DRAFT PEER REVIEW SUMMARY REPORT


FINAL

REVIEWER COMMENTS

External Peer Review Meeting on the
Toxicological Review of Chloroprene
(CAS No. 126-99-8)

Prepared for:

Allen Davis, M.P.H.

National Center for Environmental Assessment
U.S. Environmental Protection Agency
109 T.W. Alexander Drive
Research Triangle Park, NC 27711

Prepared by:

Versar, Inc.
Contract No. EP-C-07-025
Task Order 69

Peer Reviewers:

Herman J. Gibb, Ph.D., M.P.H.
Dale Hattis, Ph.D.
Ronald L. Melnick, Ph.D.
John B. Morris, Ph.D.
Avima M. Ruder, Ph.D.
Richard B. Schlesinger, Ph.D.

January 26, 2010

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External Peer Review Meeting on the Toxicological Review of Chloroprene

TABLE OF CONTENTS

I.	INTRODUCTION	1

II.	CHARGE TO THE REVIEWERS	2

III.	GENERAL IMPRESSIONS	5

IV.	RESPONSE TO CHARGE QUESTIONS	7

General Charge Questions	7

Chemical-Specific Charge Questions	14

(A)	Oral Reference Dose (RfD) for Chloroprene	14

(B)	Inhalation Reference Concentration (RfC) for Chloroprene	15

(C)	Carcinogenicity of Chloroprene	25

V.	SPECIFIC OBSERVATIONS	49

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External Peer Review Meeting on the Toxicological Review of Chloroprene

I. INTRODUCTION

The Integrated Risk Information System (IRIS) is an EPA database containing Agency
consensus scientific positions on potential adverse human health effects that may result
from chronic (or lifetime) exposure, or in select cases less-than-lifetime exposures, to
chemicals in the environment. IRIS currently provides health effects information on over
500 chemical substances. IRIS contains chemical-specific summaries of qualitative and
quantitative health information in support of two steps of the risk assessment process, i.e.,
hazard identification and dose-response evaluation. IRIS information includes a reference
dose (RfD) for noncancer health effects resulting from oral exposure, a reference
concentration (RfC) for noncancer health effects resulting from inhalation exposure, and
an assessment of carcinogenicity for both oral and inhalation exposures. Combined with
specific situational exposure assessment information, the health hazard information in
IRIS may be used as a source in evaluating potential public health risks from
environmental contaminants.

The IRIS program developed a Toxicological Review of Chloroprene, an assessment
which has not previously appeared in IRIS. Chloroprene was nominated for IRIS
assessment in 1999. The draft document contains a chronic inhalation reference
concentration (RfC) and a cancer inhalation unit risk.

Peer Reviewers:

Herman J. Gibb, Ph.D., M.P.H.

Tetra Tech Sciences
Arlington, VA 22201

Dale Hattis, Ph.D.

Clark University
Worcester, MA 01610

Ronald L. Melnick, Ph.D.

Ron Melnick Consulting, LLC
Chapel Hill, NC 27514

John B. Morris, Ph.D.

University of Connecticut
Storrs, CT 06269

Avima M. Ruder, Ph.D.

National Institute for Occupational Safety and Health (NIOSH)

Cincinnati, OH 45226

Richard B. Schlesinger, Ph.D.

Pace University
Pleasantville, NY 10570

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External Peer Review Meeting on the Toxicological Review of Chloroprene

II. CHARGE TO THE REVIEWERS

The U.S. Environmental Protection Agency (EPA) is seeking an external peer review of
the scientific basis supporting the human health assessment of chloroprene that will
appear on the Agency's online database, the Integrated Risk Information System (IRIS).
IRIS is prepared and maintained by the EPA's National Center for Environmental
Assessment (NCEA) within the Office of Research and Development (ORD). Currently
an IRIS assessment of chloroprene does not exist on the database.

The draft health assessment includes a chronic reference concentration (RfC) and a
carcinogenicity assessment. Below are a set of charge questions that address scientific
issues in the assessment of chloroprene. Please provide detailed explanations for
responses to the charge questions.

General Charge Questions:

1.	Is the Toxicological Review logical, clear and concise? Has EPA clearly synthesized
the scientific evidence for noncancer and cancer hazards?

2.	Please identify any additional studies that should be considered in the assessment of
the noncancer and cancer health effects of chloroprene.

Chemical-Specific Charge Questions:

(A)	Oral Reference Dose (RfD) for Chloroprene

1. An RfD was not derived for chloroprene. Has the scientific justification for not
deriving an RfD been clearly described in the document? Please identify and provide
the rationale for any studies that should be selected as the principal study.

(B)	Inhalation Reference Concentration (RfC) for Chloroprene

1.	A chronic RfC for chloroprene has been derived from an inhalation toxicity study
(NTP, 1998) investigating non-cancer effects in multiple organ systems. Please
comment on whether the selection of this study as the principal study is scientifically
justified. Please identify and provide the rationale for any other studies that should be
selected as the principal study.

2.	An increase in the incidence of degenerative nasal lesions in male rats, characterized
by olfactory epithelial atrophy and/or necrosis with increasing severity, was selected
as the critical effect. Please comment on the scientific justification for combining the
incidence of atrophy and necrosis and for selecting this endpoint as the critical effect.
Please identify and provide the rationale for any other endpoints that should be
considered in the selection of the critical effect.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

3. Benchmark dose (BMD) modeling was used to define the point of departure (POD)
for the derivation of the RfC. The POD was based on increased incidence of
degenerative nasal lesions in male rats at a benchmark response (BMR) of 10% extra
risk. Has the BMD approach been appropriately conducted? Is the BMR selected for
use in deriving the POD (i.e., 10% extra risk of degenerative nasal lesions of less than
moderate severity) scientifically justified? Please identify and provide the rationale
for any alternative approaches (including the selection of the BMR, model, etc.) for
the determination of the POD and discuss whether such approaches are preferred to
EPA's approach.

4. Please comment on the rationale for the selection of the uncertainty factors (UFs)
applied to the POD for the derivation of the RfC. If changes to the selected UFs are
proposed, please identify and provide a rationale(s).

1. Under the EPA's 2005 Guidelines for Carcinogen Risk Assessment
(www.epa.gov/iris/backgr-d.htm), the Agency concluded that chloroprene is likely to
be carcinogenic to humans by all routes of exposure. Please comment on the cancer
weight of evidence characterization. Is the cancer weight of evidence characterization
scientifically justified?

2. A two-year inhalation cancer bioassay in B6C3F1 mice (NTP, 1998) was selected as
the basis for derivation of an inhalation unit risk (IUR). Please comment on whether
the selection of this study for quantification is scientifically justified. Please identify
and provide the rationale for any other studies that should be selected as the basis for
quantification.

3. A mutagenic mode of carcinogenic action is proposed for chloroprene. Please
comment on whether the weight of evidence supports this conclusion. Please
comment on whether this determination is scientifically justified. Please comment on
data available for chloroprene that may support an alternative mode(s) of action.

4. Data on hemangiomas/hemangiosarcomas (in all organs) and tumors of the lung
(bronchiolar/alveolar adenomas and carcinomas), forestomach, Harderian gland
(adenomas and carcinomas), kidney (adenomas), skin and mesentery, mammary
gland and liver in B6C3F1 mice were used to estimate the inhalation unit risk. Please
comment on the scientific justification and transparency of this analysis. Has the
modeling approach been appropriately conducted? Please identify and provide the
rationale for any alternative approaches for the determination of the inhalation unit
risk and discuss whether such approaches are preferred to EPA's approach.

5. Lung tumors have been alternatively treated as systemic or portal-of-entry effects in
the modeling of cancer endpoints. Please comment on the scientific justification for
this modeling approach. Please comment on whether the rationale for this decision
has been transparently and objectively described. Please comment on data available

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External Peer Review Meeting on the Toxicological Review of Chloroprene

for chloroprene that may support an alternative method for modeling the observed
lung tumors in mice.

6. An oral slope factor (OSF) for cancer was not derived for chloroprene. Is the

determination that the available data for chloroprene do not support derivation of an
OSF scientifically justified?

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External Peer Review Meeting on the Toxicological Review of Chloroprene

III. GENERAL IMPRESSIONS
Herman J. Gibb

In general, the document lays out its arguments well. The discussion of the epidemiology,
however, should be more transparent and perhaps could be better organized (studies of a
facility where cohorts overlap or could overlap discussed together). Elaboration on the
transparency is provided in my response to Question CI. The epidemiologic studies
should be evaluated more rigorously.

Dale Hattis

Overall, the judgments made in the draft IRIS document for chloroprene are sound.
However the modeling of the cancer risk can be improved by taking into account the
existing evidence for partial saturation of metabolic activation of chloroprene in the dose
range studied in the NTP cancer bioassay. Using a simple Michaelis Menten dose
response equation to model this approach to saturation indicates that low dose cancer
risks in both the male and female mouse bioassays are likely to be 2-3 fold greater than
the risks indicated by application of a straight linear dose response model, as was done
using the Weibull equation in the current cancer slope factor analysis. For the final
assessment it would be desirable either to incorporate the Michaelis-Menten saturating
form into the Weibull model or (less desirably) to multiply the Weibull model result by a
factor derived from the Michaelis-Menten analysis of the lifetime tumor incidence
information. The former approach is preferable because it will simultaneously take into
account the time-to-tumor information and the apparent saturation of activating
metabolism indicated by the incidence data.

Ronald L. Melnick

The draft document is a well-written, comprehensive review and assessment of published
studies on the health effects of chloroprene in humans and in experimental animals. The
information is clearly presented and the conclusions are generally scientifically justified
and consistent with EPA policy. One exception is the rationale for the selection of 10%
extra risk for the benchmark response. Specific areas for improvement of this review are
described below in my response to the "chemical-specific charge questions."

John B. Morris

From my perspective as an inhalation toxicologist with expertise in rodent studies, the
Toxicological Review of Chloroprene provides an in depth review of the toxicological
literature on this compound. In many ways it is quite clear and thorough. The available
database appears to be presented accurately and objectively. The overall conclusion, that
chloroprene is an animal carcinogen whose mechanism(s) may include genotoxicity and
mutagenesis, appears well founded. In some aspects, the document is confusing and
perhaps lacks transparency. For example, information is provided in the summary and
synthesis sections that have not been discussed previously. There are some apparent

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External Peer Review Meeting on the Toxicological Review of Chloroprene

contradictions in interpretive approaches, for example the potential for systemic blood
delivery for the pulmonary but not nasal effects. The importance of some findings has
gone unrecognized. For example, the extraordinarily high pulmonary metabolism rates in
the mouse calls into question the relevance of this species with respect to pulmonary
injury. Overall, the fundamental conclusions appear sound; however, the document could
be significantly improved with respect to clarity and interpretive issues.

It is interesting that there are no charge questions relating to the toxicokinetics of
chloroprene. Since the mode of action includes activation to an epoxide as the first step,
the toxicokinetics becomes an issue of great importance. The toxicokinetic section
describes the available information, but could provide much more information.

Moreover, the toxicokinetic data is not adequately synthesized in the overall mode of
action relative to potential species differences and extrapolation to man. PBPK modeling
would be a highly appropriate way to incorporate kinetic data into the risk assessment.
The published model of Himmelstein may provide a useful structure. Because it includes
both nasal and tracheobronchial airway compartments the styrene model of Sarangapani
may be a superior approach.

Avima M. Ruder

I can only validate accuracy for the section I compared to the original papers, that on
human epidemiology. There are some key relevant references that were not cited and
some points that should have been discussed (latency, age at diagnosis, etc.) that were not
touched on (see 2.1).

The conclusions about the human hazard potential do not evaluate the role of genetic
polymorphism in genes coding for glutathione ^-transferases, epoxide hydrolase, and
other metabolic enzymes in clearing epoxide metabolites from the body. Approximately
half the human population is clears those metabolites at a much slower rate [Musak, et al.
2008], presumably making them more vulnerable to exposure. The conclusion also
should point out that the noncancer effects (page 6-1, lines 24-33) were observed at levels
lower than the current Permissible Exposure Limit.

The statements of conclusions in section 6 are less clear than those in section 4.7. It is
appropriate to include all relevant caveats about the conclusions, and all the details of the
studies that support those conclusions, but the conclusions themselves should be
succinctly stated.

Richard B. Schlesinger

The background information that is provided to support the selection of the key studies is
clearly and accurately presented. However, the derivation of some of the quantitative
factors, as noted in subsequent comments in this document, could be made more
transparent. In general, the overall conclusions appear to be sound.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

IV. RESPONSE TO CHARGE QUESTIONS
General Charge Questions:

/. Is the Toxicological Review logical, clear ami concise? Has HP. I clearly synthesized
the scientific evidence for noncancer and cancer hazards?

Herman J. Gibb

In general, the Toxicological Review is logical, clear and concise. A more rigorous and
transparent evaluation of the epidemiologic studies and an objective evaluation of how
the epidemiologic studies integrate with the rest of the data should be performed,
however. The descriptor of "likely to be carcinogenic to humans" is justified based on the
animal and genotoxicity information, but the document overstates the human evidence.

Dale Hattis

Generally, yes. I have some reservations and suggestions for incremental improvement,
as will be apparent below. But the overall evaluation in the proposed IRIS document is
sound.

Ronald L. Melnick

While the Toxicological Review is clear and comprehensive, it is not obvious why a
particular dose response model was selected for the determination of the benchmark dose
for noncancer hazards, if more than one model provided an adequate fit to the data. The
rationale for the selection of 10% extra risk for the benchmark response for non-cancer
effects is not adequately justified.

Based on the animal data, mechanistic findings, and "the reasonably consistent" evidence
of increased risk of liver cancer mortality "among workers exposed to chloroprene in
different cohorts in different continents," it is not clear why consideration was not given
to the conclusion that chloroprene is "carcinogenic to humans."

John B. Morris

In many ways, the toxicological review is logical and clear; however, the document could
be significantly improved in this regard. See my specific comments (below) for more
detail on this concern.

Avima M. Ruder

The review is logical but less clear and concise than it could be. In the section on human
carcinogenicity, the discussion should have been consolidated by population and
recommendations for additional analyses (by age at onset/death, with lags) and substudies
(nested case-control) should have been included. Such analyses should be done as very

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External Peer Review Meeting on the Toxicological Review of Chloroprene

early age at cancer onset/death has been associated with occupational exposure [Kreuzer,
et al. 1999; Ward, et al. 1988] and lagged analyses focus on exposure in time periods that
are most relevant for the development of solid tumors [Villeneuve and Steenland 2010],
All the studies on the Louisville plant should have been discussed together. The original
study includes ages at death from lung cancer for 16 workers, including four who died in
their forties [Pell 1978], but no analysis of whether the ages at onset were earlier than
expected (in another chloroprene cohort, earlier ages at onset among exposed workers
were reported [Li, et al. 1989]). The NIOSH walk-through survey of the plant, which was
not referenced in the Toxicological Review, provides useful details on plant history,
processes, and personnel, noting that "there is a complete pre-employment physical" plus
periodic re-examinations (presumably those who did not meet some standard of health
were excluded from employment; no details were presented on how the periodic re-
examinations impacted continued employment [Jones, et al. 1975], The NIOSH re-
analysis of DuPont demographic data included recommendations for improving the
epidemiologic studies by including all plant employees from 1942 on [Leet and Selevan
1982], Blood draws from 846 of the workers employed in 1977 were compared for
biochemical and hematological markers, with no significant differences in age-adjusted
analyses [Gooch and Hawn 1981] and workers and plant sites were monitored for
exposure, and workers interviewed [McGlothlin, et al. 1984](neither referenced in the
Toxicological Review).

