Route-to-Route Extrapolation of 1,2-Dichloroethane Studies 

from the Oral Route to Inhalation Using Physiologically 

Based Pharmacokinetic Models: 

Extended One Generation Toxicity Study and Reproductive 

Toxicity Studies in the Rat 

Prepared for 

HAP Task Force, Millwood, Virginia

Prepared by 

Lisa M. Sweeney, Ph.D., DABT 

Michael L. Gargas, Ph.D. 

Independent Consultants, Dayton, Ohio 

July 30, 2015 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 2 

EXECUTIVE SUMMARY 

A need has been identified for a quantitative understanding of the hazards of 1,2-dichloroethane 

(ethylene dichloride, EDC, CAS No. 107-06-2) exposure by the inhalation route. Under Section 

4 of the Toxic Substances Control Act (TSCA), the U.S. EPA issued a testing consent order 

(Enforceable Consent Agreement, ECA) to perform toxicity testing and mechanistic and 

pharmacokinetic studies to satisfy EPA’s data needs for EDC (U.S. EPA, 2003, 2009). As part 

of Tier I testing, pharmacokinetic studies were conducted (Saghir et al., 2006) and a 

physiologically based pharmacokinetic (PBPK) model was developed that describes the 

disposition of EDC in rats by the oral and inhalation routes (Sweeney et al., 2008). 

This report serves to fulfill the identified data need for route-to-route extrapolation to the 

inhalation route of results of an extended one-generation reproductive toxicity (EOGRT) study 

recently conducted via drinking water ingestion and previously identified tests of the 

reproductive toxicity of EDC. The EOGRT study serves as a substitute for the oral subchronic 

neurotoxicity and reproductive toxicity tests called for in the amended ECA (U.S. EPA, 2003, 

2009). The route-to-route extrapolation of results of the EOGRT study of EDC using the PBPK 

model was based on selection of the no adverse effects levels for the critical endpoints of 

neurotoxicity and reproductive/developmental toxicity, the most appropriate internal dose 

measures, and an appropriate point of departure. The specified points of departure were the 

NOAEL, the highest tested doses, of 169 and 155 mg/kg-day, respectively. Based on PBPK 

modeling results presented here, the NOAELs in rats exposed by drinking water in the 

neurotoxicity and reproductive/developmental are equivalent to continuous exposure of rats to 

minimums of 76 ppm and 62 ppm EDC, respectively, using total metabolism of EDC 

(normalized to body weight) as the dose metric that is equivalent in the drinking water and 

inhalation scenarios. Sensitivity analyses of the key internal dose metric at the oral and 

inhalation-equivalent NOAEL indicated that in both scenarios, the total amount of metabolized 

was sensitive to the ingested dose/exposure concentration and the maximum rate of oxidative 

metabolism of EDC. Ingested dose was well-characterized in the study, as drinking water 

consumption was measured. The maximum rate of oxidative metabolism in adult rats was wellcharacterized in the model development stage (Sweeney et al., 2008), where sensitivity analysis 

indicated high sensitivity of blood EDC concentrations to this optimized parameter. Metabolic 

rates have not, however, been characterized in lifestages other than adult rats, introducing some 

uncertainty in internal dosimetry for neonatal rats. 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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INTRODUCTION 

1,2-Dichloroethane (ethylene dichloride, EDC, CAS No. 107-06-2) is used as primarily as an 

intermediate in the production of vinyl chloride; use of EDC as a solvent has been reduced and 

largely phased out. A need has been identified for a quantitative understanding of the hazards of 

EDC exposure by the inhalation route. Under Section 4 of the Toxic Substances Control Act 

(TSCA), the U.S. EPA issued a testing consent order (Enforceable Consent Agreement, ECA) to 

perform toxicity testing and mechanistic and pharmacokinetic studies to satisfy EPA’s data needs 

for EDC (U.S. EPA, 2003); this consent order was subsequently modified with respect to timing 

(U.S. EPA, 2009). This report describes the PBPK-based extrapolation of NOAEL doses 

identified for neurotoxicity and reproduction in an extended one-generation reproductive toxicity 

(EOGRT) study conducted by the drinking water route (Charlap, 2015) to equivalent continuous 

inhalation exposures. The EOGRT replaces the separate subchronic oral neurotoxicity and 

reproductive toxicity studies specified in the amended ECA (U.S. EPA, 2003, 2009). 

Summary of Physiologically Based Pharmacokinetic (PBPK) Model 

A combination of experimental studies (D’Souza et al., 1987, 1988; Gargas et al. 1986; Spreafico 

et al., 1980; Payan et al., 1995; Reitz et al., 1982; Withey and Karpinksi, 1985; Saghir et al., 

2006) and modeling (Sweeney et al., 2008) was used to develop a physiologically based 

pharmacokinetic (PBPK) model that would allow route-to-route extrapolation from existing and 

future data describing hazards of oral EDC exposure to potential inhalation hazards. 

The Sweeney et al. (2008) model is an expansion and refinement of previous PBPK models 

(D’Souza et al., 1987, 1988; Staats et al., 1991; ENVIRON 2004) for disposition of EDC in male 

F344 rats based on EDC kinetics data following oral gavage exposure. Sprague-Dawley rats 

receiving intravenous (iv) doses of EDC (Spreafico et al., 1980) rapidly cleared EDC from the 

blood. However, the D’Souza et al. (1987, 1988) model structure was not able to replicate the 

clearance (even when applying a large rate of metabolism in the liver), suggesting that EDC also 

undergoes extrahepatic metabolism. EDC studies (Spreafico et al., 1980; Saghir et al., 2006) 

indicate that pulmonary metabolism is not significant as evidenced by the lack of GSH depletion 

in the lung; thus lung extrahepatic metabolism is not needed, although it was included in the 

D’Souza et al. (1987, 1988) model. Oral absorption of EDC was initially described using a 

simple first-order description (D’Souza et al., 1987, 1988). However, when uptake from the GI 

tract was measured in rats dosed with radiolabeled EDC, it was found that approximately 50% of 

the administered radiolabeled EDC dose remained in the GI tract at 1 hr post dosing, but only an 

additional 15% was absorbed in the following 3 hrs, indicating bi-phasic absorption (Payan et al., 

1995). The expanded model (Sweeney et al., 2008) included a two compartment GI tract where 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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EDC was rapidly absorbed from the first compartment then sent to either the portal blood or to a 

second GI compartment where additional absorption occurs, and the extrahepatic metabolism 

was modeled as occurring in a venous blood “pool” fed by the slowly perfused tissues, kidney 

and other richly perfused tissues. Updated model parameters determined from previous models 

and literature were tested against the available data. An iterative approach was used to optimize 

the model parameters (hepatic metabolism, extrahepatic metabolism, exhalation rates, and 

absorption rates); the modified model was able to provide adequate estimates of GSH-dependent 

metabolism. The revised model provided an adequate fit to all the rat data except fetal tissue (a 

3.3-fold difference was found between modeled and predicted fetal tissue concentrations). The 

model can be used to calculate specified dose metrics of interest (blood concentrations, tissue 

concentrations, or amounts metabolized) after inhalation or gavage (corn oil or water). Model 

code is provided in Appendix A. 

