysis and interpretation of anim al carcinogenicity studies. Environ Health Perspect 58:385–92.

Hasem a n, JK; Huff, JE; Rao, GN; et al. (1989) Sources of variability in rodent carcinogenicity studies. Fundam Appl Toxicol 12:793–804.

Hoyer, PB; Sipes, IG. (1996) Assessm ent of follicle dest ruction in chem ical-induced ovarian toxicity. Annu Rev Pharm acol Toxicol 36:307–331.

Hubal, EA; Bunge, A; Hanna, LM; et al. (2000) Proposed suite of m odels for estim ating dose resulting from exposures by the derm al route. Presented at: Society of Risk Analysis annual m eeting; Dec. 3–6, 2000; Arlington, VA.

ICRP (International Com m i ssion on Radiological Protection) . (1994) Hum a n respiratory tract m odel for radiological protection: a report of a task group of the Internationa l Com m i ssion on Radiological Protection. ICRP Publ. no. 66, Annals of the ICRP 24(1–3). Ta rrytown, NY: Elsevier Science.

ILSI RSI (International Life Sciences Risk Sciences Institute). (2001) W o rkshop to develop a fram e work for assessing risks to children from exposure to envir onm ental agents; July 31–Aug. 2, 2000; Stowe, VT.

IPCS (International Program m e on Chem ical Safety). (1994) Assessing hum an health risks of chem icals: derivation of guidance values for health-based exposure lim its. (E nviron. Health Criteria no. 170). Geneva: W o rld Health Organization.

IPCS. (1995) IPCS/OECD workshop on the harm onization of risk assessm ent of reproductive and developm ent toxicity. BIBRA International, Carshalt on, Surrey, UK, October, 1994, IPCS/95.25.

IPCS. (1998) Boron. (Environ. Health Criteri a no. 204). Geneva: W o rld Health Organization.

IPCS. (2001) Guidance docum ent for the use of data in deve lopm ent of chem ical-specific adjustm e nt factors (CSAF) for inter-species differences and hum an variability in dos e/concentration response assessm ent. W HO/ IPCS/01.4, draft final. Available from : <http://www.ipcsharm onize.org/CSAFsum m a ry.htm >.

R-2Jarabek, AM. (2000) Mode of action: fram e work for dosim et ry m odel developm ent. Presented at: Society of Risk Analysis annual m eeting; Dec. 3–6, 2000, Arlington, VA.

Jepson, GW ; Hoover, DK; Black, RK; et al. (1994) A partition coefficient determ ination m e thod for nonvolatile chem icals in biological tissues

. Fundam Appl Toxicol 22:519–24.

Jick, H; Porter, J; Morrison, AS. (1977) Relation be tween sm oking and age of natural m e nopause. Lancet 1:1354–55.

Kim b ell, JS; Gross, EA; Joyner, DR; et al. (1993) Appli cation of com putational fluid dynam i cs to regional dosim etry of inhaled chem icals in the upper respiratory tr act of the rat. Toxicol Appl Pharm acol 121:253–263.

Kim b ell, JS; Godo, MN; Gross, EA; et al. (1997) Com puter sim u lation of inspiratory airflow in all regions of the F344 rat nasal passages. Toxicol Appl Pharm acol 145:388–398.

Kim m e l, CA; Francis, EZ. (1990) Proceedings of the workshop on the acceptability and interpretation of derm al developm ental toxicity studies. Fundam Appl Toxicol 14:386–398.

Kim m e l, GL; W illiam s , PL; Claggett, TW ; et al. ( 2002) Response-surface analysis of exposure-duration relationships: the effects of hypertherm ia on em bryonic deve lopm ent of the rat in vitro. Toxicol Sci. 69:391-399.

Lam b , JC. (1985) Reproductive toxicity testing: evaluating and developing new testing system s. J Am Coll Toxicol 4:163–171.

Lam b , JC, IV; Maronpot, RR; Gulati, DK; et al. (1985) Re productive and developm ental toxicity of ethylene glycol in the m ouse. Toxicol Appl Pharm acol 81:169–173.