One of the more recent University of Pittsburgh papers (not referenced in the
Toxicological Review), presents SMRs for the Louisville cohort using the DuPont worker
mortality database; these are significantly elevated for all causes of death, all cancers,
respiratory cancers, and liver cancer [Leonard, et al. 2007], Kentucky cancer mortality is
significantly higher than U.S. national cancer mortality [U.S. Cancer Statistics Working
Group 2009], and the incidence of lung cancer in both Jefferson county and all of
Kentucky is almost 50% higher than the U.S. rate [Kentucky Institute of Medicine 2007],
so comparisons of a working population to the population at large will show a
pronounced healthy worker effect. Presumably an employment-based database would
control for the healthy worker effect to some extent. The most recent studies are more
comprehensive but could have included additional analyses by age at diagnosis/death,
lagged analyses, comparisons with the DuPont employee mortality database, and
inclusion of the pre-1949 PYAR [Marsh, et al. 2007a; Marsh, et al. 2007b], Some
discrepancies should be explored; for example, Jones stated that approximately 8000
hourly and 1000 salaried (one-third foremen) employees had been employed to the time
of the 1975 visit and over 1000 workers were employed in 1975; the Marsh analysis
includes 5507 employees 1949-2000 [Jones, et al. 1975; Marsh, et al. 2007a],

Some discrepancies between the report of a 1985 NIOSH walk-through of the
Pontchartrain, Louisiana, plant (neoprene production from 1968, 1264 workers to 1985)
and the recent epidemiologic studies (chloroprene from 1969, 1258 workers to 2000) also
need to be resolved [Fajen and Ungers 1985; Marsh, et al. 2007a; Marsh, et al. 2007b],

The studies of the plant in Grenoble, Isere, France, should also have been assessed
together [Colonna and Laydevant 2001; Marsh, et al. 2007a; Marsh, et al. 2007b],

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External Peer Review Meeting on the Toxicological Review of Chloroprene

As to possible human health hazards other than cancer, the two medical studies at the
Louisville plant [Gooch and Hawn 1981; McGlothlin, et al. 1984] and the recent study of
chromosomal aberrations [Musak, et al. 2008] should be included. Apparently there are
no studies of possible human reproductive effects more recent than Sanotskii's in 1976.

Richard B. Schlesinger

In general, the Review is well written and the toxicology of chloroprene is well
synthesized.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

General Charge Questions:

2. Please identify any additional studies that should be considered in the assessment of
the noncancer and cancer health effects of chloroprene.

Herman J. Gibb

The NIOSH reports by Fajen and Ungers (1985) and by McGlothin et al (1984) should be
included as background on the Pontchartrain and Louisville plants, respectively. Copies
were provided to the peer reviewers by Avima Ruder subsequent to the peer review
meeting on January 6, 2010 and are attached. Dr. Ruder also described references of
Jones et al. (1975), Gooch and Hawn (1981), and Leonard et al. (2007) in her comments.
Jones et al. (1975) and Gooch and Hawn (1981) describe conditions and the population at
the Louisville plant and should be added as background information on that facility. The
Leonard et al. paper apparently presents mortality analyses of the Louisville cohort using
a Dupont worker mortality database. These papers should be reviewed to determine what
insights they may offer to the mortality analyses by Pell (1978), Leet and Selevan (1982)
and Marsh et al. (2007a, 2007b).

I am not aware of any additional original studies or reports that should be considered. The
following reviews by Acquavella and Leonard (2001) and Bukowski (2009) should at
least be given consideration although they need not necessarily be referenced. The review
by Acquavella and Leonard (2001) appeared in the same journal as the review by Rice
and Boffetta (2001) which is cited in the current Toxicological Review.

Acquavella JF, Leonard RC. 2001. A review of the epidemiology of 1,3-butadiene and
chloroprene. Chemico-Biological Interactions 135-136 (2001) 43-52.

Bukowski JA. 2009. Epidemiologic evidence for chloroprene carcinogenicity: review of
study quality and its application to risk assessment. Risk Analysis 29(9): 1203-16.

Dale Hattis

Probably the most significant omission is an analysis by Dr. DeWoskin of EPA of the
potential to use a PBPK model for estimation of human vs. mouse and rat delivered doses
in modeling cancer dose response relationships for chloroprene. Its omission from the list
of references is surprising. The abstract of this paper I retrieved from a MEDLINE search
is:

PBPK models in risk assessment—A focus on chloroprene.

DeWoskin RS.

Chem Biol Interact. 2007 Mar 20;166(l-3):352-9. Epub 2007 Feb 8.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

US EPA/NCEA (National Center for Environmental Assessment), Mail Drop B243-01,
Research Triangle Park, NC 27711, USA. dewoskin.rob@epa.gov

Mathematical models are increasingly being used to simulate events in the exposure-
response continuum, and to support quantitative predictions of risks to human health.
Physiologically based pharmacokinetic (PBPK) models address that portion of the
continuum from an external chemical exposure to an internal dose at a target site.
Essential data needed to develop a PBPK model include values of key physiological
parameters (e.g., tissue volumes, blood flow rates) and chemical specific parameters (rate
of chemical absorption, distribution, metabolism, and elimination) for the species of
interest. PBPK models are commonly used to: (1) predict concentrations of an internal
dose over time at a target site following external exposure via different routes and/or
durations; (2) predict human internal concentration at a target site based on animal data
by accounting for toxicokinetic and physiological differences; and (3) estimate variability
in the internal dose within a human population resulting from differences in individual
pharmacokinetics. Himmelstein et al. [M.W. Himmelstein, S.C. Carpenter, P.M.
Hinderliter, Kinetic modeling of beta-chloroprene metabolism. I. In vitro rates in liver
and lung tissue fractions from mice, rats, hamsters, and humans, Toxicol. Sci. 79 (1)
(2004) 18-27; M.W. Himmelstein, S.C. Carpenter, M.V. Evans, P.M. Hinderliter, E.M.
Kenyon, Kinetic modeling of beta-chloroprene metabolism. II. The application of
physiologically based modeling for cancer dose response analysis, Toxicol. Sci. 79 (1)
(2004) 28-37] developed a PBPK model for chloroprene (2-chloro-1,3-butadiene; CD)
that simulates chloroprene disposition in rats, mice, hamsters, or humans following an
inhalation exposure. Values for the CD-PBPK model metabolic parameters were
obtained from in vitro studies, and model simulations compared to data from in
vivo gas uptake studies in rats, hamsters, and mice. The model estimate for total
amount of metabolite in lung correlated better with rodent tumor incidence than did
the external dose. Based on this PBPK model analytical approach, Himmelstein et al.
[M.W. Himmelstein, S.C. Carpenter, M.V. Evans, P.M. Hinderliter, E.M. Kenyon,

Kinetic modeling of beta-chloroprene metabolism. II. The application of physiologically
based modeling for cancer dose response analysis, Toxicol. Sci. 79 (1) (2004) 28-37;
M.W. Himmelstein, R. Leonard, R. Valentine, Kinetic modeling of beta-chloroprene
metabolism: default and physiologically-based modeling approaches for cancer dose
response, in: IISRP Symposium on Evaluation of Butadiene & Chloroprene Health
Effects, September 21, 2005, TBD—reference in this proceedings issue of Chemical-
Biological Interactions] propose that observed species differences in the lung tumor dose-
response result from differences in CD metabolic rates. The CD-PBPK model has not yet
been submitted to EPA for use in developing the IRIS assessment for chloroprene, but is
sufficiently developed to be considered. The process that EPA uses to evaluate PBPK
models is discussed, as well as potential applications for the CD-PBPK model in an IRIS
assessment.

In reading the document, I don't recall coming across an explanation for why the
implications of this model for cancer risk were not explored. It seems to me that the high
dose saturation effects that are apparent in the tumor data could be explained in part by
even a basic application of this kind of model. Explaining the high dose saturation of the

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External Peer Review Meeting on the Toxicological Review of Chloroprene

metabolic activation would, I think, (1) avoid the need to eliminate the high dose for
some data sets and (2) lead to an increase in the estimate of the linear coefficients for the
cancer dose response model. The PBPK model may well be considered not sufficiently
tested against human data for un-caveated application to human risk projection, but I
think its implications should at least be explored for sensitivity analyses.

Ronald L. Melttick

No additional studies were found that would significantly impact the overall assessment.
John B. Morris

I am aware of no additional toxicity studies relative to chloroprene. The mouse
bronchiolar airway lesions are reminiscent of those induced by naphthalene and styrene.
In this regard, comparisons to these compounds might provide some useful perspectives.

Avima M. Ruder

Two recent studies of genetic damage in workers exposed to chloroprene are relevant to
this review.

Heuser VD, de Andrade VM, da Silva J, Erdtmann B. 2005. Comparison of genetic
damage in Brazilian footwear-workers exposed to solvent-based or water-based
adhesive. Genet Tox Environ Mutat/Mutat Res 583(l):85-94.

This study compared Comet assay results for unexposed workers, workers using water-
based adhesives, and workers using solvent-based adhesives containing polychloroprene
(and, presumably, some chloroprene as a contaminant), with a significantly higher
damage index among the solvent-based adhesive users than either the unexposed or
workers using water-based adhesives.

It was not entirely clear from the article whether the solvent-based adhesive group used
adhesives (and other compounds), as stated on page 90, or produced the polychloroprene
(page 91). In either case, there are a number of additional exposures which might have
been associated with the chromosome damage. Other than the chromosome results no
health effects were reported.

Musak L, Soucek P, Vodickova L, Naccarati A, Halasova E, Polakova V, Slyskova J,
Susova S, Buchancova J, Smerhovsky Z and others. 2008. Chromosomal
aberrations in tire plant workers and interaction with polymorphisms of
biotransformation and DNA repair genes. Mutat Res 641(l-2):36-42.

This study compared lymphocyte chromosome aberrations among smoking and
nonsmoking tire workers (exposed to butadiene) and controls. In addition, participants
were genotyped for polymorphisms in genes encoding metabolic enzymes.

"Chromosomal aberrations were higher in subjects with GSTTl-null (2.4 ± 1.7%) than in

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External Peer Review Meeting on the Toxicological Review of Chloroprene

those with GSTTl-plus genotype (1.8 ± 1.4%; F = 7.2, P = 0.008)." In light of the papers
on diene (butadiene, chloroprene, isoprene) metabolism that indicate that the
detoxification of a mutagenic metabolite goes through the GST pathway [Himmelstein, et
al. 2004a; Himmelstein, et al. 2004b; Munter, et al. 2007; Munter, et al. 2003], this result
is significant. It means that the fifty percent of the human population that is GST-null
may be at higher risk from exposure; any exposure-associated carcinogenicity could be
higher in this susceptible subpopulation.

Other studies to consider:

Fajen JM, Ungers LJ. 1985. DuPont de Nemours and company, Pontchartrain
Works, LaPlace, LA, IWS-147-31. LA, LaPlace: NIOSH, Cincinnati, OH. 1-18 p.

Jones JH, Young RJ, Selevan S. 1975. du Pont de Nemours and Company, Inc.,
Louisville, Kentucky, IWS-87-10. KY, Louisville: NIOSH, Cincinnati, OH. 1-9 p.

McGlothlin JD, Meyer C, Leet TL. 1984. E.I. DuPont De Nemours And Company,
Louisville, KY, HETA-79-027-1459. KY, Louisville: NIOSH, Cincinnati, OH. 1-28 p.

These NIOSH site visits provide concise histories of processes and chemicals at the
plants, as well as descriptions of records and medical monitoring (Fajen and Jones
reports) and a Health Hazard Evaluation (McGlothlin).

Leonard RC, Kreckmann KH, Lineker GA, Marsh G, Buchanich J, Youk A. 2007.
Comparison of standardized mortality ratios (SMRs) obtained from use of reference
populations based on a company-wide registry cohort to SMRs calculated against
local and national rates. Chem Biol Interact 166(l-3):317-22.

This study calculated SMRs for the Louisville and Pontchartrain chloroprene plants using
the DuPont employee database as a reference population, rather than the U.S. national or
local population. For the Louisville plant, ".. .the SMRs based on the total U.S. DuPont
worker mortality rates for all causes of death (1.13), all cancers (1.11), and respiratory
cancers (1.37) are statistically significantly increased. The SMR for liver cancer (1.27),
although elevated, is not statistically significant."

Richard B. Schlesinger

There are none that I am aware of.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Chemical-Specific Charge Questions:

(A) Oral Reference Dose (RfD) for Chloroprene

1. An RfD was not derivedfor chloroprene. Has the scientific justification for not
deriving an RfD been clearly described in the document? Please identify and provide
the rationale for any studies that should be selected as the principal study.

Herman J. Gibb

The scientific rationale for not deriving an RfD has been clearly described.

Dale Hattis

Yes. But such a derivation would be possible if the PBPK model (or some suitable range
of models derived from sensitivity analyses) were used.

The principal study selected for analysis is fine.

Ronald L. Melnick

Yes, the lack of an adequate multiple-dose oral toxicity study on chloroprene that could
be used for a dose-response analysis and the lack of information on the disposition of
chloroprene after inhalation or oral exposure that would enable a reliable route-to-route
extrapolation justify not deriving an RfD for this chemical. Because of a likely large first-
pass liver effect after oral exposure, the systemic distribution of parent compound and
reactive metabolites could be very different after oral or inhalation exposures.

John B. Morris

An oral RfD was not derived for chloroprene. The current database is clearly described.
The rationale for the decision to not derive an oral RfD is clearly and concisely described.
The scientific justification is appropriate and the decision is well founded.

Avima M. Ruder

As the document states, there are no human data on oral exposure and only one lifetime
animal study, so clearly the justification for not deriving an RfD exists.

Richard B. Schlesinger

The decision not to derive an RfD is clearly justified in the document as based upon the
lack of appropriate datasets for oral exposure.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

(B) Inhalation Reference Concentration (RfC) for Chloroprene

1. A chronic RfC for chloroprene has been derived from an inhalation toxicity study
('NTP\ 1998) investigating non-cancer effects in multiple organ systems. Please comment
on whether the selection of this study as the principal study is scientifically justified.
Please identify and provide the rationale for any other studies that should be selected as
the principal study.

Herman J. Gibb

The selection of this study is justified. The document states that the Trochimowicz et al.
study was not chosen as the principal study "primarily due to the lack of observed effects
at similar exposure levels as the NTP (1998) study"(page 4-39, lines 19-20; page 5-2,
lines 26-29). That doesn't seem as strong an argument as the high mortality in the low
dose animals which were suffocated by the ventilation system (page 5-2, lines 13-16, 29-
31).

Dale Hattis

The principal study selected for analysis is fine.

Ronald L. Melnick

The selection of the NTP chronic inhalation toxicity study as the principal study for the
derivation of an RfC for chloroprene is scientifically justified. This was a well designed
and conducted study, which identified several non-cancer effects in multiple organs of
rats and mice exposed to a wide range of concentrations of chloroprene. A major strength
of this study is the multiple histopathological reviews of lesions identified in rats and
mice. The study clearly demonstrates the toxicity of chloroprene in multiple species and
the data are suitable for dose-response analyses.