SUMMARY OF KEY STUDY

Study Design

An EOGRT study of EDC administered in drinking water (Charlap, 2015) was conducted using 3 

EDC-dosed groups and a control group (27/sex/group) of sexually mature male and virgin female 

Crl:CD (Sprague-Dawley, SD) rats (F0 parental generation). The study design was intended to 

conform with the Organisation for Economic Development (OECD) Test Guideline (TG) 443 

(OECD, 2011). The intent of the EOGRT test is to identify the potential for test-chemical 

induced effects arising from prenatal, postnatal, and juvenile/young adult exposures. A particular 

emphasis is placed upon reproductive effects. The basic design in TG 443 includes three 

possible F1 (first generation offspring) cohorts for testing for reproductive/developmental effects 

(Cohort 1), neurodevelopmental effects (Cohort 2), and developmental immune effects (Cohort 

3). Cohort 1 is considered essential to the conduct of the study, and may be extended to a second 

generation (F2) if “triggered” by reproductive findings in the F1 generation. Cohorts 2 and 3 are 

optional, and their inclusion or exclusion is to be based on existing knowledge of the chemical 

and needs of the regulating authorities. Consistent with requirement for a subchronic 

neurotoxicity test and a reproduction study examining fertility index, gestation index, gross 

necropsy, organ weight, histopathology, estrous cycle, sperm evaluation, vaginal opening, and 

preputial separation as endpoints of interest in the ECA (U.S. EPA, 2003), Charlap (2015) 

included Cohorts 1 and 2, but not Cohort 3. 

Based on the lack of findings in previous reproductive toxicity studies (Almuot et al., 1976, Rao 

et al., 1980), Sweeney and Parker (2011) recommended that doses intended to potentially yield 

reproductive effects in rats should exceed 50 mg/kg-day in diet or drinking water. However, in 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 5 

consideration of maternal toxicity identified in previous studies and possible toxicokinetic 

saturation (a situation to be avoided, if possible, per OECD TG 443 [OECD, 2011]), they 

recommend that the highest dose should not exceed 300 mg/kg-day. Charlap (2015) therefore 

selected 50, 150, and 300 mg/kg/day as target exposure levels. 

Study Results

Actual Doses 

The actual EDC doses in the Charlap (2015) EOGRT study were less than the targeted doses 

with the exception of lactating F0 females. For the F1 generation, intake is averaged over the 

entire study period. For the F0 generation, the lowest intake among the various study phases 

(pre- or post-mating for males; premating, gestation, or lactation for females) was selected as a 

possible point of departure for route-to-route extrapolation. The applicable average daily intakes 

for the different generations, sexes, and doses are summarized in Table 1. 

Table 1. Applicable daily intakes for route-to-route extrapolation 

Generation/Sex Low dose 

(Target: 50 mg/kg/day)

Middle dose 

(Target: 150 mg/kg/day)

High dose 

(Target: 300 mg/kg/day)

F0 male 31 (after mating) 79 (after mating) 155 (after mating) 

F0 female 40 (prior to mating) 95 (prior to mating) 182 (prior to mating) 

F1 male 37 97 184 

F1 female 39 93 169 

Charlap (2015) evaluated many endpoints in the F0 animals and the two cohorts of F1 offspring. 

The many negative findings will not be enumerated here; notably, the potential breeding and 

evaluation of an F2 generation was not triggered. Charlap (2015) identified key findings and their 

associated no-adverse effect levels (NOAELs) in the “Conclusions” section (Section 1.4) of their 

report. In the summary table in this current report, the corresponding lowest-observed adverse 

effect levels (LOAELs) are also noted (Table 2). The key findings of Charlap (2015) consisted 

of decreases in body weight, body weight gain, and food consumptions. Charlap (2015) 

suggested that rats may have found EDC in drinking water to have poor palatability; drinking 

water consumption was generally decreased, relative to controls, in a dose-related manner. 

Charlap (2015) further suggested that food consumption thus may have been decreased to 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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parallel the reduced water intake. While this explanation is one possibility, it should be noted 

that decreased body weight gains have been noted in studies where EDC was delivered by other 

means. In a subchronic, 90-day study of EDC conducted by gavage in male and female SpragueDawley rats, significant decreases in body weight gain and food consumption were identified in 

males from the 150 mg/kg/day dose group (Daniel et al., 1994). Likewise, exposures of 300 ppm 

EDC for 6-7 hrs/day were found to produce adverse effects on maternal body weight or body 

weight gain in teratogenicity/developmental toxicity studies (Rao et al., 1980; Payan et al., 1995). 

As a health protective measure, we have chosen to include decreased body weight gain as a 

potential basis for route-to-route extrapolation. 

In the body of the report, additional findings (statistically significant or otherwise) beyond food 

consumption and body weight-related effects were described by Charlap (2015). These other 

statistically-significant findings were dismissed as reflecting variation within the laboratory’s 

historical ranges, or for lack of a consistent, monotonic dose-response relationship. We agree 

that these findings are not of concern with respect to identifying appropriate points of departure 

and key endpoints to serve as bases for route-to-route extrapolation of the study results. We 

have, however, noted one finding where the changes were outside of the WIL historical ranges—

relative liver weight in F0 females. As no significant histological findings were identified in the 

liver (or any other tissue), the organ weight finding may be considered non-adverse. Charlap 

(2015) reports other organ weight changes (within WIL historical ranges) that were consistent 

with the Daniel et al. (1994) findings of increased relative kidney, liver, and brain weights in the 

previously-mentioned 90-day study. 

Table 2. NOAELs, LOAELs, and key findings from Charlap (2015) EOGRT study 

Endpoint NOAEL 

(mg/kg/day) 

LOAEL 

(mg/kg/day) 

Responses at the LOAEL per Charlap 

(2015) conclusions, unless otherwise 

noted. 

F0 male 

(systemic) 

31 79 Lower mean body weights, body weight 

gains, and/or food consumption 

F0 female 

(systemic) 

40 95 Lower mean body weights, body weight 

gains, and/or food consumption 

Higher relative liver weights at 95 and 182 

mg/kg/day, exceeding historical ranges 

(not highlighted by Charlap, 2015) 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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Endpoint NOAEL 

(mg/kg/day) 

LOAEL 

(mg/kg/day) 

Responses at the LOAEL per Charlap 

(2015) conclusions, unless otherwise 

noted. 

F0 male 

(reproductive) 

155 None 

identified 

None identified 

F0 female 

(reproductive) 

182 None 

identified 

None identified 

F1 adult male 

(systemic) 

37 97 Lower mean body weights, body weight 

gains, and/or food consumption 

F1 adult female 

(systemic) 

93 169 Lower mean body weights, body weight 

gains, and/or food consumption 

F1 neonatal male 97 184 Lower body weights and body weight 

gains throughout postnatal period 

F1 neonatal 

female 

93 169 Lower body weights and body weight 

gains throughout postnatal period 

F1 male 

(reproductive) 

184 None 

identified 

None identified 

F1 female 

(reproductive) 

169 None 

identified 

None identified 

F1 male 

(neurotoxicity) 

184 None 

identified 

None identified 

F1 female 

(neurotoxicity) 

169 None 

identified 

None identified 

OTHER REPRODUCTIVE TOXICITY STUDIES IDENTIFIED IN THE ECA 

The ECA (U.S. EPA, 2003) specified that quantitative route-to-route extrapolation (oral to 

inhalation) would be conducted for the studies conducted under Tier II (neurotoxicity and 

reproductive toxicity) and the extant reproductive toxicity studies Alumot et al. (1976), Rao et al. 

(1980), and Lane et al. (1982). These existing studies did not identify any effects of EDC on 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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reproduction (Almuot et al., 1976, Rao et al., 1980, Lane et al. 1982) nor was any developmental 

toxicity for EDC observed (Payan et al., 1995, Lane et al. 1982). While the request for 

extrapolation of the findings of Alumot et al. (1976), a study conducted via dietary exposure, 

makes some sense, the other requests are somewhat puzzling. Rao et al. (1980) was a 

developmental toxicity study conducted by the inhalation route, obviating the need for 

extrapolation. The Lane et al. (1982) study was conducted in mice; but no PBPK model 

development and/or approval for mice was specified in the ECA. The Appendices of the ECA 

state that merely that the results of the newly-conducted study will be “compared” to those of the 

extant studies. 