Leopold, PW ; Rivens, ML; Leach, MO. (1997) Noninvasive diagnostic techniques in vascular disease. J Vasc Technol 11:183–186.

Lewis, SC; Lynch, JR; Nikiforov, AI. (1990) A new appr oach to deriving com m unity exposure guidelines from“noobserved-adverse-effect-levels.” Regul Toxicol Pharm acol 11:314–330.

Maull, EA; Cogliano, VJ; Scott, CS; et al. (1997) Trichl oroethylene health risk assessm ent: a new and im proved
process. Drug Chem Toxicol 20:427–442.

Meek, ME. (2001) Categorical default uncer tainty factors: interspecies variati on and adequacy of database. Hum a n Ecol Risk Assess 7:157–163.

Meek, ME; Renwick, A; Ohanian, EO; et al. (2001) Gu idelines for application of com pound specific adjustm e nt factors (CSAF) in dose/concentration res ponse assessm ent. Com m e nts on Toxicol: 7:575-590.

Morrissey, RE; Lam b , JC, IV; Morris, RW ; et al. (1989) Results and evaluations of 48 continuous breeding reproduction studies conducted in m i ce. Fundam Appl Toxicol 13:747–777.

Murray, FJ; Andersen, ME. (2001) Data derived uncertainty factors; boric acid (BA) as a case study. Hum a n Ecol Risk Assess 7:125–138.

NAS (National Academ y of Sciences). (1977) Drinki ng water and health. Safe Drinking W a ter Com m ittee. W a shington, DC: National Academ y Press.

Nau, H. (1991) Pharm acokinetic considerations in the de sign and interpretation of developm ental toxicity studies. Fundam Appl Toxicol 16(2):219–221.

R-3NRC (National Research Council). (2000) Acute exposure guidelin e levels for selected airborne chem icals, Vol. 1. W a shington, DC: National Academ y Press.

Orm e , J; Ohanian, EV. (1991) Health advisories for pestic ides. In: Richardson, ML, ed. Chem istry, agriculture and the environm ent. Cam b ridge, UK: Royal Society of Chem istry; pp. 429–443.

Pieters, MN; Kram er, HJ; Slob, W

. (1998) Evaluation of the uncertainty factor for subchronic-to-chronic extrapolation: statistical analysis of t oxicity data. Regul Toxicol Pharm acol 27:108–111.

Pinkerton, KE; Joad, J. (2000) The m a m m a lian respiratory system and critical windows of exposure for children' s health. Environ Health Perspect 108(Suppl 3):457–462.

Renwick, AG. (1993) Data-derived safety factors fo r the evaluation of food additives and environm ental
contam inants. Food Addit Contam 10:275–305.

Renwick, AG. (1998) Toxicokinetics in infants and childre n in relation to the ADI and TDI. Food Addit Contam 15(Suppl):17–35.

Renwick, AG; Lazarus, NR. (1998) Hum a n variability and non cancer risk assessm ent—an analysis of the default uncertainty factor. Regul Toxicol Pharm acol 27:3–20.

Rigas, ML; Roth, W L ; Jarabek, AM. (2000) Proposed m ode ls for estim ating relevant dose resulting from exposures by the gastrointestinal route. Presented at: Society of Risk Analysis annual m eeting; Dec. 3–6, 2000; Arlington, VA.

Rozm an, KK; Doull, J. (2000) Dose and tim e as variables of toxicity. Toxicology 144:169-178.

Rozm an, KK; Doull, J; Hayes, W J , Jr. (2001) Dose, tim e, and other factors influencing toxicity. In: Handbook of Pesticide Toxicology, vol. 1, Principles. Ed. R. Kreiger, Academ ic Press, pp. 1-93.

Schilter, B; Renwick, AG; Huggett, AC. (1996) Lim its fo r pesticide residues in infant food: a safety-based approach. Regul Toxicol Pharm acol 24:126–140.

Skowronski, G; Abdel-Rahm an, MS. (2001) Relevance of the 10X uncertainty f actor to the risk assessm ent of drugs used by children and geriatrics. Hum a n Ecol Risk Assess 7:139–152.