John B. Morris

The selection of the NTP inhalation study as the principal study is scientifically justified.
It was well conducted and subject to peer review.

Avima M. Ruder

The data files for two human studies conducted at the Louisville plant [Gooch and Hawn
1981; McGlothlin, et al. 1984] might have some information on subchronic effects.

Gooch and Hawn did biochemical and hematological assays on blood specimens from
workers characterized by their duration of chloroprene exposure. McGlothlin and
colleagues conducted medical interviews with workers who had been monitored for
chloroprene exposure (personal zone air samples). The report does not present any
tabular data on health effects. However, the lack of quantitative exposure data for Gooch

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External Peer Review Meeting on the Toxicological Review of Chloroprene

and Hawn and of quantitative medical data for McGlothlin et al. rule out their use as a
principal study. Selection of the NTP study is justified.

Richard B. Schlesinger

This study is clearly the best one to use for derivation of the RfC. It has a range of
exposure concentrations and examined two species and multiple organ systems. The other
chronic bioassay of Trochimowicz et al. has a number of problems associated with it that
in my mind preclude its use as the key study.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

(B) Inhalation Reference Concentration (RfC) for Chloroprene

2. An increase in the incidence of degenerative nasal lesions in male rats, characterized
by olfactory epithelial atrophy and/or necrosis with increasing severity, was selected as
the critical effect. Please comment on the scientific justification for combining the
incidence of atrophy and necrosis and for selecting this endpoint as the critical effect.
Please identify and provide the rationale for any other endpoints that should be
considered in the selection of the critical effect.

Herman J. Gibb

It seems reasonable to combine the incidence of epithelial atrophy and necrosis. The
rationale for choosing degenerative nasal lesions over epithelial hyperplasia or splenic
hematopoietic proliferation (page 5-10, lines 4-10) is reasonable.

Dale Hattis

I think there is no problem with the selection of these endpoints for RfC derivation.
Ronald L. Melnick

Combining the incidences of the degenerative nasal lesions, atrophy and necrosis, seems
reasonable, but does not make much difference on the overall determination - the
incidence of atrophy alone in the control and three dose groups of male rats was 6, 24, 94,
and 98%, while the combined incidence of atrophy and necrosis was 6, 26, 96, and 98%;
and the derived human equivalent POD values were essentially the same (1.1 mg/m3 for
atrophy and 1.0 mg/m3 for the combined lesions, respectively).

Nasal degeneration is the appropriate effect for determination of the POD, because this
was the most sensitive endpoint producing the lowest human equivalent POD. The
document notes that candidate endpoints considered for the critical effect were those that
were statistically increased in the lowest exposure concentration group. This limitation
should not be imposed because it could result in exclusion of sensitive endpoints
depending on the nature of the dose-response relationship. Other endpoints that should
also be considered are renal tubule hyperplasia in male rats (single and step section data)
and renal tubule hyperplasia in male mice. RfCs should also be derived and presented in
Figure 5-1 for other endpoints, including olfactory effects in female rats, male mice, and
female mice, and renal tubule hyperplasia in male rats, female rats, and male mice.

John B. Morris

Nasal degenerative lesions in the rat were selected as the critical response because the
POD-HEC derived from these data was the most protective. Several concerns could be
raised relative to this recommendation. First, the rationale for combining lesions and the
precise way in which the data were combined is poorly described. In my view, the
concept that necrosis may precede atrophy is quite straightforward. Numerous agents

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External Peer Review Meeting on the Toxicological Review of Chloroprene

induce nasal olfactory necrosis and atrophy (esters, styrene, and naphthalene to name a
few); critical evaluation of this database will provide insights into the typical progression
of lesions. The concept that atrophy precedes necrosis, however, is bewildering to me. I
am not aware of a nasal toxicant in which it has been shown that atrophy results in
subsequent necrosis. Such an example should be provided to support this concept. In the
absence of such information, it is not reasonable, in my view, to assert that atrophy
causes necrosis. I, therefore, do not concur with combining the lesions. I note that the
difference in POD-HEC between combined and uncombined data is quite small; why
invoke a poorly substantiated approach when it results in little difference? My other
concerns focus on POD issues and are provided below. In my view, the POD should not
be based on nasal lesions, making the issue of combination of lesions moot.

Avima M. Ruder

Combining the effects of atrophy and necrosis appears justified. Table 5-1 does not
provide the p-values for trend in dose response for various endpoints. However, it
appears that the trend might be stronger for the atrophy or necrosis, with percentages
affected ranging from 6 to 98% with increasing doses, than for hematopoietic cell
proliferation in the spleens of female mice, with percentages affected ranging from 26 to
78% with increasing doses.

Richard B. Schlesinger

A portal of entry effect was used as the critical effect, which is appropriate for this
chemical. The justification provided for combining these two degenerative changes as the
overall effect of interest is appropriate, even though it would be assumed that necrosis
would precede atrophy. While it appears that the chloroprene while non reactive is
metabolized in the upper respiratory tract to a reactive epoxide, there needs to be some
explanation as to why the nasal changes themselves were selected over effects in the
bronchial tree or alveolar region that were observed at the 12 ppm exposure level as well.
An explanation does appear on page 5-7 following results of modeling, but there should
have been some indication earlier on as to why the upper respiratory rather than the lower
respiratory tract endpoint was selected in the first place.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

(B) Inhalation Reference Concentration (RfC) for Chloroprene

3. Benchmark dose (BMD) modeling was used to define the point of departure (POD) for
the derivation of the RfC. The POD was based on increased incidence of degenerative
nasal lesions in male rats at a benchmark response (BMR) of 10% extra risk. Has the
BMD approach been appropriately conducted? Is the BMR selected for use in deriving
the POD (i.e., 10% extra risk of degenerative nasal lesions of less than moderate
severity) scientifically justified? Please identify and provide the rationale for any
alternative approaches (including the selection of the BMR, model, etc.) for the
determination of the POD and discuss whether such approaches are preferred to EPA '.s
approach.	

Herman J. Gibb

The BMD approach is preferred to other approaches for the given data. The arguments
made by one of the peer reviewers, Dr. Morris, to reconsider the calculation of the RfC
with regard to blood borne delivery versus airborne delivery are reasonable, and I would
recommend that the Agency evaluate both approaches prior to performing dosimetric
adjustment. If atrophy/necrosis is eventually selected as the endpoint, a BMR of 10%
extra risk is reasonable given the arguments on page 5-4 of the document.

Dale Hattis

The saturation of metabolism to the active metabolites could be clarified with the use of
the PBPK model mentioned earlier. This could facilitate dose response modeling and
perhaps lead to a somewhat lower point of departure for application of uncertainty
factors.

At the peer review meeting an issue arose as to whether the 10% benchmark response
level was appropriate in the light of the severity of the nasal lesions in some of the
animals. If counts are available on the numbers of animals showing different levels of
severity in relation to dose than this would seem to be a good case for the use of the
EPA's categorical regression software. With that system it would be possible to take the
severity information into account and estimate a somewhat lower BMDs and BMDLs
corresponding to a 10% extra risk of mildly adverse effects.

In addition, EPA might consider a modifying the benchmark dose estimation to take into
account the approach to saturation of metabolic activation derived from the cancer dose
response information (see below).

Finally I agree with some of the other reviewers that the RfC should be derived using the
procedures for a category 3 rather than a category 1 vapor.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Ronald L. Melnick

BMD modeling is the preferred approach to derive the POD because it uses all of the
dose response data and is less impacted by the group size. Some discussion is needed on
why a particular dose response model was selected for the determination of the POD in
situations where more than one model provided an adequate fit to the data. If it is EPA's
policy to select the model that yielded the lowest AIC value, then that rationale should be
explicitly noted. The characterization of chloroprene as a Category 1 gas and the
application of a dosimetric adjustment factor for portal-of-entry effects have not been
adequately justified.

The NTP study that was used to derive the RfC did not achieve a NOAEL, and the
severity of the nasal lesions was greater than minimal in the lowest exposure
concentration group. In fact, several male rats in the low exposure group (12.8 ppm) were
graded with moderate severity for olfactory atrophy and necrosis. The benchmark
response of 10% extra risk is not a NOAEL and the estimated BMDio used to derive the
RfC is approximately 60% of the lowest concentration used in the chronic toxicity study
of chloroprene. Because the NTP study included 50 animals per group, a BMR of 2% or
5% extra risk would likely provide a reliable estimate for the derivation of the POD
without substantially increasing statistical uncertainty at the POD. Thus, I strongly
recommend BMD modeling and derivation of the POD from the 2% or 5% extra risk
response; if that is not done then an additional uncertainty factor of 3 to 1 OX would need
to be applied to the human equivalent POD.

John B. Morris

I do not concur with the approach used to derive the POD-HEC. Multiple POD-HEC
values were derived for differing lesions and the most sensitive was then selected. I note
that the POD values (prior to DAF correction) for all the lesions are virtually identical,
spanning 2.1-8.3 mg/m3 range. The only reason the POD-HEC is lower for the nasal
lesions is that the DAF is so low. Thus, the selection of the nasal lesions as the most
sensitive response is simply an artifact of the DAF (RGDR) calculation and not based on
the primary experimental observations.

My concerns relative to the RGDR are described below. Essentially they are: 1) the
RGDR calculation is theoretically flawed and discordant with the inhalation dosimetry
database, and 2) there is no basis to conclude that airborne rather than blood-borne
chloroprene induces nasal olfactory lesions. The absence to consider blood-borne
delivery is particularly confusing in light of the fact that the possibility of blood-borne
delivery relative to pulmonary lesions received much attention. Why this was ignored for
the nose is perplexing. The distribution of lesions (olfactory, but no respiratory mucosal
damage) could certainly be reflective of a critical role for blood borne delivery and/or in
situ metabolic activation. The absence of nasal respiratory injury suggests the parent
compound and/or direct reactivity of the parent compound are not likely involved.
Commonly a strong anterior/posterior gradient in respiratory mucosal injury is seen for
vapors which are directly reactive. This is not the case for chloroprene, in fact, no
respiratory mucosal lesions were seen. Were blood borne delivery considered I believe

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External Peer Review Meeting on the Toxicological Review of Chloroprene

the RDGR would be 1. In my view, the assumption that chloroprene is a category 1 gas is
also flawed (see below). Given that numerous compounds produce nasal olfactory injury
following parenteral administration, the observation of nasal olfactory injury cannot be
used in support of a category 1 assignment. The partition coefficient of chloroprene is
quite small (10) from a nasal dosimetric view. It is difficult, if not impossible, to envision
a scenario in which nasal backpressure does not influence dosimetry and/or that nasal
deposited chloroprene does not penetrate to the depth of the blood. In my view,
chloroprene is a category 3 gas.

At best, the assignment of category 1 status and the exclusion of blood-borne delivery
mechanisms represent a weakness of the RfC derivation. An alternate approach would be
to select the POD on a parameter closely associated with the collected data rather than to
pick a value subject to artifact from the RGDR approach. Were this done, a differing
critical lesion would be selected - likely alveolar epithelial hyperplasia and/or
hematopoietic proliferation. Given that the subsequent text includes considerable
discussion of the possibility of blood borne delivery relative to pulmonary injury, the
selection of an inhalation based DAF of 2.3-4.1 would need to be critically discussed and
supported were lung lesions selected as the critical effect. For the cancer risk
extrapolation both inhalation based and blood-borne based DAF values were used. Why
not use both approaches for the non cancer endpoints as well? The lack of consistency is
striking.

I am supportive of using a BMD approach as the database appears sufficiently robust to
allow for this calculation. An extra risk of 10% of mild lesions is an appropriate endpoint
in my view. However, if moderate grade lesions were observed at exposure
concentrations approximating the calculated BMD 10, it would suggest the calculated
value is too permissive. As noted above, I would recommend selecting the endpoint
based on the observed data and then performing a single DAF-based calculation based on
those data. Such an approach would minimize artifacts due to complexities associated
with selection of the most appropriate DAF.

Avima M. Ruder

I don't have the expertise in risk assessment to comment on whether the modeling and
extrapolation from animal to human was appropriately conducted. However, a 10%
increase in an effect appears to be a significant enough departure from good health to
justify the calculation. Upon reflection, I agree with the argument made by Dr. Melnick
that the proposed benchmark dose does not represent a NOAEL and that it might be
better to look at a lower response level (2-5%). From the responses from EPA staff at the
review meeting it appears that a 2-5% extra risk response level was considered in internal
EPA discussions. I also think that the issues raised by Dr. Morris as to whether
chloroprene is a category 1 gas or not need to be clarified.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Richard B. Schlesinger

The BMD approach is very well suited for the large data set of the principal study being
used in this document and using chronic toxicity and carcinogenicity as endpoints. In
general when using the BMD, a 10% level of acceptable risk is used. Thus, this document
follows relatively standard procedures in this regard. However, based upon the data, this
level may be too high and it is suggested that a lower level, perhaps 5%, be used in this
case. The document could be clearer in showing the different stages in the development
of the RfC. It does provide a formula on page 5-4 but does not show the use of the
formula with actual numbers from the principal study. It would be helpful to the reader if
such a step by step actual derivation was provided. For example, it would help to see the
actual value for the PODadj (mg/m3) that was used to derive the HEC.

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(B) Inhalation Reference Concentration (RfC) for Chloroprene

Herman J. Gibb

The uncertainty factors seem reasonable.

Dale Hattis

I have no quarrel with the selection of uncertainty factors made in the document. The
analysis seems very standard. The only area of modest controversy might be the choice of
a database uncertainty factor of 3. This seems adequately justified by the absence of a
two-generation reproductive study, although the negative findings for teratogenesis and
dominant lethal effects could have been considered an adequate substitute.

Ronald L. Melnick

The selection of uncertainty factors of 10X for human variation, 3X for animal-to-human
toxicodynamic uncertainty, and 3X for database insufficiencies are reasonable and
consistent with EPA policy. However, it is not possible to know if the UFs selected for
human variability and interspecies uncertainty adequately account for the extent of these
variations. For example, human variability is greater than 10X for the activities of the
enzymes involved in chloroprene metabolism (both activation of chloroprene and
detoxification of the reactive epoxide intermediate). As noted in response #3 above, the
BMDio is a true effect level with several animals diagnosed with moderate lesion severity
(i.e., the severity level just below marked). The EPA assumption that the BMDio
represents a minimal biologically significant change that was less than moderate severity
is not correct. Thus, an additional uncertainty factor of 3-10X should be applied to the
RfC derived from a BMDi0; alternatively, the POD should be derived from a BMR or 2%
or 5% extra risk. An additional deficiency in the database includes lack of data on
potential neurodevelopmental toxicity, or other long-term effects following perinatal
exposure.

John B. Morris

The rationale for UF selection is clear and appears consistent with typical procedures.
The discussion would be greatly enhanced by inclusion of discussion of the impact and
uncertainty of selecting DAF factors based on airborne delivery. My concerns, in this
regard, are provided above. In my view, it is important to recognize that the DAF
calculation is subject to considerable uncertainty and, as such, should not be accepted as
factually based. Discussion should also be included on the basis for inclusion of a
database limitation uncertainty factor as a multi-generation study is available. It should
be stated if this is policy-based rather than scientifically-based decision.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Avima M. Ruder

The uncertainty factors appear justified. As I commented above, there is probably
considerable human variation in the metabolism of chloroprene, due to polymorphisms in
the genes coding metabolic enzymes. However, as Drs. Schlesinger, Hattis, and Melnick
suggested during the review (or as I understood them to suggest), it might be more
appropriate to change the benchmark dose response, rather than the uncertainty factors.
Their arguments should be considered.

