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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
publication
Title:
Metabolism and disposition of 1-bromopropane in rats and mice following inhalation or intravenous administration
Author:
Garner. C.E., et al.
Year:
2006
Bibliographic source:
Toxicology and Applied Pharmacology 215: 23–36

Materials and methods

Objective of study:
other: Metabolism and disposition
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the factors influencing the disposition and biotransformation of 1-BrP were examined in male F344 rats and B6C3F1 mice following inhalation exposure (800 ppm) or intravenous administration (5, 20, and 100 mg/kg). [1,2,3-13C]1-BrP and [1-14C]1-BrP were administered to enable characterization of urinary metabolites using NMR spectroscopy, LC–MS/MS, and HPLC coupled radiochromatography. Exhaled breath volatile organic chemicals (VOC), exhaled CO2, urine, faeces, and tissues were collected for up to 48 h post-administration for determination of radioactivity distribution.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): [1,2,3 13C] 1-BrP (Cambridge Isotope Laboratories (Andover, MA))
- Analytical purity: 98% pure
- Locations of the label (if radiolabelling): 1,2,3 13C
- Radiochemical purity (if radiolabelling): 99% 13C enrichment
- Other: 1Hdecoupled 13C NMR spectrum in D2O consisting of a doublet at 12.3 ppm, a doublet of doublets at 25.9 ppm and a doublet at 37.4 ppm


- Name of test material (as cited in study report): Neat 1-BrP (Aldrich Chemical Company (Milwaukee, WI))
- Analytical purity: 99% pure

- Name of test material (as cited in study report): [1-14C] 1-BrP (New England Nuclear (Boston, MA))
- Locations of the label (if radiolabelling): [1-14C]
- Radiochemical purity (if radiolabelling):>98%
- Specific activity (if radiolabelling): 42 mCi/mmol
Radiolabelling:
yes
Remarks:
[1,2,3 13C], [1-14C] and unlabelled 1-bromopropane

Test animals

Species:
other: rats and mice
Strain:
other: Fischer rats and B6C3F1 mice
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Inc. (Portage, MI)
- Age at study initiation: 10 - 12 weeks old
- Weight at study initiation: rats (200–250 g) and mice (25–35 g)
- Housing: Roth-type glass metabolism cages following administration
- Diet (e.g. ad libitum): Certified Purina Rodent Chow (5002) at libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64 - 79
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: inhalation or intravenous
Vehicle:
other: When dosing intravenously via the tail vein, 5% Alkamuls in normal saline
Details on exposure:
VEHICLE
- Choice of vehicle : When dosing intravenously via the tail vein, 5% Alkamuls in normal saline was used as the vehicle
- Amount of vehicle: 5 mg/kg total volume
Duration and frequency of treatment / exposure:
All doses were administered as a single dose. For the inhalation exposure in the urinary metabolite identification investigation, animals were exposed for 6 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
DISPOSITION STUDIES
[14C] 1-BrP at 5, 20 and 100 mg/kg i.v. (lateral tail vein, rats and mice)
[14C] 1-BrP 5 and 20 mg/kg i.v. (tethered jugular cannulae, rats only)

EFFECT OF GLUTATHIONE DEPLETION OR CYTOCHROME P450 ON THE DISPOSITION OF 1-BrP:
[14C] 1-BrP 20 mg/kg i.v. (tethered jugular cannulae, rats only)


URINARY METABOLITE IDENTIFICATION:
[1,2,3-13C]1-BrP 20 or 100 mg/kg i.v. (tethered jugular cannulae, rats only)
[1,2,3-13C]1-BrP 800 ppm inhalation (rats and mice)
No. of animals per sex per dose:
DISPOSITION STUDIES
No information regarding group sizes were available for this portion of the investigation

EFFECT OF GLUTATHIONE DEPLETION OR CYTOCHROME P450 ON THE DISPOSITION OF 1-BrP:
Four rats in each of three groups. Groups were defined in this portion of the study by their pre-treatment prior to dosing with 1-BrP

URINARY METABOLITE IDENTIFICATION:
Three methods of exposure were included in this investigation
Two groups of two rats dosed via intravenous infusion via tethered jugular cannulae at either 20 or 200 mg/kg
One group of two rats and two groups of four mice were exposed for 6 hours via inhalation to 1-BrP
Two rats were dosed with 100 mg/kg i.v after pretreatment.
Control animals:
not specified
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, VOC, individual tissues and whole carcass were also examined

