MEMORANDUM

To:	Margaret Sheppard

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

Date:	March 28, 2006

Re:	Revised Memorandum regarding RTI 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 RTI’s
Final Study Report on 1-Bromopropane, previously delivered under EPA
Contract Number 68-D-00-266, Work Assignment 4-04, Task 02 on November
30, 2005.

RTI’s investigations were conducted to improve the knowledge base on
the metabolism of nPB in animal models.  RTI analyzed the following: 

The uptake, distribution and clearance of nPB in rats and mice following
intravenous (i.v.) exposure; 

The metabolism of nPB in rats and mice; 

Sex differences in the metabolism of inhaled nPB in rats; and 

The dermal uptake, distribution and metabolism of nPB in rats.  

The data collected by RTI contrast with previous data regarding the role
of CYP enzymes in mice and rats and the role of glutathione in the
metabolism of nPB in rats.  In addition, female rats may be more
susceptible to the adverse effects of nPB at higher concentrations than
males, but metabolize nPB similarly to male rats at lower
concentrations.  

Please contact Mark Wagner at 202-862-1155 with any questions or
comments.Pharmacokinetic Studies with nPB prepared by RTI

RTI has conducted a number of studies in rats and mice to determine the
uptake, distribution, clearance, and pharmacokinetics of nPB via
intravenous (iv), inhalation, and dermal uptake.  These experiments were
conducted primarily because metabolism data on nPB in animal models was
incomplete, and much of it was dated (Barnsley, 1966; Jones and Walsh,
1979; Tachizawa et al., 1982; Ishidao et al., 2002).  Based on the data,
RTI presented the following conclusions:

nPB that is administered via injection or inhalation is eliminated
mainly via volatiles in the breath or as exhaled CO2, with excretion in
the urine as a secondary path.

Distribution and metabolism are similar across intravenous (iv) doses
spanning two orders of magnitude (up to 100 mg/kg).

Male rats and mice are similar in the distribution and clearance of
systemic nPB.

The tissue:blood ratio of 3.15 indicates that nPB preferentially
partitions into tissues and organs following exposure, and should not
remain in the bloodstream.

Metabolism of nPB appears to be primarily carried out by cytochrome P450
(CYP450) isozymes.

Rats have two systems that metabolize nPB; at low doses, oxidation
predominates, but at higher doses, metabolites shift to those indicative
of glutathione conjugation, indicating that metabolism is dose-dependent
in this species. 

Mice appear to metabolize nPB differently than rats (metabolism is
almost exclusively via oxidation by P450 enzymes and is not
dose-dependent).

Female rats may have a decreased capacity to metabolize nPB compared to
male rats;

Rodents have a decreased ability to metabolize and excrete inhaled nPB
when initial exposure concentrations are greater than 800 ppm (e.g.,
metabolism is saturated).

nPB is not appreciably absorbed in rats following dermal application.

More detailed descriptions of the experiments conducted by RTI are
provided in the sections that follow.  There are a few limitations to
the RTI studies: 1) the document does not provide any raw data from the
experiments, which prevents an independent validation of presented
results and conclusions; and 2) metabolism studies, in general, involve
very few animals (4-5 per dose in this study), which often results in
standard deviations around the mean values that overlap for many
endpoints and time points.

Uptake, Distribution and Clearance of nPB in Male Mice and Rats
Following i.v. Exposure

RTI conducted a series of experiments in male B6C3F1 mice and Fisher 344
rats using radiolabeled nPB (at the 1-carbon position) that was
administered as a single bolus iv dose.  Doses of 5, 20, or 100 mg/kg
were used.  The doses were cleared from the body in the mice primarily
as uncharacterized volatile organic compounds (VOCs) in the breath
(~39-49% of the radiolabeled dose after 48 hours), via CO2 in the breath
(~19-28% of the dose at 48 hours), and in the urine (~16-26% of the dose
after 48 hours).  Very little (<4% of the dose) was eliminated via
feces.  Approximately 2% of the dose was retained in the tissues and
carcass, with distribution primarily to the liver, muscle, skin, small
intestine, and residual carcass.  Distribution and excretion of the
radiolabel was independent of the dose.  Similar results were obtained
in Fischer 344 male rats administered 5- and 20-mg/kg dosages,
indicating that male mice and rats distribute and clear injected nPB in
a similar fashion.  Analysis of the urine revealed no bromine,
indicating that it had been reabsorbed within the time frame of the
analysis (48 hours post-dosing).  Bromine is passively reabsorbed in the
kidneys as a negatively charged ion associated with sodium that is
reabsorbed in this organ.  It has an extended half-life in the body,
which increases its bioavailability (Pavelka et al., 2005).    REF
_Ref121131986 \h  \* MERGEFORMAT  Table 1  presents the results of the
experiments in mice.