Slob, W ; Pieters, MN. (1998) A probabilistic approach for deriving acceptable hum an intake lim its and hum an health risks from toxicological studies: general fram e work. Risk Anal 18:787–798.

Sm ith, TL; Kom a n, LA; Mosberg, AT. (1994) Cardiovascul ar physiology and m e thods for toxicology. In: Hayes, AW , ed. Principles and m e thods of toxi cology. New York: Raven Press; pp. 917–935.

Swartout, JC; Price, PS; Dourson, ML ; et al. (1998) A probabilistic fram e work for the reference dose (probabilistic RfD). Risk Anal 18:271–282.

ten Berge, W F ; Zwart, A; Appelm an, LM. (1986) Concentra tion-tim e m o rtality response relationship of irritant and system ically acting vapours and gases. J Hazard Mater 13:301–309.

Terry, KK; Elswick, BA; Stedm a n, DB; et al. (1994) Developm ental phase alters dosim etry-teratogenicity relationship for 2-m e thoxyethanol in CD-1 m i ce. Teratology 49:218–227.

R-4Tzim as, G; Thiel, R; Chahoud, I; et al. (1997) The area under the concentration-tim e curve of all-trans-retinoic acid is the m o st suitable pharm acokinetic correlate to the em br

yotoxicity of this retinoid in the rat. Toxicol Appl Pharm acol 143:436–444.

U.S. EPA (Environm ental Protection Agency). (1986) Guid elines for the health risk assessm ent of chem ical

m i xtures. Federal Register 51(185):34014–34025 and <http://www.epa.gov/ncea/raf/>.

U.S. EPA. (1988) Recom m e ndations for and docum entation of biological values for use in risk assessm ent. EPA/600/6-87/008. Available from : National Technical Inform ation Service, Springfield, VA.

U.S. EPA. (1991) Guidelines for developm ental toxi city risk assessm ent. Federal Register 56(234):63798–63826 and <http://www.epa.gov/ncea/raf/rafguid.htm >

. U.S. EPA. (1992) Draft deport: a cro ss-species scaling factor for carcinogen risk assessm ent based on equivalence of m g /kg 3/4 /day. Federal Register 57(109):24152.

U.S. EPA. (1994) Methods for derivation of inhalation re ference concentrations and application of inhalation dosim etry. EPA/600/8-90/066F. Available from : Nationa l Technical Inform ation Service, Springfield, VA.

U.S. EPA. (1996) Guidelines for reproductive toxicity risk assessm ent. Federal Register 61(212):56274–56322 and <http://www.epa.gov/ncea/raf/rafguid.htm >

. U.S. EPA. (1997a) Exposure factors ha ndbook, Vols. I, II, and III. EPA/600/P-95/002Fa. Prepared by the National Center for Environm ental Assessm ent, W a shingt on, DC. Available from : <http://www.epa.gov/ncea/>.

U.S. EPA. (1997b) Sum m a ry of the U.S. EPA Colloquium on a Fram ework for Hum a n Health Risk Assessm ent, Vol. 1. EPA/600/R-99/001. Available from : <http://www.epa.gov/nceawww1/colloquium .htm >.

U.S. EPA. (1997c) Guiding principles for Monte Carl o analysis. EPA/630/R-97/001. Risk Assessm ent Forum
, W a shington, DC. Available from : National Technical Inform ation Service, Springfield, VA; PB97-188106 and <http://www.epa.gov/ncea/raf/>.

U.S. EPA. (1998a) Methods for exposure -response analysis for acute inhalation exposure to chem icals: developm ent of the acute reference exposure. EPA/600/R-98/051.

U.S. EPA. (1998b) Hazard identification: toxicology endpoi nt selection process. Office of Pesticide Program s.
U.S. EPA. (1998c) Guidelines for neurotoxicity ri sk assessm ent. Federal Register 63(93):26926–26954 and <http://www.epa.gov/ncea/raf/rafguid.htm >

. U.S. EPA. (1998d) Sum m a ry of the U.S. EPA Colloquium on a Fram ework for Hum a n Health Risk Assessm ent, Vol. 2. EPA/600/R-98/155. Available from : <http://www.epa.gov/nceawww1/colloquium .htm >.