Richard B. Schlesinger

The specific UFs chosen are well justified and appropriate for the data set used and
follow standard USEPA guidelines.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

1. Under the EPA's 2005 Guidelines for Carcinogen Risk Assessment
(www.epa.gov/iris/backgr-d.htm), the Agency concluded that chloroprene is likely to be
carcinogenic to humans by all routes of exposure. Please comment on the cancer
weight of evidence characterization. Is the cancer weight of evidence characterization
scientifically justified?

Herman J. Gibb

The characterization is clearly justified based on the animal and genotoxicity data, but the
argument for the epidemiologic data has been overstated.

The reported evidence of a liver cancer risk in the Louisville cohort studied by Marsh et
al. (2007a, 2007b) summarized on page 4-18, lines 3-5 relies heavily on a purported dose
response in 4 cumulative exposure categories. The document does not describe what the
relative risks (and confidence limits) are in each of the four exposure categories but states
that the probability of the trend is 0.09 (page 4-13, lines 13-17; page 4-71, lines 4-7)1"2.
Furthermore, the document neglects to report what the overall SMR for liver cancer is in
the Louisville cohort. Interestingly, the document concludes that there is no evidence of a
dose response relationship for respiratory cancer yet describes the relative risks and
confidence limits for respiratory cancer by all four cumulative exposure levels for all four
facilities in the Marsh et al. study (page 4-14, Table 4-9). Why isn't the reader given that
information for the liver cancer relative risks, at least for the Louisville cohort, since the
document has gone to the point of suggesting that the data indicates that there is a liver
cancer dose response? Furthermore, in the discussion of "biological gradient" on page 4-
71, no mention is made of Table 4-11 on page 4-17 showing that two studies demonstrate
evidence of a dose response for liver cancer, and two demonstrate no evidence of a dose
response. The dose response in one of the studies (Leet and Selevan 1982) would not
even exist if only deaths from liver cancer were included in the analysis since two of the
three deaths from cancer of the liver and biliary passage in the high exposure category
were due to gall bladder cancer. The other study in Table 4-11 that suggests a dose
response is Bulbulyan (1999), but the relative risks in the high and low dose are not
statistically different. The statement at the bottom of page 4-18 that there is evidence of a
dose-response relationship in different cohorts in different continents (U.S., China,

Russia, and Armenia) grossly misrepresents the evidence.

Known risk factors for liver cancer include Hepatitis B and C infection, aflatoxin
ingestion, certain inherited metabolic diseases, cirrhosis due to alcohol abuse, obesity,
and certain inherited metabolic diseases (American Cancer Society). None of these
factors with the exception of alcohol consumption (page 4-69, lines 28-29) have been

1 The document states on page 4-13, linesl5-17, and page 4-13, lines 4-71, lines 5-6 the range for the three
highest exposure levels was from 1.9-5.1 but doesn't state what the RR's for each of the four exposure
levels are nor does it provide confidence limits on the RRs.

2 If the p = 0.09 is calculated by the authors of the EPA document (as opposed to Marsh et al.), that should
be indicated.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

discussed in the review. It is interesting that in the Major Conclusions on page 6-2, lines
27-29, the document notes that "These associations (respiratory cancer) are not
considered as strong as those with liver cancer due to the inability to control for
confounding by smoking status, a strong indicator of lung cancer." What about the well-
known risk factors for liver cancer? Were they considered in the various studies? On page
4-69, lines 28-29, the document indicates that the lack of data on alcohol consumption is
a "key limitation." On lines 31-32, the document states that there is also a "high
likelihood of co-exposures which may be confounders." Nonetheless, the document goes
on to blithely state that "Despite this potential, there is little evidence of substantial
exposure to liver carcinogens in these populations." How can such a statement be made if
the study authors never considered the major risk factors?

Of particular note with respect to the Li et al. study is that the highest liver cancer rate in
the world is China (as much as 10X that in the U.S.), primarily the result of Hepatitis B
infection and aflatoxin ingestion. Given the considerable risk posed by these risk factors
in a Chinese population and that there were only 6 liver cancer deaths in the entire cohort
working in a facility where there were multiple chemical exposures, it is impossible to
conclude that the study indicates an association between chloroprene and liver cancer.

The document indicates on page 4-8 that Bulbulyan et al. (1998) found 11 deaths due to
cirrhosis. It is possible that these deaths could have been caused by chloroprene, but
alcohol and hepatitis B/C infections are the most common causes of cirrhosis which
should say something about the cohort. Liver cancer is about 50% higher in Eastern
Europe than it is in North America, and alcohol consumption in Russia is reported to be
almost double that of the U.S.

The analysis of the Bulbulyan (1999) study indicates that there was increasing incidence
of liver cancer by duration of employment and by cumulative exposure. Presumably
duration of exposure and cumulative exposure were not evaluated together in a multiple
regression by the study authors (I do not have the original paper). Given that there was an
increasing risk by duration of exposure, one cannot rule out that the increasing risk with
cumulative exposure was not due to other exposures at the facility. Presumably, there was
no analysis by intensity of exposure? If there was, what did it show?

The document should be more transparent in the presentation of the human data on liver
cancer. For example:

• The liver cancer relative risks for all four exposure categories in the Louisville cohort
studied by Marsh et al. should be reported.

• The SMR for liver cancer should be reported for the Louisville cohort studied by
Marsh et al.

• Whether Marsh et al. (2007a, 2007b) and Leet and Selevan (1982) Louisville cohorts
are independent should be addressed. If Leet and Selevan (1982) is a part of or the
same as the Marsh et al. cohort (or even very similar), then use of the Leet and
Selevan (1982) should not be described as providing independent results of dose

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External Peer Review Meeting on the Toxicological Review of Chloroprene

response, consistency, etc. The same is true of the Colonna and Leydavant (2011) and
the Marsh et al. studies of the Pontchartrain facility.

• The confounding factors for liver cancer and whether studies addressed these risk
factors should be discussed.

• The statement in the Major Conclusions on page 6-2, lines 19-20 that there was
"some evidence" of liver/biliary passage cancer risk being associated with
chloroprene exposure is followed by the statement on lines 22-23 that these measures
of association were "strong, especially in the presence of healthy worker bias" is
inconsistent.

• An association between liver cancer and chloroprene exposure being strengthened by
the healthy worker effect as indicated in the Major Conclusions is not evident in the
summary of the overall weight of evidence (some mention of HWE is made on page
4-69, lines 21-25 but does not indicate that the evidence is strengthened).

Furthermore, a healthy worker effect for liver cancer? With such a short life
expectancy following diagnosis, I would expect the healthy worker effect for liver
cancer to be minimal if it even exists.

• The small number of liver cancer deaths/cases in the studies by Li et al., Bulbulyan
(1998, 1999) and Leet and Selevan (1982) and the variability about such small
numbers should be better described, particularly in light of the limitations of those
studies with respect to calculation of the expected deaths, follow-up, etc.

As the document acknowledges on page 4-17, there is little if any evidence that
chloroprene increases the risk of respiratory cancer. The limitations of the earlier studies
(Li et al. 1989, Bulbulyan 1998, 1999) are significant with regard to whether or not they
indicate an increased risk of liver cancer from chloroprene exposure. The largest and
what appears from the document to be the best conducted study (Marsh et al., Louisville
cohort) provides little if any evidence that a liver cancer risk exists. Furthermore, the
document has not been transparent in its reasoning that there is a risk of liver cancer.

In summary, the descriptor of "likely to be carcinogenic to humans" is supported by the
animal and genotoxicity data, but not by the human data. While the descriptor is
appropriate, the document should not try to make more of the epidemiologic studies than
is warranted.

Dale Hattis

Yes. The ample information on carcinogenesis in many sites in animals, the clear
metabolism information to mutagenic metabolites, and the analogies to related chemical
carcinogens with analogous metabolic pathways to DNA-reactive metabolites all
combine to make this conclusion unequivocal. As suggested by Dr. Melnick, the final
document should consider whether the available evidence warrants an upgrade of the
classification to "carcinogenic to humans.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Ronald L. Melnick

Results from the NTP study demonstrating multiple organ carcinogenicity of inhaled
chloroprene in both sexes of rats and mice are consistent with the EPA descriptor "likely
to be carcinogenic to humans." Because the carcinogenicity of chloroprene is likely due
to its epoxide metabolites, and because cytochrome P450-mediated epoxidation of
chloroprene can occur in several organs including the liver, kidney, and lung, metabolism
of absorbed chloroprene to a mutagenic intermediate can occur by any route of exposure.
The systemic distribution of tumors in the NTP studies demonstrates that chloroprene can
induce tumors beyond the sites of initial contact. Liver toxicity of chloroprene in rats
after oral exposure (stomach tube) indicates the occurrence of oral absorption of this
chemical. Chloroprene is absorbed by the skin (Hazardous Substances Data Bank; see
page 3-1).

However, the descriptor "carcinogenic to humans" may be more appropriate based on the
multiple tumor response in two species, the fact that chloroprene is activated by CYP2E1
to a DNA reactive intermediate (chloroethenyl oxirane) by rat, mouse, or human liver
microsomes, the finding of a unique K-ras mutation (A^T at codon 61) in chloroprene-
induced lung neoplasms in mice, and the relatively consistent evidence of an association
between increased liver cancer mortality risk and occupational exposure to chloroprene.
The EPA document does not adequately justify the characterization of chloroprene as
"likely to be carcinogenic to humans" rather than "carcinogenic to humans," especially
since many of the identified methodological limitations in the epidemiologic studies (e.g.,
exposure misclassifications, healthy worker effect) would result in an underestimate of
risk. According to EPA's cancer risk assessment guidelines, the descriptor "carcinogenic
to humans" may be applied when there is less than convincing epidemiologic evidence of
a causal association between human exposure and cancer if there is strong evidence of
carcinogenicity in animals, the MOA and precursor events have been identified in
animals, and key precursor events in animals are anticipated to occur in humans and
progress to tumors. These conditions have been demonstrated for chloroprene.

John B. Morris

I concur that the weight of evidence supports the concept that chloroprene may be
carcinogenic by all routes of exposure. Multiple tumors were seen in two species in
inhalation bioassays. Additionally some data suggesting increased tumor risks in humans
is available. Tumors were seen in non-site of contact sites in the rodent studies. (In this
regard respiratory tract as well as gastrointestinal tract tumors may be considered as site
of contact because of preening activity.) Moreover, there is discussion of the possibility
of a critical role blood-borne chloroprene relative to nasal and pulmonary lesions. If there
is, indeed, a role for blood borne chloroprene, then the possibility of carcinogenicity after
multiple routes of exposure is elevated because systemic absorption and blood-borne
delivery to multiple targets is possible. (The document indicates dermal absorption may
occur.) Importantly, a potential increase in liver tumors was noted in some occupationally
exposed cohorts. In my view, these epidemiological data support the concept that
chloroprene may represent a carcinogenic hazard to man.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Avima M. Ruder

The literature supports the likely carcinogenicity of chloroprene and the mutagenicity of
its epoxide metabolites. The need for regulation of environmental (in addition to
occupational) exposure to chloroprene is justified by a report on public health in the area
where the Louisville DuPont plant and other industrial facilities, as well as residences,
are co-located. In that report, the Agency for Toxic Substances and Disease Registry
(ATSDR) stated that the volume of release of chemicals from the plants made it likely
that soil and water (groundwater and the Ohio River) had been contaminated in the past;
chloroprene air contamination was measured as 218 ppb or 789 |ig/m3 in 1956-7
downwind of the plants and 6 ppb or 2.68 |ig/m3 in 1988 at a monitoring station in
downtown Louisville not downwind of the plants [Agency for Toxic Substances and
Disease Registry 1998],

ATSDR provided a rationale for the greater vulnerability of children to toxic exposures:
they are more likely to play outdoors and bring food into contaminated areas; are shorter
and therefore closer to dust, soil, and contaminants; weigh less, resulting in higher doses
per unit body weight; and are developing rapidly [Agency for Toxic Substances and
Disease Registry 1998], The EPA's use of age-adjustment factors seems appropriate.

Richard B. Schlesinger

While the Guidelines for Carcinogen Risk Assessment are being followed in the
chloroprene assessment, even though there are limited to no data on exposure other than
inhalation, it seems that the mode of action of the chemical is such that it may not be
carcinogenic via all routes, e.g., dermal exposure. It is nonreactive chemically and
relatively insoluble in water. The weight of evidence characterization is clear and
justified. The animal toxicological data support the conclusion that it may likely be
carcinogenic to humans. While the epidemiological evidence in this regard is equivocal,
the conclusion is also supported by the fact that the MOA involves conversion to
epoxides.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Herman J. Gibb

The selection of this study is justified. The document states that the Trochimowicz et al.
study was not chosen as the principal study "primarily due to the lack of observed
neoplastic effects at similar exposure levels as the NTP (1998) study"(page 5-12, lines 5-
8). As with the response to Question 1 for the RfC above, high mortality in the low dose
animals (page 4-39, lines 19-20; page 5-2, lines 13-16, 29-31) would be a stronger
argument for not choosing the Trochimowicz study than would differences in observed
effects between studies. Differences in study results can occur regardless of how well the
individual studies are conducted.

Dale Hattis

Choice of the two-year inhalation bioassay is beyond dispute. However, as indicated
earlier, the dosimetry, in terms of active metabolite concentration AUC, could have been
informed by application of a preliminary PBPK model.

Ronald L. Melnick

The selection of the NTP 2-year inhalation carcinogenicity study of chloroprene in
B6C3F1 mice for derivation of an inhalation unit risk is scientifically justified. The NTP
study was well designed and conducted, and identified carcinogenic effects in multiple
organs of rats and mice exposed to a wide range of concentrations of chloroprene. A
major strength of this study is the multiple histopathological reviews of lesions identified
in rats and mice. As with the related human carcinogen, 1,3-butadiene, the carcinogenic
potency of chloroprene was greater in mice than in rats.

John B. Morris

In my view, the selection of the two-year inhalation bioassay done by NTP as the critical
study is appropriate. This study was well performed and peer reviewed. It is true that the
Trochimowicz study provided contradictory results, but without substantive rationale the
NTP study cannot be ignored. Inclusion of the mouse lung tumor data for dose-response
evaluation may be scientifically problematic. As is commonly observed, the mouse
metabolic activity for chloroprene is 50-fold higher (Table 3-4) than that in the human or
the rat (in which lung tumors were not increased). This fact should be discussed. It is my
view that the mouse lung data may overestimate the risk to humans. It is recognized that
exclusion of these data may be problematic, but at a minimum a discussion of this

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External Peer Review Meeting on the Toxicological Review of Chloroprene

weakness should be provided. Because the metabolism rates in the rat appear similar to
the human, the rat may offer a better species for prediction of human health risks.
Certainly the document would be improved by an explicit discussion of the relevance of
the mouse response considering its high metabolic capacity.

Avima M. Ruder

The text in section 5.4.4 explains the derivation of the inhalation risk but does not explain
why inhalation in mice was chosen over inhalation in rats from the same study. I assume
there are physiological differences which make mice a more suitable choice, but none
were provided here.