Table   SEQ Table \* ARABIC  1 .  Recovery of Radioactivity Following a
Single iv Dose of 5 mg/kg 

[14C]-nPB to Male B6C3F1 Mice at 48 hours Post-Dosinga

Route	Cumulative Percent Dose Excreted or Absorbed

Tissue Absorption

	     Liver	   0.432±0.491  

     Muscle	   0.415±0.079

     Skin	   0.332±0.046

     Small Intestine	   0.112±0.026  

     Carcass	   0.33±0.13

Subtotal for tissues	2.03

Urine	25.5±5.8

Feces	2.9±1.1

VOCs	44.7±9.6

CO2	27.5±3.5

aExcerpted from Table 5.2, RTI (2005), p. 23.

In a separate set of experiments, the researchers analyzed the clearance
of nPB as a function of dose in male rats and mice.  They showed that at
5 mg/kg (iv dose), mice excreted slightly more of the
radioactively-labeled nPB in the urine than rats, the same amount as
CO2, and less as VOCs in the breath, than rats.  When the dose was
increased to 20 mg/kg, and further to 100 mg/kg, the amount excreted in
the urine and as CO2 decreased for both animals but more dramatically
for rats, and the percentage of label in exhaled VOCs increased from 50
to 70% in the rat, but did not change appreciably in the mouse.  These
data suggested that the mouse had a greater ability to metabolize nPB
completely to CO2 compared to the rat as exposure doses increased.

RTI focused on the P450 and glutathione enzyme systems because these two
enzyme groups are primarily responsible for clearing environmental
toxicants from the body and because previous metabolism studies (Jones
and Walsh, 1979; Kim et al., 1999; Ichihara et al., 2000) implicated
these enzymes in the metabolism of nPB. The difference in metabolism of
nPB between species appears to be that in rats, metabolism is begun as
oxidation by CYP450 enzymes, with glutathione conjugation still playing
an important role.  In mice, oxidation appears to be the only
significant pathway.  This hypothesis is supported by two sets of
independent data.  The first is the change in distribution of
radioactivity in urine, VOCs, CO2, and liver following iv dosing.  For
example, nPB tends to distribute in the livers of rats more than that of
mice, even at the lowest dose of 5 mg/kg.  At this low dose, compared to
the rat, the mouse showed slightly increased excretion in urine, lower
exhalation as VOCs in breath, and equal exhalation as CO2.  However, at
this dose (and at 20 mg/kg), nPB deposition in the liver (measured as
ng-equivalents/g liver) was 7 to ~10 times higher in rats.  It is
RTI’s supposition that the first step of metabolism must be oxidation
(rather than glutathione conjugation) to make a more bio-reactive
molecule; if glutathione conjugation were the first step, the increased
water solubility of the compound would lead it to be excreted, and it
would not be further retained in the tissue otherwise the compound would
not preferentially be absorbed into tissue.  This is shown by the
decrease of radiolabeled compounds in the liver following nPB exposure
when rats are pretreated with 1-aminobenzotriazole (1-ABT), a chemical
that blocks CYP450 enzymes and prevents them from metabolizing toxicants
within the cells.  The second set of data comprises the different series
of metabolites that are found in rat and mouse urine following nPB
exposure (explained in more detail below).  Although there appear to be
species-specific differences in primary pathways for metabolism in mice
and rats, additional experiments in male and female mice and rats
indicated that both sexes of each species can metabolize an inhaled dose
of nPB at 800 ppm, with metabolism saturating only at concentrations
≥800 ppm.  It is not possible to extrapolate to the human experience
from these data except to note that cytochrome oxidation may be the
principal pathway in nPB metabolism, and that like rodents, human
metabolic pathways are likely to be saturable at high concentrations.  