Alumot et al. (1976) 

Alumot et al. (1976) describe studies in which groups of 18 rats per sex per group were fed EDCfumigated mash or a control diet. In the reproduction test, the rats consumed the fumigated diet 

for 6 weeks prior to mating. The feeding procedure, designed to minimize loss of fumigant, 

consisted of feeding the animals two portions of feed daily. In the first daily feeding, 20 percent 

of the daily allotment was provided between 11 a.m and noon, and it was quickly consumed. The 

remaining 80 percent of daily intake was made available to the rats between 7 and 9 p.m.; of this 

portion, roughly 66 percent was consumed in the first hour, with the remainder consumed during 

the second hour. The authors computed that 250 ppm EDC in diet was equivalent to 25 

mg/kg/day, so they assumed that their dose of 500 ppm EDC in diet was equivalent to 50 

mg/kg/day. The lack of findings in the Alumot et al. (1976) study is consistent with the lack of 

findings in the more extensive Charlap (2015) EOGRT study, which was conducted at higher 

ingested doses. 

Rao et al. (1980) 

Rao et al. (1980) evaluated the effects on reproduction of inhaled EDC in Sprague-Dawley rats. 

During the pre-breeding period, groups of rats (30/sex in the control group and 20/sex in each 

treated group) F0 rats were exposed to 25, 75, or 150 ppm EDC for 6 hours/day, 5 days per week. 

For the rest of the study, exposure was 6 hours/day, 7 days per week, with the exception that 

maternal animals were not exposed from gestation day 21 through the 4th day postpartum. No 

significant findings were identified in this study. 

Lane et al. (1982) 

Mice in a multigeneration study (Lane et al., 1982) were exposed to EDC in drinking water at 

concentrations intended to yield doses of 0, 5, 15, or 50 mg/kg/day. The study design included 

dominant lethal and teratology investigations. No reproductive/developmental effects were 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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identified in this study, consistent with the lack of findings in rats exposed to higher doses of 

EDC in drinking water in the Charlap (2015) EOGRT study. 

SELECTION OF CRITICAL ENDPOINTS AND DOSE MEASURES

Critical Endpoints 

The critical endpoints for systemic effects in F0 adults and adult and neonatal F1 rats in Charlap 

(2015) were decreased body weights and body weight gains. No neurotoxic, reproductive, or 

developmental effects were identified in the study. 

Critical Dose Measures

Consistent with U.S. EPA’s Guidelines for Carcinogen Risk Assessment and PBPK modeling 

guidance (U.S. EPA, 2005, 2006), the selection of an appropriate dose measure for toxicological 

endpoints of interest (both cancer and non-cancer endpoints) requires a careful consideration of 

the mode of action by which a chemical exerts an effect. Parent compound (peak, average, or 

area under the concentration vs. time curve [AUC]) and amount metabolized were suggested as 

dose metrics in the ECA (U.S. EPA, 2003). The dose metrics to be considered were expanded 

beyond those suggested in the ECA. The following additional dose metrics were considered: 

total amount of EDC metabolized in the liver (normalized to liver weight) (AMLVL), the 

minimum concentration of glutathione in the liver (GSHLmin) (per Sweeney and Parker, 2010), 

the peak concentration of EDC in arterial blood (CAmax) and the average concentration of EDC 

in arterial blood (CAavg, computed as AUCB/duration) were also evaluated, in order to provide 

a comprehensive assessment. 

The data indicate that the most appropriate dose metric for route to route extrapolation of hepatic 

effects (i.e., increased liver weights) is the minimum post exposure concentration of GSH in the 

liver (GSHLmin). Igwe et al. (1986) evaluated the potential interaction of dietary disulfiram 

(Antabuse) and EDC exposure in male Sprague-Dawley rats treated with the compounds for 30 

days. Disulfiram acts to block the metabolism of aldehydes by aldehyde dehydrogenase. One 

pathway for EDC metabolism, which competes with GSH conjugation, is oxidative metabolism 

to chloroacetaldehyde (Figure 1); co-exposure to disulfiram would block the metabolism of 

chloroacetaldehyde by aldehyde dehydrogenase (ALDH), resulting in potentially greater 

accumulation of chloroacetaldehyde and subsequent diversion of the metabolism of this 

intermediated from chloroacetic acid to the chloroacetaldehyde-GSH conjugate, with the 

consequence of more extensive depletion of GSH. Igwe et al. (1986) found that the combined 

exposure to EDC and disulfiram increased hepatotoxicity (mid-zonal necrosis), decreased spleen 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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weight, and resulted in testicular atrophy. These findings support the use of dose metrics related 

to EDC metabolism rather than parent compound as indicators of hepatotoxicity. 

Figure 1. Metabolic scheme for EDC 

Chloroacetaldehyde has been demonstrated to be hepatotoxic when administered directly to mice 

in drinking water (Daniel et al., 1992) and chloroacetaldehyde is cytotoxic to isolated rat 

hepatocytes (Sood and O’Brien, 1993). The studies by Sood and O’Brien (1993) included 

incubations with added antioxidants or GSH-depletion prior to the addition of 

chloroacetaldehyde. The greater susceptibility of hepatocytes when GSH was depleted indicates 

that the toxicity is not likely to be due to the generation of the GSH-chloroacetaldehyde 

conjugate. Incubations of hepatocytes with chloroacetaldehyde and added antioxidants produced 

delays in cytotoxicity, indicating that the overall antioxidant status is the best indicator of 

potential hepatotoxicity in chloroacetaldehyde-exposed liver cells. Thus, GSHLmin (lowest 

GSH concentration in the liver after EDC exposure) is supported as the most appropriate dose 

metric for identifying an EDC inhalation concentration that would be expected to produce 

hepatotoxicity. 

Because reproductive and developmental effects were not observed in the toxicity studies of 

EDC conducted thus far, our evaluation of potential dose measures will be based on evaluation of 

evidence regarding potential modes of action for structurally related compounds, specifically 

low-molecular-weight halogenated hydrocarbons, as was done for developmental toxicity of 

1,1,2-trichloroethane (The Sapphire Group, 2005). It was concluded that while developmental 

toxicity related to the parent compound is a plausible mode of action for halogenated 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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hydrocarbons, for 1,1,2-tricholoroethane, toxicity due to metabolic activation was considered a 

more likely mode of action for potential developmental effects (The Sapphire Group, 2005). By 

analogy, we also assume that EDC metabolites, rather than parent compound, were more likely to 

be reproductive toxicants in the rat, had such effects been identified. 

ROUTE TO ROUTE EXTRAPOLATION METHODS

Route-to-route extrapolation of the NOAELs and LOAELs for various key endpoints in the 

Charlap (2015) EOGRT study were performed using the PBPK model (Sweeney et al., 2008). 

Minor changes were made to the model code by Sweeney and Parker (2011) to convert it from 

ACSL 11.8.4 to acslX (Version 2.5.0.6) and to facilitate computation of the dose metrics to be 

considered in the subchronic route to route extrapolation, interpretation of previously-conducted 

reproductive and developmental toxicology studies (Alumot et al., 1976; Rao et al., 1980), and 

design of the extended one-generation reproductive toxicity study (Sweeney and Parker, 2011, 

Appendix A). Internal dose metrics associated with the NOAELs and LOAELs were determined 

from PBPK model simulations (Appendix B, Tables B-1, B-2, and B-3) using group-specific 

body weights averaged over the full study or, in the case of neonates, relevant life stage. 

Equivalent inhalation exposure concentrations were derived using a body weight of 250 g for 

male and female rats (with the exception of neonates, assumed to weight 25 g) and assuming 24 

hr/day exposure. Additional assumptions regarding the physiology and anatomy for neonates 

were (i.e., blood flow distribution, tissue volumes, ventilation rate, and cardiac output) were the 

same as those used by Sweeney et al. (2015) for simulation of PND 22 rats; the values used by 

Sweeney et al. (2015) were based on a data compilation prepared by Gentry et al. (2006). 

Inhalation exposure concentrations producing NOAEL- and LOAEL-equivalent internal dose 

metrics were derived by iteratively simulating various trial concentrations until a trial 

concentration approximately matching the simulated internal dose from the drinking water study 

was derived (Appendix B, Table B-5, B-6, and B-7). The level of precision of the equivalent 

exposure concentrations is two significant figures. 

