MEMORANDUM

To:	Margaret Sheppard

From:	Kara Altshuler, Charlotte Coultrap-Bagg, Reva Rubenstein, Mark
Wagner

Date:	May 9, 2006

Re:	Revised Memorandum regarding Comments made by Dr. Stelljes on
RTI’s Metabolism Study on nPB (EPA Contract Number EP-W-06-008, Task
Order 3 Task 06).



	

Attached please find a revised version of ICF’s review of comments
made by Dr. Mark Stelljes on RTI’s Final Study Report on
1-Bromopropane.

In summary, in his memorandum assessing nPB toxicity in humans, Dr.
Stelljes (2005) presents an assessment of RTI’s study using reasoning
that is poorly supported and draws insufficient conclusions.  For
instance, Dr. Stelljes argues that headache should be used as the most
sensitive endpoint for human toxicity; however, data show that
reproductive effects occur at lower doses than neurological effects. 
Dr. Stelljes also argues that male reproductive endpoints are more
appropriate endpoints than female reproductive endpoints, a view that is
not supported by other toxicological experts.  He relies on published
metabolism data for other compounds in order to draw comparisons between
the metabolism of nPB in humans and rodents and interprets RTI’s
results as suggesting that mice are not as susceptible to nPB exposure
as rats; however, RTI only concludes that mice and rats metabolize nPB
differently.

Please contact Mark Wagner at 202-862-1155 with any questions or
comments.Review of memorandum regarding RTI Metabolism Study on nPB by
Dr. Stelljes

Dr. Stelljes has prepared a lengthy memorandum to Mr. Richard Morford of
EnviroTech that presents his reasoning with regard to nPB toxicity in
humans (also duplicated in a memorandum to EnviroTech Europe at  
HYPERLINK "http://ecb.jrc.it/classlab/6702a11_IND_n-propyl-bromide.pdf" 
http://ecb.jrc.it/classlab/6702a11_IND_n-propyl-bromide.pdf  )
(Stelljes, 2005).  This document presents much unnecessary information,
does not provide sufficient, or in some cases any, data to support some
of the arguments, and abandons some of these arguments when presenting
conclusions.  The memorandum makes the following conclusions:

Female reproductive endpoints in rats, including ovarian effects such as
estrous cycle length, are not useful indicators of reproductive toxicity
(p. 5).

The reproductive toxicity of nPB is unlike that of other brominated
compounds with regard to metabolism, likely toxic mechanism, or site of
action (pp. 9, 32, 34).

The mechanism of action for nPB may be based on effects on neuroproteins
both within the nervous and reproductive systems (p. 34).

Concentrations of nPB up to 170 ppm in the workplace have not been
associated with neurological impact in humans (p. 33).

Mice are substantially less sensitive to nPB than rats (at least 5-fold
less sensitive) (pp. 8, 13).

Humans have the lowest ability to clear TCE from the body through
metabolism with CYP2E1, compared to mice and rats (p. 18).

Human testicular metabolism of TCE associated with CYP2E1 more closely
resembles that of mice than that of rats (p. 18). 

Humans will generate toxic nPB metabolites at a lower rate than rats or
mice, and therefore should be less sensitive than either of these
species (p. 36).

Information on the location of CYP2E1 and glutathione in human males,
mice, and rats and information on reproductive toxicity of other similar
brominated compounds strongly implies that humans are not more sensitive
to the toxic effects of nPB than rats or mice, and are most likely less
sensitive than rats (pp. 32-33).

Neurological effects are the only adverse effects in humans following
exposure to nPB (p. 34).

An uncertainty factor of no more than two is most appropriate for nPB
(p. 37).

The remainder of this memorandum addresses the following most
problematic points from above:

Female reproductive endpoints in rats are not useful indicators of
reproductive toxicity (p. 5).

The reproductive toxicity of nPB is unlike that of other brominated
compounds with regard to metabolism, likely toxic mechanism, or site of
action (pp. 9, 32, 34).

Mice are substantially less sensitive to nPB than rats (at least 5-fold
less sensitive) (pp. 8, 13).

Humans have the lowest ability to clear TCE from the body through
metabolism with CYP2E1 compared to mice and rats (p. 18).

Neurological effects are the only adverse effects in humans following
exposure to nPB (p. 34).