Richard B. Schlesinger

The study selected for derivation of the IUR is well justified based upon the standard
procedure used by USEPA in selecting the most sensitive animal model. However, they
may want to consider the fact that metabolic activation rate in the rat is closer to that
occurring in humans than is the situation in mice.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

3. A mutagenic mode of carcinogenic action is proposed for chloroprene. Please
comment on whether the weight of evidence supports this conclusion. Please comment
on whether this determination is scientifically justified. Please comment on data
available for chloroprene that may support an alternative mode(s) of action.

Herman J. Gibb

The hypothesized epoxide metabolite mode of action is reasonable.

Dale Hattis

Yes. The ample information on carcinogenesis in many sites in animals, the clear
metabolism information to mutagenic metabolites, and the analogies to related chemical
carcinogens with analogous metabolic pathways to DNA-reactive metabolites all
combine to make this conclusion unequivocal. I am not aware of any evidence that
comparably supports any other mode of action.

Ronald L. Melnick

Based on the fact that the predominant pathway of chloroprene metabolism is via
cytochrome P450-mediated oxidation to a DNA-reactive epoxide intermediate
(chloroethenyl oxirane), which is mutagenic in multiple strains of Salmonella, and the
finding of activating K-ras and H-ras mutations mutations in tumor tissues obtained from
mice exposed to chloroprene, including unique K-ras mutations (A—»T transversions in
codon 61) in lung tumors, the proposed mutagenic mode of carcinogenic action is
scientifically justified. This MOA is consistent with that of other epoxide-forming
carcinogens, e.g., 1,3-butadiene and vinyl chloride. There is no scientific data supportive
of any alternative mode of action. Recent experimental results presented to the Peer
Review Panel by DuPont demonstrated the induction of changes in gene expression
related to DNA damage in the lungs of mice exposed to 2.5 ppm or higher concentrations
of chloroprene (Figure 8, page 79). These data also support a mutagenic mode of
carcinogenic action for chloroprene.

John B. Morris

It should be stated that detailed assessment of mutagenic versus non-mutagenic modes of
action is somewhat beyond my expertise. With this qualification, I concur with the
proposed mutagenic mode of action of chloroprene. Chloroprene metabolite(s) are DNA
reactive and mutagenic in some bacterial strains. Data presented by DuPont suggests the
induction of DNA repair responses in chloroprene exposed animals. Mutations were
observed in vivo in lung tumors of animals exposed to chloroprene. Were a purely
cytotoxic mode of action proposed it would be important to show appropriate temporal
and dose-response data supportive of this mode. I am aware of no such data. In my view
there are insufficient data to exclude the possibility of a mutagenic mode of action. There

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External Peer Review Meeting on the Toxicological Review of Chloroprene

appears to be multiple lines of evidence in support of this mode of action and it,
therefore, appears scientifically justified. If, however, it is concluded that a metabolite
represents the ultimate toxic species, then the quantitative risk assessment should be
discussed/validated in light of the large species differences in metabolism rate.

Avima M. Ruder

The metabolic pathways detailed in figure 3-1 (and in the toxicological literature from
which this section is drawn) appear to justify this conclusion. The finding of increased
chromosome aberrations among humans with variant metabolic enzymes that clear the
epoxide metabolite more slowly [Musak, et al. 2008] also supports this conclusion.

Richard B. Schlesinger

There is much compelling evidence that chloroprene has a mutagenic mode of action due
to metabolism into reactive epoxides. While this may not be the only MO A, it clearly is
one of them.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

4. Data on hemangiomas/hemangiosarcomas (in all organs) and tumors of the lung
(bronchiolar/alveolar adenomas and carcinomas), forestomach, Harderian gland
(adenomas and carcinomas), kidney (adenomas), skin and mesentery, mammary gland
and liver in B6C3F1 mice were used to estimate the inhalation unit risk. Please
comment on the scientific justification and transparency of this analysis. Has the
modeling approach been appropriately conducted? Please identify and provide the
rationale for any alternative approaches for the determination of the inhalation unit
risk and discuss whether such approaches are preferred to EPA's approach.

Herman J. Gibb

The rationale for combining risks from different tumor sites is reasonable given a
mutagenic mode of action. It is interesting, however, that the inhalation unit risk estimate
for chloroprene is an order of magnitude higher than the inhalation unit risk estimate for
butadiene which is considered a structural analog and characterized by EPA as
"carcinogenic to humans". A reality check on the unit risk for chloroprene by comparing
it with an upper bound on the cancer risk in the Louisville cohort studied by Marsh et al.
should be performed. The Louisville cohort has the best exposure information for this
purpose. From the resulting comparison, it may be necessary to adjust the unit risk
estimate.

Dale Hattis

The approach is transparent and reasonable as far as it goes. However, I think it is not
ideal in that it fails to make explicit use of the information that there is likely to be high
dose saturation of metabolic activation.

As an alternative, at the peer review meeting I presented a series of model fits using a
dose response form that incorporates an assumption of saturating metabolism on a
systemic level (applicable to all tumors in the same way) but different effective
background rates and potencies for the causation of tumors at low doses:

Vmax; * d
-(q°j h )

P(d); = 1-e Km + d

where:

d is the external experimental concentration in ppm

P(d); is the fraction of animals with at least one tumor for a specific tissue (i)

qO; is a parameter estimated from data that is related to the background (control group)
lifetime incidence of tumors in that tissue

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Vmax is related to the maximum tumor yield over background for the specific tissue (i)

Km is the external dose that produces half the maximal tumor yield over all tissues (based
on an assumption that metabolic activation is systemic, rather than being effective for
only one tissue due to local metabolism).

This is essentially a quick and easy but approximate substitute for doing a full PBPK
model, but instead uses the tumor response nonlinearity at high doses for all the tumor
sites to quantify the approach toward saturation of the activating metabolism. Compared
to a PBPK modeling approach, this is not informative for the issue of interspecies
projection, but it does provide information about the high-dose-to-low dose projection,
assuming that the saturable activating metabolism is systemic and affects the tumor
frequency in all tissues in the same way. This sort of treatment is warranted by the fact
that, in nearly all tissues with an appreciable tumor yield in both male and female mice,
the tumor incidence over background at the highest (80 ppm) chloroprene concentration
is much less than double the tumor incidence at the next highest (32 ppm) concentration
(see plots below). Contrasting the results for the high-dose saturable metabolic activation
model with those for a straight linear model allows us to assess how large the change in
estimated low dose cancer slope might be relative to a case where there is only a term
that is linear in dose:

To maintain parallelism with the EPA analysis as much as possible, I made this
comparison excluding the anomalous high-dose point for hemangiosarcomas in female
mice. Because of this same anomaly, I choose to begin the discussion of the modeling
and the model results with the observations in male mice.

Figure 1 is a raw plot of the end of life tumor data for male mice used by EPA in its
analysis (from a comment by Dr. Melnick, I understand that tumor results adjusted for
mortality are also available in one of his papers; EPA should probably used those results
for a more refined analysis.)

A difficulty with the raw plot the tumor data is that one might object that of course there
is a flattening of the curve at higher doses and tumor incidences because no more than
one tumor can be effectively detected and recorded in any specific tissue. Thus a more
appropriate interpretation of the data is to say that each data point represents the fraction
of animals that showed at least one tumor in each specific tissue studied. A more
appropriate plot without the potential distortion due to multiple tumors per organ can be
made by using a Poisson distribution formula

P(d); = l-e"(q0i +qlid)

P0 tumors in a

where m

m an organ

m = 1 - Fraction of Animals with at Least 1 tumor = e"m
the mean number of tumor transformations per animal

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Figure 1

Plots of Raw Mouse Tumor Data by Site—Males

Lung

Hemangiosarcoma
Harderian Gland
Kidney
Forest omach

External Air Cone (ppm)

36

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Given this, we can solve for m to find

mean number of tumor transformations per animal = - ln(l - fraction of animals with at least 1 tumor)

Figure 2 is a plot of the male mouse tumor data using this transformations/animal
parameter as the dependent variable. It can be seen that even after removing the
truncation of the tumors/animal results at 1 in this way, there is still a pronounced
flattening of the curves at the higher dose levels, indicating some approach to saturation.

This is reminiscent of the vinyl chloride angiosarcoma case where there was saturation of
metabolic activation at the higher exposure levels.

One other advantage of the transformations/animal dependent variable is that we can add
up the results for the different tumor sites. Figure 3 shows a revised plot of the male
tumor data showing the sum of tumor transformations/animal at all five tumor sites. It
can be seen that the sum of tumor transformations at all five sites still shows a
pronounced convexity as one proceeds to the highest exposure levels.

The fitting of the saturable and linear models was accomplished in Microsoft Excel
workbooks designed to incorporate likelihood calculations according to the basic
structure published by Haas (1994). Copies of the final workbooks themselves will be
submitted to accompany this comment. I would be pleased to explain the detailed features
and operation of the modeling system if any EPA personnel would like to pursue this.

Basically, each workbook consists of 3 sheets: one for optimization of the maximum
likelihood estimates and two for estimation of upper and lower confidence limits on the
sum of transformations/animal at all tumor sites. The optimizations were all done with
the Excel solver tool, generally with multiple runs of hundreds to thousands of iterations
each. Because the maximum likelihood and confidence limit estimates are done on the
sum of tumor transformations per animal for all tumor sites, no Monte Carlo post-
processing analysis is needed to derive confidence limits on the total tumor risk, as was
needed for the separate Weibull model analyses done by/for EPA for the individual tumor
sites. On the other hand, a disadvantage of this modeling system is that it only
incorporated total tumor incidences observed by the end of the bioassays; not the more
detailed time-to-tumor information used in the Weibull model analysis.

Figure 4 shows the overall results of this fitting for both the saturable and linear models.

In the case of the saturable model, the parameters estimated are a Vmax and background
(zero dose) tumor risk for each organ, and a Km (external ppm needed to achieve half of
the total saturated tumor yield) common to all organs—following the hypothesis of
saturable metabolism at a systemic level followed by common exposure of all organs to
the activated metabolite(s). It can be seen that the saturable model fit corresponds very
well with the observations of total tumors per animal (the P value is 0.51, meaning that a
difference between data and model predictions as large as that observed would be
expected to be produced about half the time from chance sampling-error fluctuations).

* Haas, C. N. "Dose Response Analysis Using Spreadsheets" Risk Analysis 14:1097-1100
(1994).

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Figure 2







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Plots of Tumor Transformations/Animal—Males

Lung

Hemangiosarcoma
Harderian Gland
Kidney
F orestomach

External Air Cone (ppm)

38

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Figure 3

Plots of Tumor Transformations/Animal,
Including Total—Males

3.0

2.5

2.0

1.5

1.0

0.5

Lung

Hemangiosarcoma
Harderian Gland
Kidney
Forest omach
Sum, All 5 Sites

External Air Cone (ppm)

39

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Figure 4

Comparison of Observed Tumor Transformations/Animal
For All 5 Sites in Males with Maximum Likelihood
Expectations for Linear and Saturable Models

Sum, All 5 Sites

Male Linear Model MLE

Male Saturable Model MLE

93 transformations/ppm
>1 transformations/ppm

MLE Km = 44 ppm
MLE Vmax/Km (low dose slope) = 0.0
UCL Vmax/Km (low dose slope) = 0.1(
LCL ED10 = 0.66 ppm

MLE Slope = 0.039 transformations/ppm
UCL Slope = 0.047 transformations/ppm
LCL ED10 = 2.2 ppm

External Air Cone (ppm)

40

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External Peer Review Meeting on the Toxicological Review of Chloroprene

The linear model fit somewhat less well at P = 0.06, although still barely within the
conventional P = 0.05 criterion based on estimation of one fewer parameter (10, rather
than 11, corresponding to a background rate and a transformations/ppm parameter for
each tumor site).

The results in Figure 4 indicate a half saturation point (Km) of about 44 ppm, and an
approximately 2-3 fold greater cancer potency at low doses for the saturable, compared to
the linear model, depending on whether one makes the comparison based on MLE slopes
or lower confidence limit EDIO's. Thus the indication is that a simple linear formulation,
as incorporated into EPA's Weibull model is likely to considerably understate the low
dose potency indicated by the data for males.

Figure 5 shows a plot of the female tumor data comparable to Figure 2. The same
tendency for flattening at high exposure levels is apparent. Figure 6 shows the results a
similar comparison of saturable and linear model fits for the female tumor data
(excluding, as did EPA, the high dose point for the hemangiosarcomas). The overall fit in
this case is less successful than for the male tumor data, with a P value of about 0.02, but
the saturable model still fits a great deal better than the linear model with a P value of

about 9X10 .In this case the indicated Km is slightly lower (30 ppm) indicating a
slightly greater effect of the indicated saturation of metabolic activation, and the saturable
model again suggests a low dose cancer potency a few fold greater than expected with the
linear model formulation.

In summary results lead me to five conclusions/recommendations:

• The tumor data are better fit by models incorporating systemic saturable
metabolism.

• Saturable models lead to 2-3 fold increases in expected low dose risks compared
to simple linear models.

• However, the current saturable models do not incorporate available time-to-tumor
information.

• The best way forward would therefore be to add a saturable component to the
Weibull time-to-tumor model.

• A second-best approach would be to multiply the expected ratio of saturable vs.
linear model-predicted low dose risk by the existing Weibull linear model
coefficient (or make a similar adjustment downward in the Weibull model
estimated ED 10 or LED 10).

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Figure 5

Plots of Tumor Transformations/Animal
Excluding Hemangiosarcomas—Females

2

Lung
Liver
Skill
Breast

Harderiaii Gland
Forestomach
Zymbal's Gland

External Air Cone (ppm)

42

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Figure 6

Comparison of Observed Tumor Transformations/Animal
For All 8 Sites in Females with Maximum Likelihood
Expectations for Linear and Saturable Models

Sum, All 8 Sites (Incl. Hemang)
Fem Lin Model MLE (Incl Hemang)
Fern Sat Model MLE (Incl Hemang)

MLE Km = 30 ppm

MLE Vmax/Km = 0.155 transformations/ppm
UCL Vmax/Km = 0.191 transformations/ppm
LCLED10 = 0.55 ppm

MLE Slope = 0.058 transformations/ppm
UCL Slope = 0.069 tranformations/ppm
LCLED10 =1.53 ppm

—r-

60

External Air Cone (ppm)

43

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Ronald L. Melnick

Yes, all of the induced tumor sites in mice should be used to estimate the inhalation
cancer unit risk; an assessment based on separate modeling of each tumor type would
underestimate the carcinogenic potency of chloroprene. Cancer potency estimates are
increased only about 2-fold by combining all sites in the assessment compared to
estimates based on only the most potent response in either male or female mice. Because
of the reduced mortality of exposed mice due to induction of malignant tumors, a
multistage Weibull time-to-tumor model that accounts for differences in survival among
groups is most appropriate. The chloroprene document should provide discussion on why
no uncertainty factor (other than early-life susceptibility) for human variability was
applied to the cancer unit risk estimate. There are certainly substantial differences in
human metabolism of chloroprene and its reactive epoxide metabolite and in human
susceptibility to chloroprene-induced cancer.

The suggestion by Dale Hattis to apply a model that accounts for saturable metabolism of
chloroprene to its epoxide intermediate should be pursued and incorporated into the
estimate of the inhalation cancer unit risk. This analysis should use survival-adjusted
tumor incidence values. The blood time-course data for chloroprene presented by DuPont
(Figure B-l, page 99) to the Peer Review Panel clearly demonstrates saturable
metabolism of chloroprene in mice at exposures between 13 and 90 ppm.