ROUTE TO ROUTE EXTRAPOLATION RESULTS

The consideration of an array of dose metrics yielded ranges of potential NOAEL and LOAEL 

equivalents for F0 and F1 rats, reported in Appendix B for systemic, reproductive, neurotoxic, and 

neonatal effects. Only those for the reproductive/developmental and neurotoxic endpoints are 

reported in this section, because those were the endpoints specified for study in the ECA (U.S. 

EPA, 2003). 

For reproductive/developmental effects, the highest tested oral doses were designated NOAELs 

by Charlap (2015) (155 and 184 mg/kg/d for F0 and F1 males, respectively; 182 and 169 mg/kg/d 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 12 

for F0 and F1 females, respectively). The internal dose metric which yielded the lowest 

equivalent inhalation concentrations was the total amount of EDC metabolized, normalized to 

body weight. This continuous exposure equivalent 62 ppm for F0 males, the lowest inhalation 

equivalent for the 2 generations and sexes. 

Neurotoxicity testing in the Charlap (2015) EOGRT study was conducted on an F1 cohort. The 

highest doses were identified as NOAELs—184 mg/kg/d for male rats, 169 mg/kg/d for female 

rats. As with the extrapolation of the reproductive/developmental toxicity testing results, use of 

amount of EDC metabolized (normalized by body weight), yields the lowest equivalent 

continuous inhalation concentrations, 76 and 78 ppm for males and females, respectively. More 

commonly, however, neurotoxic effects of solvents are attributed to peak concentrations of the 

parent compound. Use of CAmax as the basis of the extrapolation would yield continuous 

inhalation concentrations of 160 ppm for males, 128 ppm for females. 

SENSITIVITY ANALYSIS

The route-to-route extrapolation of the EOGRT of EDC in rats involved the consideration of 

multiple endpoints and internal dose metrics consistent with supportable modes of action, as 

computed using a PBPK model (Sweeney et al., 2008). 

Reproductive/Developmental Toxicity Extrapolation 

The sensitivity of the critical dose metric for reproductive/developmental toxicity, the simulated 

total amount EDC metabolized (AMETBW), to changes in model parameter values was 

evaluated for scenarios involving drinking water ingestion or continuous inhalation. In the 

drinking water scenario, a NOAEL of 155 mg/kg-day to male rats, was used as the basis of the 

analysis. The continuous inhalation equivalent for male rats, 62 ppm for 24 hrs/day, was used as 

the basis of the inhalation sensitivity analysis. 

The sensitivity analysis was conducted by changing the value of an input parameter and noting 

the change in the model prediction of interest (AMETBW), normalized to the baseline prediction 

of that dose metric. The fractional increase in the prediction divided by the fractional increase in 

the parameter was defined as the normalized sensitivity coefficient (NSC). A negative value 

indicates that when the value of the input parameter increases, the output decreases in value. A 

value of 1 indicates a positive linear relationship between the input and output (e.g., a 1% 

increase in the input parameter produces a 1% increase in the output of interest). The results are 

summarized in Table 3. For the drinking water scenario, AMETBW was moderately sensitive to 

the dose, the time over which the dose was assumed to be ingested, and the rate of oxidative 

metabolism in the liver (VMAXLC). Ingested dose was well-characterized in the study, as 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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drinking water consumption was measured. The maximum rate of oxidative metabolism in adult 

rats was well-characterized in the model development stage (Sweeney et al., 2008), where 

sensitivity analysis indicated high sensitivity of blood EDC concentrations to this optimized 

parameter. Metabolic rates have not, however, been characterized in lifestages other than adult 

rats, introducing some uncertainty in internal dosimetry for neonatal rats. Under the inhalation 

scenario, AMETBW was most sensitive to the exposure concentration and ventilation rate. 

Table 3. Sensitivity coefficients for total amount of EDC metabolized (AMETBW): 

reproductive/developmental toxicity extrapolation 

Parametera 

Drinking 

water dose 

(155 mg/kgday) Inhalation exposure 

(62 ppm) 

Drinking water dose (CDOSEC) 0.48 Not applicable 

Dose ingestion duration (TING) 0.38 Not applicable 

Inhaled concentration (CONC) Not applicable 0.91 

Alveolar ventilation rate (QPC) -0.12 0.78 

Body weight (BW) -0.14 -0.27 

Blood:air partition coefficient (PB) |NSC| < 0.1 0.14 

Maximum rate of oxidative metabolism of EDC in the 

liver (VMAXLC) 0.50 0.13 

Maximal rate of extrahepatic EDC metabolism, as a 

fraction of maximum rate of oxidative metabolism of 

EDC in the liver (XLRATIO) 0.12 |NSC| < 0.1 

a Parameters not listed had |NSC|<0.1 for both scenarios. 

Neurotoxicity Extrapolation 

The sensitivity of two relevant dose metrics for neurotoxicity, the more mechanistically realistic 

metric peak concentration of EDC (CAmax) and the most conservative metric, total EDC 

metabolism (AMETBW), to changes in model parameter values was evaluated for scenarios 

involving drinking water ingestion or continuous inhalation. In the drinking water scenario, a 

NOAEL of 155 mg/kg-day to F1 male rats, was used as the basis of the analysis. The continuous 

inhalation equivalents for male rats, continuous exposure to76 ppm for AMETBW and 160 ppm 

for CAmax, were used as the basis of the inhalation sensitivity analyses. 

The sensitivity analyses were conducted as described for reproductive/developmental toxicity. 

The results are summarized in Tables 4 and 5. For AMETBW, the neurotoxicity sensitivity 

analyses were very similar to the reproductive/developmental toxicity sensitivity analyses, and 

occurrence that was not surprising given that the dose metric and NOAELs were very similar. In 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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contrast, the maximum arterial blood concentration of EDC (CAmax) was sensitive to more 

parameters than AMETBW, and was sensitive to a much greater degree. It appears that EDC 

metabolism is saturated in these scenarios, since increases in dose or exposure concentration 

produce much greater than linear increases in CAmax (NSC >1), and increases in the capacity for 

oxidative metabolism likewise produce greater than linear decreases in the peak concentration. 

An increase in the blood:air partition coefficient (PB) also increases CAmax because of a 

decreased tendency for EDC clearance via exhalation. Under a drinking water scenario, 

ventilation functions only as a clearance mechanism, so the sensitivity coefficient is negative. 

Under an inhalation exposure, increase QPC yields a greater than linear increase in CAmax, but 

not as much of an increase as is produced by a similar increase in inhaled exposure concentration 

(CONC). 

Table 4. Sensitivity coefficients for total amount of EDC metabolized (AMETBW): 

neurotoxicity extrapolation 

Parametera 

Drinking 

water dose 

(184 mg/kgday) Inhalation exposure 

(76 ppm) 

Drinking water dose (CDOSEC) 0.50 Not applicable 

Dose ingestion duration (TING) 0.41 Not applicable 

Inhaled concentration (CONC) Not applicable 0.87 

Alveolar ventilation rate (QPC) -0.11 0.73 

Body weight (BW) -0.15 -0.27 

Blood:air partition coefficient (PB) |NSC| < 0.1 0.15 

Maximum rate of oxidative metabolism of EDC in the 

liver (VMAXLC) 0.51 0.18 

Maximal rate of extrahepatic EDC metabolism, as a 

fraction of maximum rate of oxidative metabolism of 

EDC in the liver (XLRATIO) 0.12 |NSC| < 0.1 

a

Parameters not listed had |NSC|<0.1 for both scenarios. 