Metabolism of nPB in Rats and Mice

In order to determine which enzyme systems were primarily responsible
for metabolizing nPB, RTI exposed male rats to iv nPB after exposing the
rats to an oral dose of a glutathione inhibitor (buthionine sulfoximine,
BSO) or a cytochrome P450 inhibitor (1-aminobenzotriazole, 1-ABT).  When
compared to naïve rats (i.e., rats that were not pre-exposed to BSO or
1-ABT) treated only with nPB, 1-ABT exposure decreased the amount of
radioactive label found in CO2 by ~80%, and decreased the amount of
radioactive label in urine by ~40%, while BSO only decreased the amount
of label in CO2 and urine by ~10 and 4%, respectively.  These data
suggested that CYP450 enzymes, and not glutathione, were primarily
responsible for the metabolism of nPB in rats, because the CYP450
inhibitor was associated with a much greater decrease in metabolized
nPB.  Further, tissue:blood ratios of nPB decreased from a control value
of 3.12 in male rats treated only with nPB to 2.64 in BSO-pretreated
rats, and a low of 1.00 in 1-ABT-pretreated rats.  A lower tissue to
blood ratio occurs where more of the nPB is cleared from the body and
less is absorbed into body tissue.  The results suggest that reactive
metabolites, which should be absorbed more by the liver, were formed
primarily by CYP450 rather than glutathione.

The researchers performed a series of HPLC analyses of thawed breath and
urine samples taken at different time points from the rats injected with
bolus doses of radiolabeled nPB at 5, 20, and 100 mg, rats pretreated
with either 1-ABT or BSO and rats exposed to nPB via inhalation at 800
ppm.  Parent nPB was not detected in any urine sample at any dose. 
After a 20-mg/kg dose, radioactivity was associated with 8 major and 2
minor metabolites; 20% of the label co-eluted with a spike of
N-acetyl-S-propylcysteine.  At 100 mg/kg (4 hours post-dosing),
approximately 80-90% of the radiolabel is N-acetyl-S-propylcysteine,
although the other metabolite peaks evident at the lower dose are also
present.  The urinary profile of a rat at 8 hours following a 100-mg
dose resembles that of a 20-mg dose. The proportion of urinary
metabolites in the rat 6 hours following inhalation of 800 ppm nPB is
similar to that 8 hours following iv dosing with 100 mg/kg, indicating
the similarity of metabolism across exposure routes.  When male rats
were pretreated with 1-ABT, the majority of metabolites diminished,
except for N-acetyl-S-propylcysteine, which accounted for >90% of the
radioactivity.  With BSO pretreatment, the N-acetyl-S-propylcysteine
decreased slightly, but the other metabolites increased.  Taken
together, these data indicate that cytochrome oxidation of nPB is the
primary metabolic route in rats, accounting for perhaps as much as 70%
of the metabolism of the compound at lower doses (20 mg/kg) and that
when the dose is increased, metabolism via this pathway is saturated,
and glutathione conjugation becomes more important.