An uncertainty factor of no more than two is most appropriate for nPB
(p. 37).

The other conclusions mentioned above are not discussed because they are
related to the points below, but have less scientific relevance or are
secondary to the points discussed.

Female reproductive endpoints in rats are not useful indicators of
reproductive toxicity.

Dr. Stelljes contends that female reproductive effects, particularly
ovarian effects, are unreliable and should not be used as measurable
endpoints to define a dose response; in his view, effects on male sperm
health are more appropriate reproductive endpoints (p. 5).  Dr. Stelljes
dismisses estrous cycle issues on the basis of an NTP review of selected
13-week studies that did not find conclusive evidence supporting estrous
cycle length as a strong indicator of reproductive toxicity (Morrissey
et al., 1988).  Dr. Stelljes incorrectly quotes Morrissey and coauthors
by stating that their conclusion was that “stages of estrous cycle are
so variable that they are not useful in assessing potential reproductive
toxicity” (Stelljes, 2005, p. 5).  The article actually states that
“[m]ore data from breeding studies in which female estrous cycle
length is measured are needed to assess fully the association of cycle
length with reproductive outcome; stages of the estrous cycle are so
variable that they may not be useful in assessing potential reproductive
toxicity.”  The Morrissey article was a review of 50 NTP 13-week
studies on compounds in which 46 had included measurement of estrous
cycle data in the final week of exposure.  The authors stated that very
few of the compounds had been investigated in “definitive reproductive
toxicology protocols.”  Given the dated nature of the studies
reviewed, the recent development by EPA of guidelines on evaluating
reproductive toxicity, and improved standardization of reproductive
studies such as multi-generation reproductive studies, it is not
appropriate to declare use of estrous cycle length uninformative or
irrelevant on the basis of this 1988 review.

Contrary to Dr. Stelljes’ conclusion, there are hard data to support
the estrous cycle changes in both F0 and F1 females exposed to nPB (WIL,
2001); further, these changes are part of a spectrum of effects that
increase with dose and end in complete loss of fertility at the highest
exposure concentrations.  Further, his viewpoint on female reproductive
effects is not supported by experts in the field.  Dr. George Daston of
Proctor & Gamble, Drs. Ralph Cooper and Sally Darney of the Reproduction
Toxicology Division of the National Health and Environmental Effects
Research Laboratory, US EPA, Dr. Ulrike Luderer, currently at the
University of California at Irvine, and Dr. Jodi Flaws at the University
of Maryland, all provided the latest thinking with regard to
reproductive effects in animal models and their relevance to humans in
support of their peer review of ICF’s derivation of the AEL for nPB. 
All five are recognized experts in the field of reproductive and
developmental toxicology, and Drs. Daston, Darney and Luderer were among
the coauthors of the Center for the Evaluation of Risks to Human
Reproduction (CERHR) Report on Reproductive Toxicity of nPB (CERHR,
2002).  All believed that female reproductive effects in rodents were
not only relevant to the human experience, but predictive of potential
adverse outcomes in humans. Further, all agreed with ICF’s approach to
model rat estrous cycle length as the most sensitive indicator of
toxicity following subchronic exposure to nPB.  

The reproductive toxicity of nPB is unlike that of other brominated
compounds with regard to metabolism, likely toxic mechanism, or site of
action (pp. 9, 32, 34).

This statement is not supported by comparison of nPB to isopropyl
bromide (iPB).  Dr. Stelljes presents data that suggest that iPB and nPB
are metabolized differently.  While their metabolic pathways appear to
be different, both compounds target ovarian follicle development,
disrupting ovarian function in rats (Yamada et al., 2003; Yu et al.,
1999; Sekiguchi et al., 2001).  Further, iPB disrupted ovarian function
in female workers exposed for an average of 11 months to a solvent
containing the compound (Koh et al., 1998).  Yamada and coworkers
speculate that iPB attacks follicles at all developmental stages,
including primordial follicles in contrast with nPB, which they found to
primarily affect maturing follicles.  Though the mechanism of action may
be dissimilar, the studies show that the compounds have a similar site
of action in the female rodent (the ovary).  Further, changes in the
follicle development induced by both compounds increased estrous cycle
length in the rodents.  Therefore, based on the effects of iPB on female
workers, it is reasonable to assume that similar changes may be
identified in female workers exposed to nPB, if the appropriate studies
were performed.    