Table 5. Sensitivity coefficients for peak arterial blood concentration of EDC (CAmax): 

neurotoxicity extrapolation 

Parametera 

Drinking water 

dose (169 mg/kgday) 

Inhalation 

exposure (128 

ppm) 

Drinking water dose (CDOSEC) 2.72 Not applicable 

Dose ingestion duration (TING) -2.67 Not applicable 

Rate of absorption of EDC from the intestines (KAI) 0.13 Not applicable 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 15 

Parametera 

Drinking water 

dose (169 mg/kgday) 

Inhalation 

exposure (128 

ppm) 

Inhaled concentration (CONC) Not applicable 2.28 

Alveolar ventilation rate (QPC) -0.75 1.63 

Body weight (BW) 0.78 |NSC| < 0.1 

Blood:air partition coefficient (PB) 0.77 0.65 

Maximum rate of oxidative metabolism of EDC in 

the liver (VMAXLC) -1.72 -1.36 

Michaelis constant for EDC oxidation (KM) |NSC| < 0.1 0.11 

Maximal rate of extrahepatic EDC metabolism, as a 

fraction of maximum rate of oxidative metabolism of 

EDC in the liver (XLRATIO) -0.41 -0.35 

Maximum rate conjugation of EDC with GSH in the 

liver (VMAXLGC) -0.27 -0.12 

Michaelis constant for EDC for GSH conjugation 

(KMG) 0.21 0.11 

a

Parameters not listed had |NSC|<0.1 for both scenarios. 

CONCLUSIONS

The route-to-route extrapolation of results of the EDC EOGRT study using the PBPK model was 

based on selection of critical endpoints, an array of internal dose measures, and appropriate points of 

departure. For reproductive/developmental toxicity, a dose metric related to the most credible mode 

of action for chemically similar compounds was chosen. For neurotoxic effects, a conservative dose 

measure and the most plausible metric (on a mechanistic basis) were selected from among the 

options. The rat reproductive/developmental toxicity NOAEL of 155 mg/kg-day by drinking water 

was determined to be equivalent to continuous (24 hr/day) exposure of rats to 62 ppm EDC, when 

using the conservative dose metric of total EDC metabolism normalized to body weight. The male 

rat neurotoxicity NOAEL of 184 mg/kg-day by drinking water was determined to be equivalent to 

continuous (24 hr/day) exposure of rats to 76 ppm EDC, when using the conservative dose metric of 

total EDC metabolism normalized to body weight, and 160 ppm when using peak arterial blood EDC 

concentration, which is more reflective of a likely EDC neurotoxic mode of action. 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 16 

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EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 19 

APPENDIX A: PBPK MODEL CODE 

edc4.csl 

PROGRAM: GLUTATHIONE DEPLETION MODEL FOR EDC (EDC4.CSL) 

!Initial parameter values for the rat (DSouza et al. 1987, 1988) 

!Retyped, documentation modified by LMS 7/19/01 

!revised February 23, 2005 

!Add kidney, update tissue volumes and flows per Brown et al., 1997 

!Adjust flow and volume calcs to ensure mass balance 

!Add chamber for closed chamber simulations, add iv administration 

!implement GSH delay per Environ report 

!revise GSH parameter calculations to ensure ss in unexposed 

!gsh turnover rates from Potter and Tran, tissue specific values 

!Input pcs as tissue:air and calculate tissue:blood 

!revised February 23, 2006 

!add two compartment GI, repeat gavage dosing, GST conj of parent in liver 

!turn off lung metab for now 

!revised June 2006 extrahepatic metabolism, reformulate reactions 

!for oxidative metabolite! 

!2010-06-11 edited by Lisa M. Sweeney for acslX 

!2010-06-17 added feeding scenario to accommodate simulation of Alumot et al. 

1976 

!and extended one gen ingestion scenario 

INITIAL 

!SPECIAL FLOW RATES! 

CONSTANT QPC=15 $!alveolar ventilation rate L/hr/kg^0.74! 

CONSTANT QCC=15 $!Cardiac output L/hr/kg^0.74! 

!FRACTIONAL BLOOD FLOW TO TISSUES! 

CONSTANT QLC = 0.18 $!Fractional blood flow to liver! 

CONSTANT QFC = 0.07 $!Fractional blood flow to fat! 

CONSTANT QSC = 0.34 $!Fractional blood flow to slow! 

CONSTANT QKC = 0.14 !Fractional blood flow to kidney 

 QRC = 1.0-(QLC + QFC + QSC + QKC) $!Fractional blood flow to rapid! 

!BODY WEIGHT! 

CONSTANT BW = 0.22 $!Body weight (kg)! 

!FRACTIONAL TISSUE VOLUMES! 

CONSTANT VPC = 0.005 $!Fraction lung tissue! 

CONSTANT VLC = 0.037 $!Fraction liver tissue! 

CONSTANT VFC = 0.07 $!Fraction fat tissue! 

CONSTANT VSC = 0.59 $!Fraction slow tissue (skin and muscle)! 

CONSTANT VKC = 0.0073 !Fraction kidney 

constant vvc = 0.05 

VRC = 0.95- (vpc + vlc + vfc + vsc + vkc + vvc) $!Fraction rapid tissue! 

!PARTITION COEFFICIENTS! 

CONSTANT PPA = 33 $!Lung/air partition coefficient! 

CONSTANT PLA = 33 $!Liver/air partition coefficient! 

CONSTANT PFA = 340 $!Fat/air partition coefficient! 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 20 

CONSTANT PSA = 23 $!Slowly perfused tissue/air partition coefficient! 

CONSTANT PRA = 33 $!Richly perfused tissue/air partition coefficient! 

CONSTANT PKA = 33 $!Kidney/air partition coefficient! 

CONSTANT PB = 29 $!Blood/air partition coefficient! 

 pp=ppa/pb 

 pl=pla/pb 

 pf=pfa/pb 

 ps=psa/pb 

 pr=pra/pb 

 pk=pka/pb 

CONSTANT MW = 98.96 $!Molecular weight (g/mol)! 

!KINETIC CONSTANTS! 

CONSTANT VMAXlC = 3.15 !!Maximum velocity of oxidative metabolism (mg/hrkg^0.7)! 

CONSTANT KM = 0.25 $!Michaelis-Menten constant (mg/L)! 

CONSTANT vmaxlgc=480.0 $!enzy conj rate const gsh w parent(mg/(hr-kg^-0.7))! 

constant kmg = 2000 !Km with respect to EDC mg/L (not rate limiting at relev 

conc?) 

constant kmgg = 125 !Km with respect to liver GSH in uM (rate limiting when 

gsh depleted) 

CONSTANT KGSM =2.0 $!gsh conj rate const w metab (1/(uM-hr))! 

CONSTANT KFEE=4600 $!conj rate const w non gsh (1/hr)! 

CONSTANT Kdgsyn=0.32 $!GSH synthase breakdown (1/hr)! 

CONSTANT Kdgshl=0.14 $!GSH turnover rate--liver! 

CONSTANT KDGSHP =0.011 !Gsh turnover rate--lung 

CONSTANT KSL = 200 $!Maximum GSH production control (liver)! 

CONSTANT KSp = 200 $!Maximum GSH production control (lung)! 

CONSTANT Tau = 2 $!Time delay constant (hr)! 

CONSTANT GSOl = 5500 $!Initial GSH concentration (uM)! 

constant gslmit = 1200 !mitochondrial GSH, does not conjugate or turnover 

CONSTANT GSOp = 1200 $!Initial GSH concentration (uM)! 

CONSTANT PLRATIO=0.0 $!MFO ratio lung/liver! 

constant racpgp=0 

CONSTANT xLRATIO=0.0 !MFO ratio extrahepatic/liver 

constant kdet = 0.025 !P450 turnover constant (1/hr) 

constant kdi = 0.0 !second order reaction rate for liver P450 inactivation 

by oxid metab 

 ! 1/((mg-equiv/l)-hr) 

!DOSING INFO! 

CONSTANT CONC = 0 $!Inhaled concentration (ppm)! 