John B. Morris

The modeling approaches for the quantitative risk evaluation of chloroprene
carcinogenicity were transparently described. Cancer unit risks are calculated
individually for specific tumor types and an overall unit risk was calculated. Presumably
the overall unit risk was calculated in concordance with accepted EPA procedures. It is
beyond my expertise to comment on the generalized appropriateness of combining
tumors in this way relative to overall cancer unit risk calculation. If tumors are to be
combined then the human relevance of each tumor type must be considered. As noted
above, in my view, some skepticism is appropriate relative to the quantitative importance
of mouse bronchiolar tumors. The mode of action includes metabolic activation as the
first step. The metabolic activation rates in the mouse exceed those in other species by
50-fold (Table 3-4). Clearly this is a critical observation relative to quantitative risk
extrapolation. This pattern of mouse vs. human bronchiolar metabolism is certainly not
unique to chloroprene. The large differences in mouse vs. human relative to pulmonary
activation raise questions as to the relevance of the mouse lesions. At the very least, this
issue needs to be discussed. Exclusion of the mouse lung tumors would influence the
final overall unit risk estimate indicating this is not a trivial concern.

It should be noted that the epidemiological data suggests the liver at the primary target,
although this may be the result of statistical issues related to the high incidence of lung
tumors in humans obscuring a response. Nonetheless, a discussion of the site discordance
would strengthen clarity of the text. I don't know if it is possible, but some comparison of
the unit risk versus the observed tumor risks in the worker populations would seem
warranted. Is it possible to estimate an upper bound risk from the human data?

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Alternatively, is it possible to project human occupational risks from the unit risk factor
to determine if the unit risk factors are consistent with epidemiologic observations? I
recognize that only crude comparisons could be made, but a large discordance would be a
cause of concern.

Avima M. Ruder

The assumption of tumor independence (p 5-20), based on the National Research Council
risk assessment document, appears justified. However, the results of the animal studies
should be evaluated to determine if there is a distinction (genetic, epigenetic, or other)
between animals which get one tumor versus those which get more than one.

Richard B. Schlesinger

The derivation of the IUR could be made somewhat clearer in the text.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

5. Lung tumors have been alternatively treated as systemic or portal-of-entry effects in
the modeling of cancer endpoints. Please comment on the scientific justification for
this modeling approach. Please comment on whether the rationale for this decision has
been transparently and objectively described. Please comment on data available for
chloroprene that may support an alternative method for modeling the observed lung
tumors in mice.

Herman J. Gibb

It makes sense that lung tumors could develop from a systemic as well as a portal-of-
entry effect. The extent that the lung tumors occur by systemic vs. portal of entry effects
may not be possible to determine, but the text should provide more elaboration for the
reader so that they can better understand the approach.

Dale Hattis

The early results for the saturation modeling described in section 4 above strongly
suggest that the lung tumors for both male and female mice are completely compatible
with the systemic saturable metabolic activation model with a half-saturation point
similar to that derived with data for other tumor locations. Therefore, I think the lung
tumors should not be treated as if they depended on local metabolism and other portal-of-
entry specific processes.

Ronald L. Melnick

Both treatments of the lung tumor data are appropriate because these tumors may have
arisen from metabolites formed in the lung, or in other organs, particularly the liver, and
subsequently distributed to the lung. No data are available to distinguish the extent of
these possibilities. The EPA document did note that the induction of tumors in multiple
organs after inhalation exposure to chloroprene demonstrates the systemic distribution of
carcinogenic metabolites by this route of exposure.

John B. Morris

The importance of portal of entry versus systemic delivery of chloroprene is not known.
A reasonable approach would be to make estimates using both approaches and then make
a determination of whether or not it is of quantitative importance. Naturally, the default
approach would be to select the more health protective approach. In my view, the
fundamental issue in this regard is actually based on the assignment of category 1 status
to chloroprene. This assignment is not appropriate (see my other comments), and at the
very least needs to justified. Chloroprene should be determined to be a category 3 vapor
in my view. It is a low partition coefficient vapor that does not appear to be highly
reactive. Indeed, were it highly reactive it would be impossible to measure a partition
coefficient. Moreover, the pattern of nasal injury (olfactory but not respiratory mucosal

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External Peer Review Meeting on the Toxicological Review of Chloroprene

damage) is inconsistent with a highly reactive vapor. Finally the modeling efforts of
Himmelstein would not have been successful were chloroprene highly reactive in tissues.
True it is metabolized, but the provided data do not indicate it is metabolized to such an
extent that it should behave as a category 1 vapor. If category 1 vapors do not penetrate to
the blood in any sufficient degree and if they should be scrubbed very efficiently in the
nose, then why are distal lung tumors and non-respiratory tract tumors observed? Were
chloroprene to be determined to be a category 3 vapor, then I believe the whole issue of
portal of entry versus system delivery will be moot because a DAF=1 would be assumed
for both cases. The regional injury pattern in the respiratory tract (olfactory and
bronchiolar injury) is suggestive for a critical role of local metabolic activation. It is
possible however that active metabolite is formed in and then escapes from the liver.

Avima M. Ruder

If chloroprene is indeed rapidly absorbed in mice, it makes sense that a systemic effect
from the metabolite as well as a portal-of-entry effect could occur. From the text (p 5-21)
I could not determine whether it is postulated that the portal-of-entry effect is from the
parent compound or the metabolite; this could be made clearer.

Richard B. Schlesinger

Since it is not clear, as noted in the Document, the extent to which chloroprene induces
cancer via direct contact with the lungs or via systemic contact of lungs with metabolites,
the approach used is valid. However, the application of this approach is not clear from the
discussion in the document.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

(>. . \n oral slope factor (i)Si') for cancer uv/.s not derived for chloroprene. Is the
determination that the available data for chloroprene do not support derivation of an
()Si' scientifically justified?

Herman J. Gibb

The determination is justified. There were no data on which to base an OSF and the
PBPK model developed by Himmelstein (2004) (description on page 3-7) did not seem
adequate to allow route-to-route extrapolation.

Dale Hattis

Not completely. With a PBPK model formulation, an oral slope factor could be
estimated.

Ronald L. Melnick

Yes, the lack of an adequate multiple-dose oral carcinogenicity study on chloroprene and
the lack of information on the disposition of chloroprene, including the AUC for the
DNA-reactive epoxide intermediate, after inhalation or oral exposure that might enable
reliable route-to-route extrapolation justify not deriving an oral slope factor for this
chemical. Because of a likely large first-pass liver effect after oral exposure, the systemic
distribution of parent compound and reactive metabolites could be very different after
oral versus inhalation exposures.

John B. Morris

I concur with the determination that the available data do not support derivation of an
oral slope factor.

Avima M. Ruder

As there are no quantitative data on effects of oral administration (p 5-1), the
determination appears justified.

Richard B. Schlesinger

The lack of oral exposure data clearly justifies not deriving an OSF.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

V. SPECIFIC OBSERVATIONS

Herman J. Gibb

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Page 4-3, line 1: Delete "also"

Page 4-3, line 8: Delete "number"

Page 4-3, lines 8-9: Delete "of these"

Page 4-3, line 14: Delete the second "were"

Page 4-5, lines 1-2: The document indicates that a limitation of Li et al. is that only three
years of local area data were used to estimate the expected numbers of deaths which may
not be representative with regard to the period of follow-up of the cohort. An issue not
considered is the stability of the expected rates based on local data.

Page 4-5, line 5: This discussion is unclear. If the general population had a higher
mortality for a given disease during the periods not examined, then there would have
been a higher number of expected deaths and the SMR for that disease would have been
overestimated for the period of time that was considered, not underestimated. If the
mortality was lower, then the SMRs would have been overestimated. In any case, the
discussion is not clear.

Page 4-6, line 18: Change "1979-1993" to "1979 to 1993".

Page 4-6, line 22: Insert "the" before "general".

Page 4-8, line 19: Change "1979-1988" to "1979 to 1988".

Page 4-9, line 12: There is an inconsistency in how the SIR is reported on line 12 and in
Table 4-6. Line 12 reports as 327 with 95% CI of 147 and 727; Table 4-6 reports as 3.27
with 95% CI of 1.47 and 7.27. The epidemiology section has several examples of
changing back and forth between the convention of using the convention of multiplying
by 100 and the ratio. Need to make consistent.

Page 4-9, line 23: Change "suggested" to "suggest"

Page 4-9, line 23: What are "highly exposed operators"? High cumulative exposure?
Intensity of exposure? Duration of exposure? It makes a difference in the interpretation.

Page 4-10, line 29: Insert "in the group employed" before "prior". Presumably the author
is describing those employed prior to 1977 and not those who developed cancer prior to
1977.

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Page 4-10, line 33: The document states that "all of the SIRs exceeded 100" yet Table 4-
7 indicates no SIR is over 100. Again, the authors need to use a consistent convention
(report as a multiple of 100 or not report as a multiple of 100).

Page 4-11, line 10: Change "cancers" to "cancer"

Page 4-11, line 15: Is there any indication of how many workers died or left the area
prior to 1979? Does the author have an idea of how much impact this would have on
results or is it part of a laundry list of study faults? The power of the study was low
regardless of whether workers died or left.

Page 4-14, lines 16-24 and Page 4-15, lines 1-3: It is not difficult to understand why
Marsh et al. would conclude that their study provided no evidence of cancer risk
associated with chloroprene exposures. Table 4-9 on page 4-14 shows little evidence of a
dose response. It is inappropriate to conclude as is done in lines 1-3 on page 4-15 that
Marsh et al.'s explanations are "not entirely consistent with the data presented". The
authors of this document have chosen one interpretation; the authors of the study have
chosen another interpretation.

Page 4-15, lines 24-35: Some of the criticisms are too harsh. For example, how often are
causes of death verified by histological confirmation or review of medical records? Nice
if it can be done, but the vast majority of mortality studies would fall in the same boat.
Incomplete enumeration of incident cases is a criticism that could be leveled at many
incident studies. The statement that despite the lack of quantitative exposure information,
occupational studies are still able to contribute to the overall qualitative weight of the
evidence considerations (lines 31-33) states the obvious, but the statement should not be
used as license to draw conclusions on studies that have serious limitations.

Page 4-16, Table 4-10: All SMRs are reported as the multiple of 100 except for
Bulbulyan et al. (1998). "Sullivan" should be "Selevan". It would be more logical to have
the intermediate exposure column first, followed by the high exposure column, followed
by the total cohort column.

Page 4-17, Table 4-11: The relative risk is reported as a multiple of 100 for the high and
intermediate exposures in the Leet and Selevan (1982) study but not for the other studies.
"Sullivan" should be "Selevan." It would be more logical to have the intermediate
exposure column first, followed by the high exposure column, followed by the total
cohort column.

Page 4-18, lines 7-8: The limited number of cases (one in each cohort) "precluding
meaningful examination" states the obvious.

Page 4-18, line 19: "these cancers"? Should this be "an increased liver cancer risk"?

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Page 4-19, line 8: "No workers experienced loss of hair." This is the first place where
loss of hair is mentioned. Since that is an unusual effect, it would be better to report the
results of the distillation workers after the results of the polymerization workers.

Page 4-63, line 13: What is "horizontal activity"?

Page 4-66, line 30: Delete "based on available data".

Page 4-67, Table 4-38: "Sullivan" should be "Selevan"

Page 4-69, lines 6-8: "Although not statistically significant, these findings were
comparable to results (RR range 2.9-7.1) detected in two other studies for high and
intermediate cumulative exposures (Bulbulyan et al., 1999, 1998)." Given that there
could have been considerable differences in exposure, follow-up, duration of exposure,
etc. between the studies, such a statement is not justified.

Page 4-69, lines 23-26: "only Bulbulyan et al. (1999) observed a statistically significant
association between chloroprene exposure and liver cancer mortality." The preceding
sentence suggests that this was done by an internal analysis, but the increase in liver
cancer mortality was observed from an external analysis.

Page 4-69, lines 29-30: "... .although there is no direct evidence that alcohol is related to
the exposure of interest (i.e., chloroprene) " There may be no "direct evidence that
alcohol is related to the exposure of interest"; there is no direct evidence that is not either.
More convincing that alcohol did not play a confounding role would have been clear
evidence of a dose response to chloroprene since it would be unlikely that alcohol
consumption would correlate with chloroprene exposure. Evidence of a dose response,
however, is equivocal (see Table 4-11 on page 4-17).

Page 4-70, lines 7-10: Criticizing mortality studies for not doing a medical record review
or histological examination to confirm cause of death is extreme. Almost all mortality
studies could be faulted for not doing that.

Page 4-71, lines 21-24: What "current understanding" allows one to state that specificity
is "one of the weaker guidelines"? Reference?

Page 6-1, line 22: Replace "th" with "the".

Dale Hattis

1. Table 3.2 should express results in fraction of total metabolites rather than relative to
butanol standard. Or it could be expressed in terms of absolute rates per unit time per unit
microsomal protein. Recalculate?

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2. p. 3-5, lines 5-7: "Estimates for Vmax and Km for oxidation of chloroprene in liver
microsomes ranged from 0.068-0.29 [j,mol/hour/mg protein and 0.53-1.33 [xM,
respectively."

The meaning of the ranges should be described. If these are in fact the ranges of all
observations, then the number of observations should be given; also, there should be
some way of describing the dependencies of the estimates of Vmax and Km values.

3. Presentation of metabolic data in Table 3-4 is inadequate. No error bars or statements
of how many animals tested independently (or pooled?), or more crucially, how many
humans and how they differ in Vmax/Km for various organs (obtain original papers on
metabolism).

Source: Himmelstein et al. (2004a).

Himmelstein, MW; Carpenter, SC; Hinderliter, PM. (2004a) Kinetic modeling of beta-
chloroprene metabolism: I. In vitro rates in liver and lung tissue fractions from mice, rats,
hamsters, and humans. Toxicol Sci 79(1): 18-27..

4. Table 3.5: Again, no error bars or description of the number of animals studied or
experimental errors.

5. p. 3-7, lines 4-5: "The clearance of these thioethers reached a threshold at 24 hours
after dosing, indicating that elimination was rapid."

Use of the word "threshold" here is unclear and ill-advised. If what is meant is that there
was no further increase in thioether excretion, then that should be said explicitly.

6. Table 3-6: Why are values not provided for the major physiological parameters (body
weight, cardiac output, and alveolar ventilation)?

7. Epi data discussion: The authors do qualify the discussion of the epidemiological data
with the healthy worker effect. However, they do not as yet include suitable caveats for
the "internal" comparisons by mentioning the distortions expected from the healthy
worker survivor" effect — that longer exposed workers with higher cumulative exposures
have lower mortality than shorter term workers. This must be incorporated into the
analysis. Some language I have adapted from prior work (Hattis and Goble 2007) is:

"The "healthy worker survivor" effect is a known phenomenon that produces
established distortions in relationships between measured risks and measures of
cumulative exposure, as shorter term workers suffer greater mortality than
workers who work at exposure-producing jobs for longer periods of time
(Steenland et al., 1996; Kolstad and Olsen, 1999; Garshick et al. 2004; Siebert et
al. 2001; Steenland and Stayner 1991). Adjustments for this effect are at the
cutting edge of current practice for the treatment of human epidemiological data,
but they are vital for achieving the best possible analysis of those data. Even if the
data will not support the more complex analyses [and analyses of this sort are

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notoriously complex (Robins 1986; Arrighi and Hertz-Picciotto 1996; Hertz-
Picciotto, personal communication)], EPA could provide at least some discussion
of how large the distortions might be by citing such previous cases as the cancer
risks from diesel particles (Garshick et al. 2004; 2008) and the approach that
California risk assessors (and possibly others) have taken to risk analysis where
the healthy worker survivor effect is even more prominent than it may be in this
case. (For diesel particulates, initial estimates of the relative risk vs. cumulative
dose curve even had a negative, rather than a positive slope.)"