Mice are substantially less sensitive to nPB than rats (at least 5-fold
less sensitive).

This speculation is not supported by the results of the NTP 13-week
inhalation toxicity studies (NTP, 2003) in rats and mice in which both
male and female mice exhibited increased early mortality following
exposure to 500 ppm.  By contrast, male and female rats exhibited no
increased mortality at concentrations up to 1000 ppm.  Further, no
significant differences in histopathology between species were noted in
either port of entry tissues (nasopharyngeal tissues) or potential
target organs (liver, CNS, peripheral nerves).  ICF has not found any
studies of nPB in mice other than the NTP and RTI studies.

Humans have the lowest ability to clear TCE from the body through
metabolism with CYP2E1 compared to mice and rats (p. 18).

Dr. Stelljes’ review of the RTI metabolism study focuses on metabolism
of nPB following inhalation exposures in both rats and mice and draws a
conclusion that the metabolism of nPB in humans resembles that of mice
more than that of rats.  Because essentially no data currently exist to
indicate similarities between humans and rodents with regard to nPB
metabolism, Dr. Stelljes relies on published metabolism data for other
halogenated compounds.  He also discusses data from other laboratories
with regard to which detoxification mechanism (oxidation or conjugation)
is more important in rats, mice, and humans with regard to these
different compounds.  Dr. Stelljes interprets the RTI metabolism report
to suggest that mice are not as susceptible to nPB exposure as rats
because the response of rats was affected at a lower exposure
concentration than mice (p. 8).   

Dr. Stelljes argues that humans are more similar to mice than to rats in
their metabolism of other halogenated compounds, particularly TCE, and
therefore, it is expected that humans will be more similar to mice in
the metabolism of nPB.  He supports this argument by discussing CYP2E1
metabolism of several environmental toxicants, including iPB and TCE. 
Dr. Stelljes makes the case for structurally dissimilar compounds having
different metabolic pathways in rodents and humans by discussing iPB. He
then contradicts his own argument regarding the importance of CYP
metabolism of nPB in both rodents and humans when he states that, in
contrast to iPB, nPB is primarily metabolized by glutathione (p. 13). 
He also discusses CYP metabolism of TCE; however, metabolism of TCE is
irrelevant to consideration of possible metabolism and mechanisms of
toxicity of nPB because TCE is not sufficiently structurally similar to
nPB.  If it was Dr. Stelljes’ aim to provide examples of compounds
that may be used as proxies for nPB, there are several
structurally-similar compounds, including 1-chloropropane, that could
have been used.  

If we accept RTI’s interpretation of their results (discussed below)
that cytochromes play the largest role in nPB detoxification in mice and
also rats, then further knowledge of how humans compare to mice and rats
with regard to similarity in cytochrome gene number and function would
be informative.  It is known that humans have variability within groups
of CYP genes (e.g., P4502A:  Pearce et al., 1992; P4502E1 and others:
Dorne et al., 2004), meaning that differences in these genes with regard
to amino acids translates into differences in specificity and activity
within the same type of gene product between people.

Although this variability forms the basis for many disease states,
little is published about similarities or differences in these genes
across different species.  Nelson et al. (2004) recently published data
regarding the similarity in the cytochrome P450 gene superfamily in mice
and humans.  The authors report that the seven CYP clusters are expanded
in the mouse compared to the human, with 72 functional genes in the
mouse, and only 27 functional genes in the human.  Without any knowledge
as to the efficacy of the functional genes in each species, the
difference in number suggests that the mouse would be a heartier species
with regard to robustness of the CYP family of proteins, and thus, in
its ability to clear nPB.  Therefore, one should be very cautious in
comparing a human to a mouse with regard to CYP metabolic capability. 
Given that Dr. Stelljes did not present any empirical data comparing the
mouse response to the human response following exposure to an
environmental toxicant, ICF finds his argument highly speculative and
wholly unsupported.  There are no data indicating that humans would
respond in a manner more similar to mice than to rats following exposure
to nPB.