CONSTANT VCHC = 9.1 !Total chamber volume (L) 

CONSTANT NRATS = 0. !number of rats in closed chamber 

 Vch = vchc-nrats*bw !net chamber volume 

CONSTANT KLC = 0.0 !loss rate to chamber 

 AI0 = conc*vch*mw/24450.0 !initial mass of chemical in chamber 

constant odosec = 0 !gavage dose at t=0 in mg/kg 

constant kas=1 !oral absorption rate from stomach 

constant kt = 1 !transport rate from stom to intestines 

constant kai = 1 ! oral absorption rate from intestines 

constant ivdosec = 0. !iv dose at t=0 in mg/kg 

constant tinf=0.01 !duration of iv injection (hr) 

riv=ivdosec*bw/tinf 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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constant FDOSEC =0. ! daily dose in feed (Alumot study) in mg/kg 

constant CDOSEC = 0. ! daily dose for continuous ingestion (water or feed) 

mg/kg/d 

constant TING = 12. ! ingestion duration for continuous ingestion scenario 

(hrs) 

!TIMING PARAMETERS! 

CONSTANT TSTART =0. $!Start of exposure (hrs)! 

CONSTANT TPER=24 

CONSTANT TSTOP=120 

CONSTANT POINTS=1200 $!Number of points in plot! 

 CINT = TSTOP/POINTS $!Communication interval! 

constant TCHNG=6 

!SCALED PARAMETERS! 

 QC = QCC*BW**0.74 

 QP = QPC*BW**0.74 

 QL = QLC*QC 

 QF = QFC*QC 

 QS = QSC*QC 

 QK = QKC*QC 

 qr = qc-(ql+qf+qs+qk) 

 VP = VPC*BW 

 VL = VLC*BW 

 VF = VFC*BW 

 VS = VSC*BW 

 VR = VRC*BW 

 vk = vkc*bw 

 vv = vvc*bw 

!Liver metabolism! 

 VMAXl0=VMAXlC*BW**0.7 

 vmaxlg = vmaxlgc*bw**0.7 

 vmaxp= vmaxl0*plratio 

 vmaxx=vmaxl0*xlratio 

 AGSOl=GSOl*VL 

!Lung metabolism! 

 AGSOp=GSOp*VP 

 P1=0 

 P1R=0 

 P2=0 

 P2R=0 

 P3=100 

 P3R=100 

KgshprodL0=agsol*kdgshl !baseline liver gsh synthesis rate umol/hr 

 r10=kgshprodl0 

 r20=kgshprodl0 

 kgshprodtdl0=kgshprodl0 

kgshprodp0=agsop*kdgshp !baseline lung gsh synthesis rate umol/hr 

 rp10=kgshprodp0 

 rp20=kgshprodp0 

 kgshprodtdp0=kgshprodp0 

kgsynprodl0=kgshprodl0*kdgsyn !baseline prod gsh synthetase umol/hr/hr 

kgsynprodp0=kgshprodp0*kdgsyn !base prdxn of gsh synthetase in lung 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 22 

END $!End of initial! 

DYNAMIC 

ALGORITHM IALG=2 $!Gear method for stiff systems! 

DERIVATIVE 

 RAI = nrats*qp*(CA/pb - ci) -klc*ai 

 AI = INTEG(RAI, AI0) 

 cch= ai/vch 

 cp = cch*24450/mw 

!CI=Concentration in inhaled air (mg/l)! 

 CI=cch*pulse(tstart, tper, tchng) 

 RAinh=qp*ci 

 Ainh=INTEG(RAINH,0.) 

 !Algebraic solution for CA1 after gas exchange! 

 !CA1 is arterial blood concentration after gas exchange! 

 CA1= (QC*CV +QP*CI)/(QC + QP/PB) 

 CX = CA1/PB 

 raexh=qp*cx 

aexh=integ(raexh,0.) 

 !Mass balance for the lung tissue compartment! 

 RAP = QC*(CA1-CA) - RAMpu 

 AP = INTEG(RAP,0.0) 

 Clung = AP/VP 

 AUCP=INTEG(CP,0.0) 

 CA=Clung/PP 

 AUCB=INTEG(CA,0.0) 

 !UPTAKE BY ORAL ROUTE! 

 RFEEDA = 

FDOSEC*BW*(0.528*(PULSE(0.0,24.,1.))+0.272*(PULSE(1.,24.,1.))+0.2*PULSE(16.,24

.,1.)) !Alumot study 

 RCDOSE= CDOSEC*BW/TING*(PULSE(0.0, 24.,TING)) 

 RING=RFEEDA+RCDOSE 

 AING = INTEG(RING, 0.) 

 RSTOM = -(KAs+kt)*STOM+RING $!dSTOM/dT! 

 days=t/24 

 ODOSE =odosec*bw*(1.0 + (int(days))) !repeat dosing 

 STOM =INTEG(RSTOM,0.0) +ODOSE $!amount in stomach (mg)! 

 rintest = kt*stom - kai*intest 

 intest = integ(rintest, 0.) 

 !AS = Amount in slowly perfused tissues (mg)! 

 RAS = QS*(CA-CVS) 

 AS = INTEG(RAS,0.0) 

 CVS = AS/(VS*PS) 

 CS = AS/VS 

 !AR = Amount in richly perfused tissues (mg)! 

 RAR = QR*(CA-CVR) 

 AR = INTEG(RAR,0.0) 

 CVR = AR/(VR*PR) 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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 CR = AR/VR 

 !AK = Amount in kidney (mg)! 

 RAk = Qk*(CA-CVk) 

 Ak = INTEG(RAk,0.0) 

 CVk = Ak/(Vk*Pk) 

 Ck = Ak/Vk 

 !AF = Amount in fat tissues (mg)! 

 RAF = QF*(CA-CVF) 

 AF = INTEG(RAF,0.0) 

 CVF = AF/(VF*PF) 

 CF = AF/VF 

 !CV = Mixed venous blood concentration (mg/l)! 

 rinf=riv*pulse(0., 2000., tinf) 

 ivdose=integ(rinf,0.) 

 RAVx = rinf + (QS*CVS + QR*CVR+ qk*CvK)-(qs+qr+qk)*CVx - ramx 

 

 AV = integ(ravx, 0.) 

 cvx=Av/vv 

 cv = QFc*CVF + QLc*CVL + cvx*(qsc + qrc + qkc) 

 !AL = Amount in liver tissue (mg)! 

 RAL = QL*(CA-CVL)-RAMl + KAs*STOM + kai*intest 

 AL = INTEG(RAL,0.0) 

 CVL = AL/(VL*PL) 

 CL = AL/VL 

 AUCL = INTEG(CL,0.) 

 !LIVER METABOLISM! 

 !AM = Amount metabolized liver (mg)! 

 RAMl= RAMox + RAMCon 

 RAMox =(VMAXl*CVL)/(KM+CVL) 

 ramcon = vmaxlg*(GSl/(gsl+kmgg))*(CVL/(cvl + kmg)) 

 AMl=INTEG(RAMl,0.) 

 AMPl=AMl*1000/MW 

 RAMPl=RAML*1000/MW 

!time-dependence of hepatic P450 oxidation due to inactivation by oxid 

metabolite 

 rvmaxl = (vmaxl0-vmaxl)*kdet - vmaxl*kdi*CML 

 vmaxl = INTEG(rvmaxl, vmaxl0) 

 !CMl=OXIDATIVE METABOLITE LIVER mg/L)! 

 RAMMl= ramox -RACMGl*MW/1000-RACMEEl*MW/1000 

 AMMl=INTEG(RAMMl,0.) 

 CML=AMMl/VL 

 AUCML=INTEG(CML,0.) 

 !ACPGl = AMT PARENT CONJUGATED WITH GSH LIVER (uMOLES)! 

 RACPGl=ramcon*1000/MW 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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 ACPGl=INTEG(RACPGl,0.) 

 !ACMGl=AMT METABOLITE CONJUGATED WITH GSH LIVER (uMOLES)! 