8. The discussions of both liver and lung cancer might benefit from some attempt at
integrative meta-analysis, combining the effects of multiple studies for reasonably
comparable levels of exposure. This, however, likely depends on obtaining some
disaggregated data from the individual investigators, and that might not be possible. Even
if the combination is somewhat speculative, it might be informative to make some
attempt to combine the human evidence for comparison with the projections from animal
studies.

9. Chronic NTP exposures: For later modeling, the authors should report integrated
average exposures that were measured, rather than the nominal target exposures. The
difference may well be small, as indicated in the discussion, but the measurements should
be used in preference to the target levels in the dose response modeling which appears
later in the document.

10. p. 4-54, lines 16-18: "Estimates for Vmax and Km for oxidation of chloroprene (into
(l-chloroethenyl)oxirane) in liver microsomes ranged from 0.068-0.29 |imol/hour/mg
protein and 0.53-1.33 [xM, respectively."

Again, what is the meaning of these ranges? Simple ranges of all best estimates for all
species? 5%-95% confidence limits? What is the number of experiments based on how
many different individuals in which species, particularly for humans?

Undescribed ranges of this type are absolutely useless for understanding the uncertainty
and variability of the data, or for drawing inferences for subsequent steps in the risk
analysis.

11. p. 4-61, lines 5-7: "A comparative report of the carcinogenicity of these compounds
highlights the qualitative and quantitative concordance of their tumorigenic effects
(Melnick and Sills, 2001). The female mouse lung was the most sensitive site of
carcinogenicity for both chloroprene and butadiene."

It would be useful to have some quantitative comparison of cancer potency in rodents for
these compounds. The full abstract is:

Comparative carcinogenicity of 1,3-butadiene, isoprene, and chloroprene in rats and
mice.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Melnick RL, Sills RC.

Chem Biol Interact. 2001 Jun 1; 135-136:27-42.

National Institute of Environmental Health Sciences, National Institutes of Health, PO
Box 12233, Research Triangle Park, NC 27709, USA. melnickr@niehs.nih.gov

1,3-Butadiene, isoprene (2-methyl-1,3-butadiene), and chloroprene (2-chloro-l,3-
butadiene) are high-production-volume chemicals used mainly in the manufacture of
synthetic rubber. Inhalation studies have demonstrated multiple organ tumorigenic effects
with each of these chemicals in mice and rats. Sites of tumor induction by these epoxide-
forming chemicals were compared to each other and to ethylene oxide, a chemical
classified by the National Toxicology Program (NTP) and by the International Agency
for Research on Cancer (IARC) as carcinogenic to humans. For this group of chemicals,
there are substantial species differences in sites of neoplasia; neoplasia of the mammary
gland is the only common tumorigenic effect in rats and mice. Within each species, there
are several common sites of tumor induction; these include the hematopoietic system,
circulatory system, lung, liver, forestomach, Harderian gland, and mammary gland in
mice, and the mammary gland and possibly the brain, thyroid, testis, and kidney in rats.
For studies in which individual animal data were available, mortality-adjusted tumor rates
were calculated, and estimates were made of the shape of the exposure-response curves
and ED10 values (i.e. exposure concentrations associated with an excess risk of 10% at
each tumor site). Most tumorigenic effects reported here were consistent with linear or
supralinear models. For chloroprene and butadiene, the most potent response was for the
induction of lung neoplasms in female mice, with ED10 values of 0.3 ppm. Based on
animal cancer data, isoprene and chloroprene are listed in the NTP's Report on
Carcinogens (RoC) as reasonably anticipated to be a human carcinogen. Butadiene is
listed in the RoC as known to be a human carcinogen 'based on sufficient evidence of
carcinogenicity from studies in humans, including epidemiological and mechanistic
information', with support from experimental studies in laboratory animals.

Epidemiology data for isoprene and chloroprene are not considered adequate to evaluate
the potential carcinogenicity of these agents in humans.

I believe the similarity of ED 10s for lung tumors is potentially helpful for the reader,
however, a more comprehensive summary of potencies for other and/or all tumors would
provide important background for the quantitative cancer risk analysis. Table 4-37 should
be supplemented with a table giving quantification of the indicated potency for multiple-
and all sites.

12. p. 4-69, lines 13-19: "One of the strengths of several of the more recent
epidemiologic studies was improved exposure assessment data. These studies utilized
industrial hygiene information to determine which areas or jobs were most likely to have
received higher chloroprene exposures. This allowed for examination of various exposure
contrasts and helped reduce the potential for exposure misclassification. As such, valid
internal analyses were conducted which were less impacted by bias due to the healthy
worker effect. Despite these improvements, several study limitations added to the

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uncertainty in addressing the weight of evidence of the epidemiologic data."

The discussion following this paragraph should include the healthy worker survivor
effect.

13. Table 5-2: DAFs greater than 1 for lung and less than 1 for nasal epithelium deserve
specific discussion.

14. Page 5-20, top: Variability (uncertainty?) in slope factors follows a normal
distribution? Try lognormal.

15. Cancer modeling: In view of the saturation of the generation of active metabolite,
and the need to drop high doses in some cases, there should be investigation of a
Michaelis Menten transformation of dose, in lieu of a full PBPK model. Demonstrate
results of this for the incidence of tumors in mice (without the Weibull factor for time
dependent tumor observations).

References

Arrighi HM, Hertz-Picciotto I. 1996. Controlling the healthy worker survivor effect: an
example of arsenic exposure and respiratory cancer. Occup Environ Med. 53(7):455-462.

Garshick E, Laden F, Hart JE, Rosner B, Smith TJ, Dockery DW, Speizer FE. 2004.
Lung cancer in railroad workers exposed to diesel exhaust. Environ Health Perspect.
112(15): 1539-43.

Garshick E, Laden F, Hart JE, Rosner B, Davis ME, Eisen EA, Smith TJ. 2008. Lung
cancer and vehicle exhaust in trucking industry workers. Environ Health Perspect.
116(10): 1327-32.

Hattis, D. and Goble, R. L. "Uncertainties in Risk Assessment for Carcinogenesis: A
Road Map Toward Practical Improvements" White paper for the U.S. Environmental
Protection Agency, May, 2007.

Kolstad HA, Olsen J. 1999. Why do short term workers have high mortality? Am J
Epidemiol. 1999 Feb 15;149(4):347-52.

Neumeyer-Gromen A, Razum O, Kersten N, Seidler A, Zeeb H. 2009. Diesel motor
emissions and lung cancer mortality—results of the second follow-up of a cohort study in
potash miners.Int J Cancer. 124(8): 1900-6.

Robins J. 1986. A new approach to causal inference in mortality studies with a sustained
exposure period—application to control of the healthy worker survivor effect. Mathemat
Modeling 7:1393-1512.

Siebert U, Rothenbacher D, Daniel U, Brenner H. 2001. Demonstration of the healthy

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External Peer Review Meeting on the Toxicological Review of Chloroprene

worker survivor effect in a cohort of workers in the construction industry. Occup Environ
Med. 2001 Dec; 58(12):774-779.

Steenland K, Deddens J, Salvan A, Stayner L. 1996. Negative bias in exposure-response
trends in occupational studies: modeling the healthy workers survivor effect. Am J
Epidemiol. 143(2):202-210.

Steenland K, Stayner L. 1991. The importance of employment status in occupational
cohort mortality studies. Epidemiology. 2(6):418-423.

Ronald L. Melttick

Page 3-2 to 3-5. The discussion on chloroprene metabolism is deficient in its
consideration of species differences in glutathione conjugation, catalyzed by glutathione-
S-transferase, in the detoxification of (chloroethenyl)oxirane.

Page 3-7 to 3-8. Discussion is needed on likely differences in chloroprene clearance
among species. Factors influencing the clearance of chloroprene include fat:air partition
coefficients, % of body weight as fat (mouse: 5%; rat: 7%; human 21%), metabolic
elimination, etc.

Page 4-13. It seems odd that of the 652 cancer cases in the Louisville facility, only 1 case
was unexposed (Table 4-8). This might suggest that a large percentage of individuals
classified as exposed were essentially unexposed. The document should provide greater
emphasis on the potential impact of exposure misclassifications.

Page 4-16 to 4-17. Use common units for SMR and RR values in Tables 4-10 and 4-11.
On some cases the actual ratios are given, while in other cases the ratios are expressed as
per cent.

Page 4-22. Contrary to the statement on lines 2-6, the data in Table 4-14 show incidences
of ovarian or mammary tumors in control female rats.

Page 4-47, lines 5-7. Additional analyses are needed before dismissing the finding of
increased resorptions in the 10 and 25 ppm exposure groups.

Page 4-60. Delete lines 12-15. The hypothesis that chloroprene would only produce
tumors in directly exposed tissues has been disproved by the NTP studies which
demonstrated the multiple organ carcinogenicity of this chemical.

Page 4-63, line a6. Severities were minimal to moderate, not minimal to mild.

Page 4-73, line 7. The document specifies a mutagenic MOA involving the reaction of
epoxide metabolites formed at target sites. Until studies are conducted evaluating blood
levels of epoxide intermediates it would not be appropriate to impose this target site

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limitation. It is not known if epoxide formation occurs in all of the tumor target sites
identified in the rodent carcinogenicity studies.

Page 5-19, Table 5-7. The unit risk value for hemangiosarcomas/hemangiomas is
incorrect - it should be 2.8 x 10"5, not 8.3 x 10"5.

John B. Morris
Pages 3-1 - 3-6

The data on partition coefficient should be discussed more completely. It is true that it is
possible to infer information on tissue distribution from such data. It is also possible to
make inferences on regional respiratory tract absorption from these numbers. A vapor
with a blood:air partition coefficient less than 10 is not likely to be scrubbed efficiently
from the airstream in the upper airways. This is an important point because an inhalation
cancer potency factor will be derived assuming category 1 status.

More detail should be provided on the metabolism kinetics for chloroprene. The
information on elucidation of putative metabolites is clear and concise, but the data on
kinetics is incompletely presented data and is very difficult to interpret fully. The
information in Table 3-1 needs to be more fully described. Is this table cited in the text?
Precisely how were these data obtained, what is the meaning of these data, particularly
with respect to rodent-human extrapolations? The relative level of metabolite 1 in the
humans was approximately 10-fold lower than the F344 rat and mouse. The level of
metabolite in the Wistar rat and hamster was lower as well. Were these quantitative
differences synthesized into a coherent explanation of species differences in response?

Similar issues could be related relative to Tables 3-4 and 3-5. The text should precisely
indicate how the estimates for Vmax/Km for lung metabolism were obtained. The mouse -
human comparison for lung metabolism is particularly important, a fact that was not
adequately considered in the risk evaluation. The presented data indicate the activity in
human lung is 50-fold lower than in mouse lung (Table 3-4). The liver activities in the
mouse and man are much more similar. Since metabolic activation is the first step in the
mode of action and lung tumors in mice drives the risk extrapolation, this comparison
becomes particularly important. Exactly how was the value of 1.3 for Vmax/Km in the
human obtained? What is the reliability of this number? Can it or can it not but used for
quantitative species extrapolations? An explicit rationale for not using these data in the
data synthesis sections needs to be provided. It should be noted that this type of species
difference (mouse to human pulmonary metabolism) is hardly unique to chloroprene. For
example, consider styrene.

Pages 4-1 - 4-18

The section on human exposures to chloroprene appears to be objectively and concisely
presented. Epidemiology is not within my area of expertise. My only comment is the
thought that it would be useful if as much information as possible on occupational
exposure levels would be presented in the text. At least to me, information on exposure
concentrations in addition to cumulative (ppm-year) would be of value. If available,

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recent published reviews of the epidemiological data relative to chloroprene should be
cited.

Page 4-25

Clarity would be enhanced if the table also provided information on the magnitude of
injury in Table 4-16 and subsequent tables. A footnote might be adequate. Alternatively,
the average injury score might be provided parenthetically in each column. The wording
of the text infers there was no observed histopathological damage in the lungs of mice in
the 16 day study. Clarity would be enhanced if this were explicitly stated.

Page 4-28

Clarity would be enhanced if it were explicitly stated that lesions were not observed in
the nasal respiratory mucosa in the 13-week study. All lesions in Table 4-19 were in
olfactory mucosa, the reader must make the inference that respiratory mucosa damage
was absent. This is an important issue relative to data interpretation.

Page 4-29

Clarity might be enhanced if it is stated that preening behavior might have lead to direct
gastrointestinal exposure to chloroprene. If this is not thought to be the case, then it
should be explicitly stated.

Pages 4-30 - 4-43

It is noted that all nasal lesions in Table 4-16 are presented under the heading of
"olfactory," implying that no nasal respiratory mucosal lesions were observed. This needs
to be explicitly stated. The subsequent text is quite ambiguous in this regard. For
example, in the absence of any respiratory mucosal lesions, why include speculation on
the relative expression of CYP450 in olfactory versus respiratory mucosa of the rat nose?
(I did a quick scan of the NTP report to confirm, at least superficially, the absence of
respiratory mucosal lesions.) All subsequent descriptions of these data, e.g. chronic nasal
inflammation (p5-2) should be qualified to state chronic nasal olfactory inflammation (if
this is, in fact, true). Site specificity of nasal lesions is a critical aspect in the evaluation
of nasal response.

Subsequent portions of the text refer to time to tumor data. Where are these data and
derivation described? Should some discussion of maximum tolerated dose and whether it
was exceeded be included in the text?

Clarity would be enhanced if the text provided more detail on how the survival adjusted
neoplasm rates in Table 4-28 were calculated.

The description of the Trochimowicz et al. 1998 study indicates there was less chronic
respiratory disease in exposed than controls. Perhaps more information should be

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provided on the lesions that were present in control animals. This would seem to be a
relevant issue with respect to interpretation.

Page 4-54

The text (line 20) indicates epoxide hydrolysis was faster for the human and hamster than
rat or mouse. Where are these data presented?

Page 4-45

The test (lines 27-32) indicates "some activity" was observed in strains TA97A and
TA98. Subsequently (p. 4-65), it is stated the epoxide mutagenicity is "positive in all
strains." Are these two parts of the text concordant?

Page 4-61. Table 4-37

This table is very confusing. What was the basis for including data from the rat relative to
"sites of increased incidence" of neoplasms? Listed are many sites in which statistically
significant results were not enumerated in previous portions of the text. Obviously, clarity
needs to be improved.

Pages 4-62 - 4-65

In general, this "synthesis" of the inhalation exposure data is not a synthesis but merely a
reiteration of the results. Rather than repeat the results study by study, it might be much
preferable to organize this section on the basis of target organ. It could, for example,
discuss the olfactory lesion data in toto, followed by the liver, etc. On page 4-62 line 15,
it is stated that chloroprene is associated with reproductive and developmental effects, yet
the earlier portions of the text concluded otherwise.