It is possible that metabolism of nPB by CYP2E1 produces compounds that
are biologically more active than the parent compound.  If this is the
case, then metabolism that occurs primarily, or exclusively, via
cytochrome oxidation may put a species at increased risk for nPB-induced
toxicity.  One may speculate that this is the case with mice and rats. 
For example, mice in the NTP 13-week inhalation toxicity study exhibited
increased mortality at 500 ppm, with 3 of 10 males dying or sacrificed
as moribund within the first week of exposure, and one male sacrificed
as moribund the second week of exposure compared to none in the control
group.  Three of 10 female mice at 500 ppm also died and two others were
sacrificed as moribund in the first week, compared to none in the
control group.  Necropsy of these mice revealed marked liver necrosis in
all mice exhibiting early mortality.  By contrast, no rats exhibited
early mortality at concentrations up to 1000 ppm for 13 weeks.  The
possibility exists that the study involved a subset of unhealthy mice;
however, it is unlikely that all the unhealthy mice would all be
allocated to the highest exposure groups in this study. 

Neurological effects are the only adverse effects in humans following
exposure to nPB 

Dr. Stelljes states that neurological effects are the only adverse
effects that have been reported in humans (p. 34).  This is not
accurate; reproductive effects have not been reported often as related
to occupational exposure to nPB in the case reports or National
Institute for Occupational Safety and Health (NIOSH) Health Hazard
Evaluations (HHEs).  Ichihara and coworkers (2002) reported that two of
three women at a cushion factory in NC experienced temporary disruption
of menstrual cycles, following exposure to nPB for 5-12 months. 
However, no details regarding how many disrupted cycles or the time of
the disruption were provided.  The case reports and HHEs are limited by
low worker participation in either questionnaire completion or
biological analyses that might provide more conclusive information
regarding reproductive health in exposed individuals.  Therefore, the
available occupational data are insufficient to support a conclusion
that nPB is not a reproductive toxicant in humans at concentrations seen
in the workplace.

  

An uncertainty factor of no more than two is most appropriate for nPB.

Dr. Stelljes suggests that the total uncertainty factor should be as low
as 2.  He asserts that the metabolism of mice is more like that of
humans than that of rats.  Based on the test in the RTI study in which
rats showed saturation of metabolism at lower exposure concentrations
than mice, Dr. Stelljes concludes that mice can metabolize nPB better
than rats at high concentrations.  Therefore, he concludes, if humans
are more like mice in their superior ability to metabolize nPB, then
humans should be able to metabolize nPB better than rats.  Dr. Stelljes
uses this as a basis to recommend dropping the interspecies uncertainty
factor accounting for differences between rats and humans.  However, the
interspecies uncertainty factor of 3 that currently exists is to take
into account any differences in pharmacodynamics between rodents and
humans, not to account for differences in pharmacokinetics.  The data
that Dr. Stelljes cites address similarities and differences in
pharmacokinetics, which is the activity or fate of chemicals in the
body, including the processes of absorption, distribution, localization
in tissues, biotransformation, and excretion.  Pharmacodynamics is
defined as the biochemical and physiological effects of chemicals in the
body and the mechanism of their actions.  In order to reduce the UF for
pharmacodynamics, information on which organs nPB targets and the
mechanism of action in multiple species, including humans, would be
required.

ICF disagrees with Dr. Stelljes’s conclusion that the uncertainty
factor for animal to human extrapolation can be dropped.  As discussed
above, the remaining UF of 3 accounts for differences in
pharmacodynamics between the species and is still necessary because
inadequate information exist with regard to the mechanism of action of
nPB in different species and across differing endpoints related to
reproductive effects in different species.  Reproductive health has not
been adequately addressed in the population of workers exposed to nPB. 
Current case reports and NIOSH Human Health Evaluations have been either
silent on reproductive health or have had sample sizes that were too
small to determine if adverse changes in reproductive endpoints were
related to exposure to elevated concentrations of nPB.

Further, Dr. Stelljes suggests that an uncertainty factor for
variability within the working population of 2 is sufficient (pp.
36-37).  Dr. Stelljes asserts that the variability of the working
population is not expected to be wider than that in test animals, and
references Lipscomb et al., 1997.  However, Lipscomb and coworkers found
significant variability in CYP 2E1 metabolism of TCE in a limited sample
of liver enzyme preparations from 23 individuals.  Further, the Km
values for metabolism of TCE from these liver samples were not normally
distributed, showing that humans were non-uniform in their ability to
metabolize TCE.  In addition, no data are available to determine whether
CYP or glutathione would be primarily responsible for metabolism on nPB
in humans.  Thus, these data do not support Dr. Stelljes’ recommended
UF of 2.   