 RACMGl=KGSM*GSl*CMl*1000/MW*VL 

 ACMGl=INTEG(RACMGl,0.0) 

 !ACMEEl=AMT METABOLITE CONJUGATED WITH EVERYTHING ELSE LIVER! 

 !uMOLES! 

 RACMEEl=KFEE*VL*CMl*1000/MW 

 ACMEEl=INTEG(RACMEEl,0.) 

!LUNG METABOLISM! 

 !AMp=Amount metabolized lung (mg)! 

 RAMpu=RAMpox + rampcon 

 rampox=(VMAXp*CA)/(KM+CA) 

 rampcon=RACPGp*MW/1000 

 AMp=INTEG(rampu,0.) 

 AMPp=AMp*1000./MW 

 RAMPp=RAMpu*1000./MW 

!CMp=OXIDATIVE METABOLITE (mg/L)! 

 RAMMp= rampox -RACMGp*MW/1000-RACMEEp*MW/1000 

 AMMp=INTEG(RAMMp,0.) 

 CMp=AMMp/VP 

!ACMGp= AMT METABOLITE CONJUGATED WITH GSH LUNG (uMOLES)! 

 RACMGp=KGSM*GSp*CMp*VP*1000/MW 

ACMGp=INTEG(RACMGp,0.) 

!ACMEEp=AMT METABOLITE CONJUGATED WITH EVERYTHING ELSE LUNG (uMOLES)! 

 RACMEEp=KFEE*VP*CMp*1000/MW 

 ACMEEp=INTEG(RACMEEp,0.) 

!ACPGp=AMT PARENT CONJUGATED WITH GSH LUNG (uMOLES)! 

 ! RACPGp=KGS*GSp*Ca*VP*1000/MW 

 ACPGPp=INTEG(RACPGp,0.) 

!extrahepatic METABOLISM! 

 !AMx = Amount metabolized extrahepatic (mg)! 

 RAMx= RAMxox + RAMxCon 

 amx=integ(ramx, 0.) 

 RAMxox =(VMAXx*CVx)/(KM+CVx) 

 amxox = integ(ramxox, 0.) 

 ramxcon=0 

 amxcon=integ(ramxcon, 0.) 

!gsh synthesis rate--time dependent 

 kgshprodl=integ(kgsynprodl0*(ksl+gsol)/(ksl+gsl) & 

 -kdgsyn*kgshprodl,kgshprodl0) 

 kgshprodp=integ(kgsynprodp0*(ksp+gsop)/(ksp+gsp) & 

 -kdgsyn*kgshprodp,kgshprodp0) 

!time delay (Bishchoff et al. 1971) 

 r0=kgshprodl 

 rr1=(r0-r1)/tau 

 r1=integ(rr1,r10) 

 rr2=(r1-r2)/tau 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

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 r2=integ(rr2,r20) 

 rr3=(r2-kgshprodtdl)/tau 

 kgshprodtdl=integ(rr3, kgshprodtdl0) 

 rp0=kgshprodp 

 rrp1=(rp0-rp1)/tau 

 rp1=integ(rrp1,rp10) 

 rrp2=(rp1-rp2)/tau 

 rp2=integ(rrp2,rp20) 

 rrp3=(rp2-kgshprodtdp)/tau 

 kgshprodtdp=integ(rrp3, kgshprodtdp0) 

!GSl = GlUTAHIONE LIVER (uM)! 

 RAMGSl=kgshprodtdl-Kdgshl*GSl*VL-RACMGl-RACPGl 

 AMGSl=INTEG(RAMGSl,AGSOl) 

 GSl=AMGSl/VL 

 gshl = gsl + gslmit 

!GSp=GLUTAHIONE LUNG (uM)! 

 RAMGSp=Kgshprodtdp-Kdgshp*GSp*VP-RACMGp-RACPGp 

 AMGSp=INTEG(RAMGSp,AGSOp) 

 GSp=AMGSp/VP 

!PCTGSH-PERCENT GSH COMPARED TO CONTROL! 

 PCTGSHP=GSp/GSOp*100 

!mass balance 

anet = AING+ odose + ivdose + ainh - aexh 

atissue=ap + as+ ar + al+ ak + af +av 

agi = stom + intest 

amet = aml + amp + amx 

AMETBW = AMET/BW !total amount metabolized, normalized to BW 

AMXBW = AMX/BW !extrahepatic metabolism normalized to BW 

AMLVL = AML/VL !liver metabolism normalized to BW 

termt(t.ge.tstop) 

END $!End of derivative! 

END $!End of dynamic! 

END $!End of program 

Rat.m 

% rat.m 

% 2010-06-11 created from edc4.cmd proced rat by Lisa M. Sweeney 

% physiol parameters from Brown et al. 1997 

QPC=15; QCC=15; 

QLC=0.18;QFC=0.07; QSC=0.34; QKC=0.14; 

PB=29; 

PLA=33; PPA=10; PFA=340;PRA=33; PSA=23; PKA=33; 

VMAXLC=4.0; KM=0.25; PLRATIO=0.0; XLRATIO=0.3; 

%PLRATIO WA0.026 

VMAXLGC=4.80; KMGG=530; KMG=20.0; 

KGSM=2.0;KFEE=4600.0; 

KDGSYN=0.324;KDGSHL=0.14; KDGSHP=0.011; 

GSOL=5500;GSOP=1200; TAU=0.89; 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 26 

KSL=1200; KSP=240; 

NRATS=0; KLC=0; CONC=0; IVDOSEC=0; ODOSEC=0;FDOSEC=0.;CDOSEC=0; 

VCHC=9.1; KAS=8; KT=2; KAI=0.15; % Default absorption parameters are for corn 

oil gavage 

TSTART=0; TPER=24; TSTOP=24; TCHNG=24; TINF=0.01; 

Sdrat.m 

% sdrat.m 

% updated 5/19/2006 by Lisa M. Sweeney 

% updated 6/9/2006 LMS 

% 2010-06-10 sdrat m file created from edc4.cmd files by Lisa M. Sweeney 

%proced sdrat 

%fat from schoeffner et al. 1999 

%pcs from Dsouza et al., 1987 

use rat 

VFC=0.065; 

PB=28; PLA=30; PSA=22; 

BW=0.267; %default weight for a male SD rat in a subchronic study (EPA, 1988) 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 27 

APPENDIX B 

PBPK-Derived Internal Dosimetry and Route-to-Route Extrapolations for EDC-Exposed 

Rats 

Values of the dose metrics of arterial blood EDC (peak and average concentrations), liver GSH 

concentration and daily metabolism occurring the liver (normalized to liver weight or body weight) 

were predicted using the PBPK models. Simulations were conducted at the relevant NOAELs and 

LOAELs for both sexes, using study specific body weights. The outputs are provided for F0 rats 

(Table B-1), F1 rats from birth to PND 123 (Table B-2), and F1 rats from birth to PND 21 (Table B3). The values of these same metrics in rats exposed to an array of inhaled concentrations of EDC 

(0-1000 ppm) 24 hrs/day were computed for rats of standard weight for a subchronic study (U.S. 