Table 4-38

Table 4-38 is somewhat confusing. Why was lung cancer mortality listed under "rare
tumors?" The table includes a reference to time to tumor, yet such data were not
presented earlier in the text.

Page 4-72

Lines 11-12 include a listing of increased incidences of tumors, yet the basis for inclusion
in this listing is unclear. Some organs are listed in which the tumor incidence was not
significantly increased. The discussion of species differences (lines 27-31) should include
reference to possible species differences in epoxide hydrolysis rates. Such data are
presented earlier and its absence here is confusing. This section fails to include the most
important species difference - the appearance of lung tumors in mice but not rats. An in
situ pulmonary metabolic basis might be provided, given that the metabolic activation
rate in mice appears to be 50-fold higher than the rat but that in the liver differs by only

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2-fold (Table 3-4 and 3-5). This would also serve to emphasize the potential role of
metabolism relative to carcinogenicity. Epoxide formation is thought to be important
relative to the respiratory tract toxicity/carcinogenicity of naphthalene and styrene and
the same species differences (lung tumors in mice but not in rats) is seen for these vapors.
Line 32 includes a reference to Dong et al 1989; this study was not described previously.

Page 4-75

The statement that in vivo uptake of chloroprene involved the balance between epoxide
formation and detoxification is confusing. Certainly the toxicity depends on the balance,
but it is unlikely that uptake does. Uptake rates depend on the blood and tissue
concentration of parent, downstream conversion of metabolite is not necessarily
important in diffusion-based uptake. Greater clarity is needed.

Page 4-76

It is stated on lines 3-4 that there is remarkable similarities in the potency and shape of
the dose response between butadiene and chloroprene. Such data are not presented in
earlier portions of the text.

Page 4-77

It is stated that Melnick et al. (line 18) performed a 6 month exposure-6 month follow-up
study. Where are these data presented?

Page 5-3, top

The text needs to clearly describe how the atrophy and necrotic data were combined. I am
not certain there are any data indicating nasal olfactory atrophy leads to necrosis (as
stated on lines 5-6). The concept that necrosis may lead to atrophy is quite
straightforward however.

Page 5-5

In my view, chloroprene is not a category 1 gas (see also my comments above). Its
partition coefficient is only 10, clearly backpressure in nasal tissues controls the uptake
process. The presence of non-respiratory tract tumors clearly indicates it is absorbed into
the bloodstream. This vapor does not possess the physical chemical characteristics
required of category 1 gases; in my view, it is a category 3 gas. The text needs to
rigorously support this conclusion with respect to the physical chemical characteristics of
chloroprene relative to those required of category 1 gases. The presence of olfactory
lesions is NOT evidence that the toxicant was delivered via the airstream. Numerous
compounds produce selective olfactory injury after parenteral administration. Indeed, the
presence of olfactory but not respiratory nasal mucosal injury might be considered to
provide data in support of a blood-borne mechanism. Naphthalene is one example of this
phenomenon. Importantly, the subsequent text describes in great detail how the lung

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lesions may be due to blood-delivered rather than air-delivered chloroprene. The text
needs to be consistent. Redistribution of chloroprene from fat stores during non-exposure
periods is one potential mechanism for a role of blood borne chloroprene in inducing
olfactory lesions.

The RfC methodology is fatally flawed with respect to RGDR calculation. The
derivations of these equations are based on the faulty assumption that the mass transfer
coefficient is uniform throughout the nose. Dosimetry predictions from RGDR-based
evaluations are totally discordant with the data. For example, the RGDR-predictions are
counter to the theoretically sound modeling and experimental data obtained for
formaldehyde and vinyl acetate. The RGDR-based estimates of species differences in
dosimetry are discordant with the database on acetaldehyde dosimetry in multiple
laboratory animal species. While application of a flawed methodology may be consistent
with EPA policy, it certainly is not consistent with the scientific state-of-the-art. Perhaps
it is felt that chloroprene is truly a category 2 gas, but it is assigned category 1 status
because of difficulty in implementing RGDR calculations for category 2 gases. If so, it
should be explicitly stated. As noted above, its low partition coefficient and the existence
of distal organ effects indicate chloroprene is likely a category 3 gas.

The mode of action is assumed to include metabolic activation to the epoxide. The
RGDR of 0.28 indicates the humans will receive roughly 4-fold more toxicant (1/0.28)
than the rat. Is it meant to imply that the metabolic activation rate in the human nose is 4-
fold higher than the rat? Is there a single example of this being the case? The use of the
RGDR needs to be discussed in light of the metabolically-based mode of action.

Page 5-8

I recognize that it may be policy to include a database limitation factor due to the lack of
a two generation study, but I do not feel it is scientifically justified in this case. A
multigeneration study does exist. The rationale for the selection of this uncertainty factor
should include this study.

Table 5-3

Table 5-3 does not include a row in the consideration column for database limitation.
Table 5-4

This table provides time to tumor data, but such data have not been presented.

Page 5-21

Would it be possible to compare the tumor risk calculations with the human workplace
experience? This might provide a useful "reality check." Even if the occupational
exposure levels were only crudely known, it might be possible to determine if the
estimated cancer risks were at least somewhat reflective of reality.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Page 5-25

The cross-species scaling section is deficient in that it does not include consideration of
metabolism rate. The first step in the mode of action is metabolic activation to an epoxide
and the toxicokinetic data indicate the mouse lung activity exceeds that in the human by
50-fold (Table 3-4). Clearly, this is highly relevant. Moreover, magnitude of species
difference in metabolism is not unique, consider styrene or naphthalene. One might
convincingly argue that the enormous metabolic activation rate in the mouse coupled
with the low epoxide hydrolysis rate renders this species inappropriate relative to
extrapolation of lung tumors. The authors of the document may not agree, but a critical
discussion and rationale for using the mouse data needs to be included.

Page 6-5

The sentence on lines 18-19 is confusing. Lesions were specific to the olfactory mucosa,
what is the relevance of cytochrome P450 in respiratory mucosa in this regard?

Avima M. Ruder

Page 2-1 line 12. volume produced or volume used?

Page 2-1 line 18. Is Mg a million grams? Not in List of Abbreviations.

Page 2-1 line 22. Starting material for chloroprene synthesis is butadiene in the U.S.

Page 2-2 line 15. Suggest rewording to: The polymerization process has been
discussed...

Page 3-2 line 5. Suggest inserting "that of' between "similar to" and "vinyl chloride"

Page 3-4 Figure 3-1 and caption. Why these numbers? Why not consecutive in
key/caption? Why no 2, 3, 6, etc.?

Throughout section 4, SMRs and SIRs should consistently use base 1 or base 100, not
vary (cf pp 4-10 and 4-11). The adjectives low-exposure and high-exposure are not
consistently hyphenated (cf p 4-2 lines 18 and 19 versus line 25, p 4.7 table 4-4 title vs.
header for column 3). Deaths can be in excess but cannot be elevated (cf p 4-3 line 13).
SMRs can be elevated. Deaths in and of themselves cannot be statistically significant;
SMRs can be (cf p 4-3 line 13). Mortality is a rate and therefore "Mortality rate" (cf page
4-6 line 22) is redundant. Check citations! Leet and Selevan becomes Leet and Sullivan
in tables 4-10 and 4-11.

Page 4-1 line 2. occupationally exposed should not be hyphenated, "during" not "from"
the period ...

Page 4-1 line 8. delete "and" at beginning of line

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Page 4-1 line 20. Need comma after 1957. Similarly page 4-3 lines 24-25, page 4-4 line
13, etc.

Page 4-2 line 14. Change "both internal... and" to "either internal... or"

Page 4-2 lines 24-25. Needs commas after SMR and liver.

Page 4-2 line 31. Lack of adjustment (data were available) or lack of ability to adjust
(data were unavailable)?

Page 4-3 line 8. A total.. .was observed

Page 4-3 line 13. Suggest rewording to: "observed cancer deaths were also in excess
(SMR = 140) but the SMR was not statistically significant.

Page 4-3 line 14. Change last phrase to "and four deaths due to lung cancer"

Page 4-3 lines 15-17. Suggest rewording to: "With five observed cancers of the urinary
system (3 bladder and 2 kidney) the SMR was significantly elevated (300 compared to
the DuPont population and 250 compared to the U.S...."

Page 4-3 line 23. Suggest "accrued" instead of "worked for"

Page 4-3 line 24. Should be "was identified" (subject is "a cohort")

Page 4-4 line 3. Were exposures determined or estimated?

Page 4-4 lines 8-10. The sentence as written doesn't actually state that males had
increased exposure. Suggest "Males had statistically significant (p<0.005) greater
exposure to chloroprene than females based on.

Page 4-4 line 11. Subgroup has not been defined.

Page 4-4 line 13. "their dates of death"

Page 4-4 line 15. Suggest "sixteen reported cancer deaths occurred among.

Page 4-5 Table 4-2, row "researcher". All cause cell needs slash between 21 and 176.

Page 4-5 line 1. Suggest "One limitation of the Li et al. (1989) study was insufficient
comparison mortality data"

Page 4-5 line 2. "years were not"

Page 4-5 line 4. "time periods"

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Page 4-5 line 6. Suggest. .during the time periods with no rates available,..."

Page 4-5 line 8. "there were no data..."

Page 4-5 line 17. "age at death was 12.7 years younger"

Page 4-6 line 7. Not clear whether "lasting and making" is one or two departments
Page 4-6 line 10. Locations or departments?

Page 4-6 lines 11-12. Suggest: "year. They therefore devised a relative exposure system.
Workers in the high-exposure departments were assigned.

Page 4-6 lines 19-20. Suggest: "Thirty-seven percent of cohort members (female/male
distribution was not provided) contributing 26,063 person-years.

Page 4-6 line 22. Suggest: "Mortality of the general population of Moscow was used for
comparison."

Page 4-6 line 24. Suggest "available only"

Page 4-6 line 25. "the rate of expected deaths"

Page 4-6 lines 29-31. Need to specify that SMRs were elevated, not just statistically
significant. What are "cancer-specific SMRs for liver cancer and leukemia" as opposed to
"SMRs for liver cancer and leukemia"?

Page 4-7 line 4. "low number". Is this a statistically significant decrease? Or provide
expected.

Page 4-7 line 8. Delete comma after leukemia.

Page 4-4 Table 4-4 header. All cases or just high-exposure cases?

Page 4-7 lines 10-11. Suggest: ".. .analysis by categories of duration of employment in
high-exposure jobs (1-9..."

Page 4-7 line 12. Need new paragraph starting with "The cumulative.."

Page 4-7 line 15. "Kidney cancer was increased in all categories..." Are these categories
of duration of employment as in lines 10-11 or tertiles or quartiles of cumulative
exposure?

Page 4-8 line 13. "Similar to the Li et al. study..."

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Page 4-8 line 14. Suggest: . .values if mortality during these years was not
representative..."

Page 4-8 line 20. "Death certificates were coded by using the ICD-9..."

Page 4-9 line 9. Suggest: "Cancer incidence data were available for 1979-1999..."

Page 4-9 line 10. "...were identified, with six liver.."

Page 4-9 line 13. "lung cancer in both the total..."

Page 4-9 line 20. "noted in analyses using..."

Page 4-9 lines 21-22. ".. .five cases in the highest cumulative exposure category of.

Page 4-10 line 7. "adjusted for in either mortality..."

Page 4-10 line 12. "time" of employment—era of employment or time of first
employment?

Page 4-10 line 23. "... estimated daily exposure..." ?

Page 4-10 lines 29-30 states that 32 cancers occurred prior to 1977. How is that possible
if the registry began in 1979? Does this mean 32 cancers occurred among those exposed
prior to 1977?

Page 4-10 line 32 states all SIRs exceeded 100. Table 4-7 presents SIRs using base 1.
Page 4-11 Table 4-7 header 3rd column. Cases Exposed before 1977?

Page 4-11 lines 2-3. "lung cancers occurred in workers with >20 years of exposure..., 3
in those with 11-20 years.. .and 1 in those with <10..."

Page 4-11 line 10. "the lung cancer excess..."

Page 4-11 line 11. "... smoking and alcohol consumption were..."

Page 4-11 line 18. Suggest: ".. .using external regional rates and internal comparisons..."

Page 4-11 line 20. ".. .both chloroprene and a potential..."

Page 4-12 throughout. As done in some places, but not consistently, label data with
plant initials instead of providing a string of numbers and then stating "respectively". For
example, line 9, change "1.54 and 0.094 ppm, respectively" to "1.54 (L) and 0.094 (M)".
Similarly in lines 11, 24, 25.

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Page 4-12 lines 4-6. Suggest: "Kentucky, and Ponchartrain (P), Louisiana. The third one
was the Maydown (M) plant in Northern Ireland and the fourth facility was the Enichem
Elastomer plant in Grenoble (G), France."

Page 4-12 line 8. Suggest "occurred at" instead of "existed in"

Page 4-12 line 14. "cohorts" (as in line 10)

Page 4-12 line 23 states 266, 48, 12, 10 for lung cancer deaths; table 4-8 has these
numbers for all respiratory cancer deaths. Were all respiratory cancer deaths lung
cancers?

Page 4-12 line 26. Suggest: "deaths than expected from liver cancer were..."

Page 4-12 line 29. Suggest: "when compared to expectations based on the general
population. When.

Page 4-13 line 2. "trends across quartiles of exposure were examined"

Page 4-13 line 14. "included" instead of "contained"

Page 4-13 line 23. Delete "the" at end of line

Page 4-14 line 4. "...work status was so highly..."

Page 4-14 line 7. "They found inverse associations..."

Page 4-15 lines 7-8. "cohorts had fewer than 1000 workers, while the remaining cohorts
had fewer than 6000."

Page 4-17, line 8. "...Louisville, Kentucky, plant."

Page 4-18 line 16. "found in workers who..."

Page 4-18 line 32. "... cohorts on different...

Page 4-19 line 7. "...much lower numbers..." or "many fewer numbers"

Page 4-20 line 1. "... 19-23 employed..."

(I did not read section 4.2 as closely as the preceding section; there may be errors and
ambiguities I did not catch.)

Page 5-15 line 3. Delete period preceding 1st word in line
Page 5-17 line 26. "multisfage-Weibull..."

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External Peer Review Meeting on the Toxicological Review of Chloroprene

Page 5-21 line 21. EPA 1994A or EPA 1994B?

Page 7-3 lines 19-20. Only articles by the same author (which these are not) should be
labeled 2001a and 2001b.

Page 7-5 line 33. "...life table analysis.

Page B-2 Figure B-l. Abbreviations should be explained in a caption (similarly for other
figures). What is the metric for the doses (horizontal axis)?

Richard B. Schlesinger

Section 4.6. The first paragraph of this section should have a subsection 4.6.1. Human
Studies and the Animal Studies should be renumbered as 4.6.2.

Section 4.7. This section could be better organized. The summary in section 4.7.1 should
probably be moved to the end of the entire section on carcinogenicity. The human data
are discussed separately in an Evidence for Causality section, yet this is not provided for
the animal studies. A true synthesis would discuss Evidence for Causality across studies
in all species. This could be integrated with the discussion in Section 4.7.3.3 on Mode of
Action to provide a stronger rationale for effects of chloroprene

67

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