13C-NMR and mass spectrum analyses were performed on urine samples from
two male rats exposed to nPB via iv injection (two 100-mg/kg doses of
13C-nPB, labeled at the 1-carbon position, given 4 hours apart) and from
two rats exposed to 800 ppm [1,2,3-13C] nPB (labeled at all carbons) in
an inhalation chamber.  Additional inhalation metabolism experiments
were performed with wild-type mice and mice deficient in CYP 2E1 (4
males per group) also exposed to 800 ppm radiolabeled nPB.  Studies were
also done using liver homogenate from one male rat to try to elucidate
intermediate metabolites that were not detected in urine from exposed
rats.  The similarity of HPLC traces of urinary metabolites for rats
exposed via iv or inhalation indicated that qualitatively, the
metabolism of nPB is not route-dependent.  NMR spectra of urine from the
rats given nPB alone via iv injection revealed several 13C multiplets
(or peaks).  When 1-ABT was given to the rats administered nPB via iv,
the NMR spectrum was simplified to two compounds,
N-acetyl-S-propylcysteine and N-acetyl-S-propylcysteine-S-oxide
(determined by comparing to NMR spectra of synthetic standards of these
compounds).  This result indicated that cytochrome P450 inhibition
eliminates all metabolites of nPB save the ones listed above. The latter
compound was detected following 100 mg/kg injections and inhalation
exposures, but was minor at the low (20 mg/kg) iv dose.  Inspection of
NMR scans of mouse urine indicated that there were one major multiplet,
and two minor multiplets, that accounted for >90% of the 13C multiplets
present above the natural background.  Comparison of the spectra to rat
urinary metabolites confirmed the major metabolite to be
N-acetyl-S-(2-hydroxypropyl)cysteine, and the minor ones were
N-acetyl-S-propylcysteine and 1-bromo-2-propanol-O-glucuronide. 
Evaluation of the NMR data from CYP 2E1 (-/-) mice indicated that fewer
2-hydroxylated metabolites were present in urine.  The data for
metabolites in rats and mice are presented in   REF _Ref130719776 \h  \*
MERGEFORMAT  Table 2  below, and those data showing differences in
wild-type and CYP 2E1 null mice are shown in   REF _Ref130719784 \h  \*
MERGEFORMAT  Table 3 .

Table   SEQ Table \* ARABIC  2 : Primary Metabolites of nPB in Rats and
Mice

Metabolitea	Acronym	Relative Abundanceb



Rat 

(% of administered dose)	Mouse 



N-acetyl-S-propylcysteine	APC	37%	Minor

N-acetyl-S-(2-hydroxypropyl)cysteine	AHPC	16%	Major

N-acetyl-S-propylcysteine S-oxide	APCO	5%	Secondary

1-bromo-2-propanol-O-glucuronide	BPG	9%	Minor

N-acetyl-S-(2-oxopropyl)cysteine	APS	12%	Secondary

N-acetyl-S-(2-oxopropyl)cysteine-S-oxide

Minor

	aThree additional trace metabolites were detected in rats, but not
identified by RTI (2005).

bAs measured in urine following 6 hour nPB inhalation at 800 ppm.

Source: RTI (2005), Stelljes (2005).

Table   SEQ Table \* ARABIC  3 . Recovery of Metabolites from Wild-Type
and CYP 2E1 (-/-) Mice Following Inhalation Exposure to 800 ppm [13C]
nPB

Metabolite	Wild-Type	CYP 2E1 (-/-)	% Change (Approximate)

	(mmol recovered)

	N-acetyl-S-(2-hydroxypropyl)cysteine	0.034	0.021	-38

1-bromo-2-propanol-O-glucuronide	0.005	0.002	-64

N-acetyl-S-propylcysteine	0.008	0.024	190

Ratio 2-hydroxylated:directly conjugated	4.77	0.97

	Source: RTI (2005), Table 6.3.

The data indicate that mice with no capacity for CYP 2E1 metabolism of
nPB have a ~60% decrease in the 1-bromo-2-propanol-O-glucuronide and a
190% increase in the directly conjugated metabolite of nPB,
N-acetyl-S-propylcysteine, when compared to wild-type mice.  Thus, it
appears that wild-type mice metabolize nPB primarily via a P450
mechanism that begins with hydroxylation at the 2-position carbon.  

   

These analyses indicate that following inhalation in the rat, the
primary pathway of oxidation is the hydroxylation of nPB at the 2
position to form 1-bromo-2-propanol.  This compound was not detected in
urine following nPB exposure, but the data discussed above indicate that
a majority (>50%) of urinary compounds following low-level iv or
inhalation exposures are metabolized from this compound.  The
1-bromo-2-propanol is then either metabolized further or conjugated. 
When higher doses are administered, the CYP 2E1 system becomes
saturated, cannot metabolize the nPB, and the glutathione-dependent
pathway becomes critical for elimination of the compound.  In mice, at
all administered doses, glutathione conjugation was a very minor
contributor to nPB metabolism; the major metabolite was the mercapturic
acid of the P450 oxidation product, 1-bromo-2-propanol.  Therefore, the
metabolism of nPB differs in mice compared to rats.