EPA, 1988) by Sweeney and Parker (2010); those values are reproduced here for the convenience of 

the reader. 
Table B-1. Internal dose metrics corresponding to the NOAELs and LOAELs for F0 rats in the Charlap (2015) rat EOGRT 

study, as computed using a PBPK model (Sweeney et al., 2008; Sweeney and Parker, 2011) 

Dose metric 

F0 male systemic toxicity 

F0 female systemic 

toxicity 

F0 male 

reproductive 

toxicity 

F0 female 

reproductive 

toxicity 

NOAEL 

31 mg/kg/d 

(554 g) 

LOAEL 

79 mg/kg/d

(524 g) 

NOAEL 

40 mg/kg/d 

(306 g) 

LOAEL 

95 mg/kg/d

(295 g) 

NOAELa 

155 mg/kg/d 

(500 g) 

NOAELa 

182 mg/kg/d 

(288 g) 

Total amount of EDC metabolized 

daily, normalized by body weight 

(AMETBW) (mg/kg body 

weight/day) 30.3 72.6 39.1 87.2 109 128 

Amount of EDC metabolized in liver 

each day, normalized by liver weight 

(AMLVL) (mg/kg liver/day) 726 1590 934 1907 2396 2802 

Maximum concentration of EDC in 

arterial blood (CAmax) (mg/L) 0.0870 1.02 0.0994 1.09 7.27 7.40 

Time-weighted average EDC in 

arterial blood (CAavg) (mg EDC/L 

blood) 0.0415 0.409 0.0470 0.434 2.95 3.04 

Minimum concentration of 

glutathione in the liver (GSHLmin) 

(μM) 5089 3723 4713 3348 2805 2473 

a

 Highest tested dose 
EDC Ext. One-Gen. Route-to-Route July 30, 2015 

Page 29 

Table B-2. Internal dose metrics corresponding to the NOAELs and LOAELs for F1 rats (birth to PND 123) in the Charlap 

(2015) rat EOGRT study, as computed using a PBPK model (Sweeney et al., 2008; Sweeney and Parker, 2011) 

Dose metric

F1 male systemic toxicity F1 female systemic toxicity

F1 male reproductive 

 toxicity and neurotoxicity

F1 female reproductive 

toxicity and neurotoxicity

NOAEL 

37 mg/kg/d

(270 g) 

LOAEL 

97 mg/kg/d

(261 g) 

NOAEL 

93 mg/kg/d

(173 g) 

LOAEL 

169 mg/kg/d

(162 g) 

NOAELa 

184 mg/kg/d 

(240 g) 

NOAELa 

169 mg/kg/d 

(162 g) 

AMETBWb 36.2 89.3 88.2 134 132 134 

AMLVL 895 1957 1965 2913 2889 2913 

CAmax 0.0809 1.03 0.554 4.20 6.79 4.20 

CAavg 0.0387 0.411 0.231 1.70 2.79 1.70 

GSHLmin 4779 3297 3303 2434 2417 3303 

a

 Highest tested dose 

b For definitions and units, see Table B-1. 
Table B-3. Internal dose metrics corresponding to the NOAELs and LOAELs for neonatal 

F1 rats (birth to PND 21), 25 g, in the Charlap (2015) rat EOGRT study, as computed 

using a PBPK model (Sweeney et al., 2008; Sweeney and Parker, 2011) 

Dose metric

F1 male neonatal toxicity F1 female neonatal toxicity

NOAEL 

97 mg/kg/d

(27.4 g) 

LOAEL 

184 mg/kg/d

(24.9 g) 

NOAEL 

93 mg/kg/d

(26.0 g) 

LOAEL 

169 mg/kg/d

(23.8 g) 

AMETBWa 94.5 171 90.7 160 

AMLVL 2304 3882 2223 3667 

CAmax 0.1386 0.804 0.124 0.5594 

CAavg 0.0646 0.333 0.0578 0.237 

GSHLmin 3011 2121 3081 2211 

a

For definitions and units, see Table B-1. 

Table B-4. Internal dose metrics corresponding to 24-hr exposure of adult rats to a range 

of inhalation concentrations (ppm), as computed using a PBPK model (Sweeney et al., 

2008); reproduced from Sweeney and Parker (2010) 

 Males 

Concentration 0 1 10 100 1000

AMETBWa 0 1.88 18.6 162 288

AMLVL 0 20.6 225 3323 6619

CAmax 0 0.00965 0.106 2.42 97.4

CAavg 0 0.00965 0.106 2.41 97.5

GSHLmin 6700 6673 6403 3491 1721

 Females 

Concentration 0 1 10 100 1000

AMETBWa 0 2.02 20.0 174 308

AMLVL 0 22.0 240 3572 7113

CAmax 0 0.00960 0.106 2.39 97.5

CAavg 0 0.00960 0.106 2.38 97.3

GSHLmin 6700 6671 6383 3339 1660 a

For definitions and units, see Table B-1. 
Table B-5. Inhaled concentrations (ppm) for 24-hr/day exposures to EDC that yield simulated internal doses (Sweeney et al., 2008; 

Sweeney and Parker, 2011) equivalent to NOAEL and LOAEL doses from the EOGRT study (Charlap, 2015) in 250 g F0 rats 

Dose metric

F0 male systemic toxicity F0 female systemic toxicity 

F0 male reproductive 

toxicity 

F0 female reproductive 

toxicity 

NOAEL 

31 mg/kg/d 

LOAEL 

79 mg/kg/d 

NOAEL 

40 mg/kg/d 

LOAEL 

95 mg/kg/d 

NOAELa 

155 mg/kg/d 

NOAELa 

182 mg/kg/d 

AMETBWa 16 40 21 48 62 74 

AMLVL 26 50 32 58 70 80 

CAmax 8.4 60 9.4 63 160 166 

CAavg 4.2 31 4.7 32 110 111 

GSHLmin 44 85 54 102 142 184 

a

For definitions and units, see Table B-1. 

Table B-6. Inhaled concentrations (ppm) for 24-hr/day exposures to EDC that yield simulated internal doses (Sweeney et al., 2008; 

Sweeney and Parker, 2011) equivalent to NOAEL and LOAEL doses from the EOGRT study (Charlap, 2015) in 250 g F1 rats (birth to 

PND 123)

Dose metric

F1 male systemic toxicity F1 female systemic toxicity

F1 male reproductive 

 toxicity and neurotoxicity

F1 female reproductive 

toxicity and neurotoxicity

NOAEL 

37 mg/kg/d

LOAEL 

97 mg/kg/d

NOAEL 

93 mg/kg/d 

LOAEL 

169 mg/kg/d 

NOAELa 

184 mg/kg/d 

NOAELa 

169 mg/kg/d 

AMETBWb 19 50 49 78 76 78 

AMLVL 31 59 59 83 83 83 

CAmax 7.8 61 39 128 160 128 

CAavg 3.9 31 20 83 107 83 

GSHLmin 52 105 105 190 193 190 

a

 Highest tested dose 

b For definitions and units, see Table B-1. 
Table B-7. Inhaled concentrations (ppm) for 24-hr/day exposures to EDC that yield simulated 

internal doses (Sweeney et al., 2008; Sweeney and Parker, 2011) equivalent to NOAEL and 

LOAEL doses from the EOGRT study (Charlap, 2015) in neonatal F1 rats (birth to PND 21)

Dose metric

F1 male neonatal toxicity F1 female neonatal toxicity

NOAEL 

97 mg/kg/d

(27.4 g) 

LOAEL 

184 mg/kg/d

(24.9 g) 

NOAEL 

93 mg/kg/d

(26.0 g) 

LOAEL 

169 mg/kg/d

(23.8 g) 

AMETBWa 25 47 24 43 

AMLVL 38 57 36 55 

CAmax 12 49 11 37 

CAavg 5.9 25 5.3 19 

GSHLmin 63 101 61 95 

a

For definitions and units, see Table B-1. 

The values of the “equivalent” inhaled concentrations were determined for all generations, endpoints, 

and life stages, and are reported in Tables B-5, B-6, and B-7. The “equivalent” values were highly 

dependent on the choice of internal dose metric. For example, the inhalation equivalent of 31 

mg/kg-day via drinking water ingestion by male rats ranged from 4.2 ppm, based on the average 

concentration of EDC in arterial blood to 44 ppm, based on the minimum level of GSH in the liver. 

The endpoints of primary interest for this study, reproductive toxicity and neurotoxicity, had higher 

NOAELs that the example previously noted. At these higher NOAELs, the ranges of equivalent 

values span ~3-fold, rather than the >10-fold range identified at lower ingested doses. The most 

conservative doses metrics also shift from average arterial blood concentration (at lower doses) to 

amount metabolized in the liver (normalized to body weight) at higher doses.