U.S. EPA. (1999a) Guidelines for carcinogen risk assessm ent [review draft]. NCEA -F-0644. Risk Assessm ent Forum
, W a shington, DC. Available from : <http://www.epa.gov/ncea/raf/cancer.htm >.

U.S. EPA. (1999b) Toxicology data requirem e nts for asse ssing risks of pesticide exposure to children’s health: report of the Toxicology W o rking Group of the 10X Task Force [April 28, 1999 draft]. Available from : <http://www.epa.gov/scipoly/sap/1999/index.htm # m a y>.

U.S. EPA. (1999c) Exposure data requirem e nts for assessing risks of pesticide exposure to children’s health: report of the Exposure W o rking Group of the 10X Task Force [March 8, 1999 draft]. Available from : <http://www.epa.gov/scipoly/sap/1999/index.htm # m a y>.

R-5U.S. EPA. (2000a) Supplem entary guidance for conducti ng health risk assessm ent of chem ical m i xtures. EPA/630/R-00/002. Available from : <http://cfpub.epa.gov/ncea/cfm / >.

U.S. EPA. (2000b) Science policy handbook on risk char acterization. EPA/100/B-00-002. Available from : <http://www.epa.gov/ORD/spc/2riskchr.htm >.

U.S. EPA. (2000c) Benchm ark dose technical guidance docum ent [external review draft]. EPA/630/R-00/001. Available from : <http://www.epa.gov/ncea/bnchm rk/bm d s_peer.htm >.

U.S. EPA. (2001a) Scientific applicability for use of an alternative test guideline for derm al sensitivity. FIFRA Scientific Advisory Panel review. Available from : <http://www.epa.gov/scipoly/sap/2001/index.htm >

. U.S. EPA. (2001b) Toxicological review of boron and com pounds (CAS No. 7440-42-8) [external review draft]. National Center for Environm ental Assessm ent, W a shington, DC. Available from : <http://www.epa.gov/ncea/boron.htm >

. U.S. EPA. (2001c) Press release: Agency requests Nationa l Academ y of Sciences input on consideration of certain hum an toxicity studies; announces interim policy. Decem ber 14

. (Contact David Deegan, 202-564-7839; deegan.david@epa.gov).

U.S. EPA (2001d) W a ter Quality Criterion for the Protec tion of Hum a n Health: Methylm e rcury. EPA-823-R-01- 001, January. Office of Science and Technology, Office of W a ter, W a shington, DC.

U.S. EPA. (2002a) Fram ework for cum u lative risk assessm ent [external review draft]. Risk Assessm ent Forum
, W a shington, DC. Available from : <http://cfpub.epa.gov/ncea/cfm / nceahom e.cfm >

. U.S. EPA. (2002b) Determ ination of the appropriate FQPA sa fety factor(s) for use in the tolerance-setting process. Office of Pesticide Program s. Available fro m : <http://www.epa.gov/oppfead1/trac/science/#10_fold>.

U.S. EPA. (2002c) Integrated risk inform ation system (IRIS). National Center for Environm ental Assessm ent, W a shington, DC; <http://www.epa.gov/iris/>.

U.S. EPA. (2002d) Health assessm ent of 1,3- butadiene. EPA/600/P-98/001F. Available from : <http://cfpub.epa.gov/ncea/cfm / nceahom e.cfm >

. Versar, Inc. (2001a) Report on exploration of aging and toxic response issues. Risk Assessm ent Forum
, W a shington, DC. EPA Contract 68-C-99-238. Available from : http://cfpub.epa.gov/ncea/raf.

Versar, Inc. (2001b) Report on exploration of perina tal pharm acokinetic issues. Risk Assessm ent Forum
, W a shington, DC. EPA Contract 68-C-99-238. Available from : http://cfpub.epa.gov/ncea/raf.

W e ller, E; Long, N; Sm ith, A; et al. (1999) Dose-rate e ffects of ethylene oxide exposure on developm ental toxicity. Toxicol Sci 50:259–270.

W e st, GB; Brown, JH; Enquist, BJ. (1997) A general m odel fo r the origin of allom e tric scaling laws in biology. Science 276(5309):122–126.

R-6