Sex Differences in Metabolism of Inhaled nPB

ant (λz), and elimination half-lives (t1/2).  The researchers
determined that in general, males were able to metabolize nPB more
rapidly than females as the estimated half-lives for nPB in female rats
were roughly twice those of the male rats (in mice, however, the speed
of metabolism was not consistently different between the sexes and was
only different at 70 and 2700 ppm).  However, both male and female rats
metabolized comparable amounts of nPB when measured 6.5 hours following
exposure.  The authors estimated Vmax values (a measure of the maximum
initial rate of an enzyme-catalysed reaction) for male and female rats
of 0.227 and 0.143, respectively. In mice, the Vmax values were 0.329
for male mice and 0.234 for female mice.  Taken together, these data do
suggest the possibility that there may be sex-related differences in
metabolism in nPB.  The small numbers of animals and the exposure
methodology used do not allow strong conclusions to be drawn regarding
whether sex-based differences are expected at lower concentrations
administered over a sub-chronic or chronic duration.  Further, the fact
that both male and female animals were able to metabolize comparable
amounts of nPB (up to 2700 ppm) indicates that sex-based differences may
not be observed when exposures are limited to the concentrations and
exposure durations more typical of occupational environments.

Dermal Uptake, Distribution and Metabolism of nPB in Rats

Six male rats were administered approximately 96 mg [14C]-nPB/kg
bodyweight dermally to a shaved portion of the back (scapular area)
using a device equipped with a charcoal filter that did not prevent the
nPB from evaporating.  The rats were exposed within a metabolism chamber
for at least 48 hours.  This device thus allowed the researchers to
simulate an environment similar to that of an occupational worker.  If
the experiment had been conducted as a typical dermal toxicity
investigation, the treated area would have been occluded with plastic or
some similar non-permeable material, and the compound would not have
been able to evaporate.  The charcoal filter was intended to trap nPB;
however, concentrations of the compound in the chamber were measured at
4 and 10 ppm, at 1 and 6 hours post-dosing, respectively, thus
indicating the potential for exposure via inhalation of the volatilized
nPB.

Radioactivity levels were measured in the following media at 48 hours: 
urine, feces, air in the chamber, tissues at the dose site, carcass,
other tissues, and in the application devices, skin washes, and other
sinks.  Approximately 37% of the dose was obtained in volatiles derived
from the breathing of the rats, while 35.7% of the dose was located on
the application devices and skin washings.  A minimal 1.2% of the dose
was located in urine, and 1.7% was exhaled as [14C]-CO2.  Radioactivity
in tissues measured 0.28% of the administered dose, of which skin and
ears (0.103%) and liver (0.102%) had the highest percentages.  The study
authors measured free bromide in the plasma of the dermally-exposed rats
at 48 hours post-dosing and noted that the values were comparable to
those measured at 7 hours following iv exposure to 20 mg/kg nPB.  Using
this free bromide level, and the terminal rate constant for elimination,
the authors calculated that the free bromide concentrations reached in
plasma following dermal exposure were 27% of those following iv
exposure.  Therefore, they hypothesized that only 3-27% of the dermal
dose was absorbed relative to the iv dose and the impact of inhaling
volatilized nPB could not be quantified. 

ICF Conclusions

Based on the data from the metabolism studies, it appears that CYP
enzymes may be responsible for the majority of metabolism of nPB in mice
and also in rats.  However, in rats, glutathione conjugation is an
important clearance pathway that is susceptible to depletion even at low
exposure doses (5 mg/kg).  This finding is in contrast to what previous
researchers concluded (Jones and Walsh, 1979).  Nevertheless, the
results indicate that both CYP and glutathione conjugation are necessary
pathways for clearance, and decreasing hepatic levels of one leads to a
shift toward the other enzyme for metabolism.  