References

Center for the Evaluation of Risks to Human Reproduction (CERHR).  2002.
 NTP-CERHR Expert Panel Report on the Reproductive and Developmental
Toxicity of 1-Bromopropane.  National Toxicology Program. 
NTP-CERHR-1-BP-02.  March 2002.

Dorne JL, Walton K, Renwick AG.  2004.  Human variability for metabolic
pathways with limited data (CYP2A6, CYP2C9, CYP2E1, ADH, esterases,
glycine and sulphate conjugation).  Food Chem Toxicol.  42(3):397-421.

Koh J-M, Kim C-H, Hong SK, Lee K-U, Kin YT, Kim OJ, and Kim GS. 1998. 
Primary ovarian failure caused by a solvent containing 2-bromopropane. 
Eur J Endocrin 138:554-556.

Ichihara G, Miller JK, Ziolkowska A, Itohara S, Takeughi Y. 2002
Neurological disorders in three workers exposed to 1-bromopropane. 
Journal of Occupational Health 44:1-7 (2002).

Lipscomb JC, Garrett CM, and Snawder JE.  1997.  Cytochrome
P450-dependent metabolism of trichloroethylene:  interindividual
differences in humans.  Toxicol Appl Pharm 142:311-8.

Morrissey RE, Schwetz BA, Lamb JC 4th, Ross MD, Teague JL, Morris RW. 
1988.  Evaluation of rodent sperm, vaginal cytology, and reproductive
organ weight data from National Toxicology Program 13-week studies. 
Fundam Appl Toxciol 11:343-58.

National Toxicology Program (NTP). 2003.  National Toxicology Program
Database Search Application.  Search criteria for 1-bromopropane. 
Available at
<http://ntp-apps.niehs.nih.gov/ntp_tox/index.cfm?fuseaction=shorttermbio
assaydata.datasearch&study_no=C20011&cas_no=106-94-5&chemical_name=1-bro
mopropane&study_length=13%20Weeks&test_type=Short-Term>.

Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM, Nebert DW.  2004.
 Comparison of cytochrome P450 (CYP) genes from the mouse and human
genomes, including nomenclature recommendations for genes, pseudogenes
and alternative splice-variants.  Pharmacogenetics.  14(1):1-18.

Pearce R, Greenway D, Parkinson A.  1992.  Species differences and
interindividual variation in liver microsomal cytochrome P450 2A
enzymes:  effects on coumarin, dicumarol, and testosterone oxidation. 
Arch Biochem Biophys.  298(1):211-25.

Sekiguchi S, Asano G, Suda M, and Honma T. 2001.  Influence of
2-bromopropane on reproductive system—short-term administration of
2-bromopropane inhibits ovulation in F344 rats.  Toxicol Ind Health
16(7-8):277-283.

Stelljes, ME.  2005.  Mechanistic Hypothesis for n-Propylbromide and
Ramifications for Occupational Exposure Limit in the United States. 
Technical Memorandum to EnviroTech International.  7 September.

Yamada T, Ichihara G, Wang H, Yu X, Maeda K-I, Tsukamura H, Kamijima M,
Nakajima T, and Tadeuchi Y. 2003.  Exposure to 1-bromopropane causes
ovarian dysfunction in rats.  Toxicol Sci 71:96-103.

Yu X, Kamijima M, Ichihara G, Li W, Kitoh J, Xie Z, Shibata E, Hisanaga
N, Takeuchi Y. 1999.  2-Bromopropane causes ovarian dysfunction by
damaging primordial follicles and their oocytes in female rats.  Toxicol
Appl Pharmacol 159(3):185-93.

WIL, 2001.  An Inhalation Two-Generation Reproductive Toxicity Study of
1-Bromopropane in Rats.  Conducted by Stump D.G. at WIL Research
Laboratories, Inc., Sponsored by Brominated Solvents Consortium.  May
24, 2001.

 See ICF, 2006.  “Revised Memorandum regarding RTI Metabolism Study on
nPB.”  EPA Contract Number EP-W-06-008, Task Order 3 Task 06 for a
review of RTI’s Final Study Report on 1-Bromopropane.

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