The data indicate that at very high concentrations (2700 ppm and
greater), females may be more susceptible to the adverse effects of nPB,
based on lower metabolism of the compound in female rats. But at lower
concentrations, females and males seem equally capable of clearing the
compound.

It would be useful to make conclusions regarding human metabolism of nPB
based on these studies.  One could speculate that women might not be
able to metabolize nPB as quickly as men; it is not unusual for there to
be sex-related differences in the amount of detoxification enzymes
(e.g., lesser amounts of alcohol dehydrogenase).  Case studies appear to
involve more women than men, but this is likely to be the result of more
women carrying out the work of spraying nPB-containing adhesives in
foam-cushion manufacturing facilities (Majersik et al., 2004).  These
case reports cannot be used to make broad generalizations about which
sex is more susceptible.  Data from the NTP (2003) 13-week inhalation
studies do not strongly support a finding of increased susceptibility in
female rodents.  

posure period.  Therefore, it is impossible to extrapolate the results
following a one-time acute exposure at ≥ 240 ppm to potentially
chronic exposures.  

It is reasonable to suspect that repeated dosing may result in the
depletion of cellular glutathione levels, based on reported findings in
the RTI studies, as well as the published literature on nPB.  It may
also result in saturation of an oxidative system (P450).  However,
despite the animal evidence, it is not possible to know how quickly
enzyme depletion would occur following repeat exposure, how extensive
the depletion would be, and how quickly an alternative pathway of
metabolism would be activated.  This study indicates that 80% decreases
in GSH enzyme were observed even at the lowest exposure concentrations.

The authors of the RTI report only speculate that rats may be more
susceptible to the toxicity of nPB than mice, based on the different
metabolic pathways.  They do not comment on what the results might mean
for human exposure to nPB.  

References

Barnsley EA, Grenby TH, Young L.  1966.  Biochemical Studies of Toxic
Agents.  The Metabolism of 1- and 2-Bromopropane in Rats.  Biochem. J.
100: 282-288.

Ichihara G, Yu X, Kitoh J, Asaeda N, et al.  2000.  Reproductive
Toxicity of 1-Bromopropane, a Newly Introduced Alternative to Ozone
Layer Depleting Solvents, in Male Rats.  Toxicol Sci 54: 416-423.

Ishidao T, Kunugita N, Fueta Y, Arashidani K, Hori H.  2002.  Effects of
inhaled 1-bromopropane vapor on rat metabolism.  Toxicol Lett
134:237-243.

Jones AR, Walsh DA.  1979.  The oxidative metabolism of 1-bromopropane
in the rat.  Xenobiotica.  9:763-772.

Kim Y, Park J, Moon Y.  1999.   Hematopoietic and Reproductive
Toxicity of 2-Bromopropane, a Recently Introduced Substitute for
Chlorofluorocarbons.  Toxicol. Lett. 108:309-313.

Majersik JJ, Steffans JD, Caravati EM.  2004.  Chronic Exposure to
1-Bromopropane Associated with Spastic Paraparesis and Distal
Neuropathy: A Report of Six Foam Cushion Gluers. Salt Lake City, Utah. 
A study presented October 5, 2004, at the 129th annual meeting of the
American Neurological Association in Toronto. 

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>.

Pavelka S, Babicky A, Vobecky M.  2005.  Biological half-life of bromide
in the rat depends primarily on the magnitude of sodium intake.  Physiol
Res.  (Epub ahead of print).

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al Memorandum to EnviroTech International.  7 September.

Tachizawa H, MacDonald TL, Neal RA.  1982.  Rat liver microsomal
metabolism of propyl halides.  Mol Pharmacol 22:745-751.

  Cytochrome P450 (CYP450) isozymes are similar varieties of the CYP450
enzymes.  These enzymes play an important role in the metabolism of
compounds in many organs/tissues.

 Glutathione (g-glutamylcysteinylglycine, GSH) is a sulfhydryl (-SH)
water soluble antioxidant and antitoxin. It is found in animals, plants,
and microorganisms.  Glutathione conjugation is the process by which
enzymes called glutathione S-transferases bind glutathione to compounds
in the body to increase their water solubility so that they may be
excreted. 

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