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine
- Method type(s) for identification: HPLC and NMR

Details on sampling and analysis are presented under the field "Any other information on material and methods incl. tables". The methodology is presented under the individual investigations.
Statistics:
In cases where a test of statistical significance was necessary, Student's t test was used

Results and discussion

Main ADME resultsopen allclose all
Type:
excretion
Results:
The micromoles of bromide released relative to micromoles released via oxidation to 14CO2 in rats showed that the proportion of 1-BrP dose metabolized via oxidation relative to other bromide releasing pathways was dose dependent.
Type:
excretion
Results:
Rats and mice exhaled a majority of the administered dose as either VOC (40–72%) or 14CO2 (10–30%).

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
- Contribution of cytochrome P450 and glutathione on BrP metabolism
Hepatic levels of 1-bromopropane equivalents were also determined in rats and mice 48 h following doses of 5, 20, and 100 mg/kg and at 20 mg/kg 1-BrP following pretreatment of rats with metabolic inhibitors (Table 2). Liver to blood tissue radioactivity ratios were similar (ca. 3) regardless of dose. However, residual 1-BrP derived radioactivity was not proportional to dose. In rat, dose normalized 1-BrP ng equivalents per gram of liver decreased from 315 (ng eq/g)/(mg/kg) to 59 (ng eq/g)/(mg/kg) as nominal dose increased from 5 to 100 mg/kg. At 48 h following i.v. administration of [14C]1 -BrP at 20 mg/kg, approximately 0.6% of the dose remained in the liver. Pretreatment with ABT dropped hepatic radiochemical content nearly 10-fold, from 4.1 to 0.5 μg eq per g tissue (P < 0.05), and tissue to blood ratios were reduced three-fold to 1.0 (P < 0.05). Pretreatment with BSO reduced hepatic residues by approximately half to 2.3 μg eq/g time (P < 0.05) though liver/blood ratios were not significantly reduced. In the mouse, dose normalized 1-BrP ng equivalents per gram of liver were lower than those measured in rat. Residuals in mouse liver were decreased by half from 44 (ng eq/g)/(mg/kg) to 21 (ng eq/g)/(mg/kg) as nominal dose increased from 5 to 100 mg/kg, contrasting with a nearly seven-fold drop in rat over the same dose range.
Details on excretion:
- Disposition of radiolabel following administration of [14C]1-BrP
Disposition of radioactivity in rats and mice following intravenous administration of [14C]1-BrP is summarized in Table 1. The administration of [14C]1-BrP for all mouse studies and for the 100 mg/kg rat study was conducted such that radioactivity exhaled as VOC could be collected during the tail vein administration. For the 3 and 16 mg/kg studies, dosing was performed via the tail vein (no VOC were collected during administration) and then repeated in a subsequent study using rats implanted with jugular cannulae with dosing performed while housed in the glass metabolism cages (all VOC collected). Recovery of the administered dose was >88% for all mouse studies. For the studies in rats, the recovery of the administered dose varied from 82% to 103%, and the lower percent recoveries appear to be related to loss of exhaled radioactivity during dose administration.
For rats and mice at all doses (in the absence of metabolic inhibitors), the majority of VOC and CO2 was captured 0–4 h post-dose (time-point not shown), demonstrating that 1-BrP is rapidly cleared from the blood by exhalation. For mice, ∼39–48% of the dose recovered by 48 h was attributed to VOC and ∼19–26% as CO2 (Table 1). Radioactivity recovered in urine accounted for 13–23% of the administered dose, and only a small fraction (<4%) was retained in the carcass and tissue or in feces (<4%).
For studies in rats in which VOC were collected during dosing for up to 48 h following dose administration, 50–71% of the dose was exhaled as VOC, and 10–30% was exhaled as 14CO2 (Table 1). For all studies in rats, the percentage of the dose recovered in urine ranged from 13 to 19%, while a smaller percentage was recovered in feces (≤2%) or retained in the tissues and carcass (≤6%). There was a statistically significant decrease in the portion captured as 14CO2 as dose increased from 3.4 to 24 (P < 0.001) and then to 104 mg/kg (P < 0.001), suggesting a dose dependence in oxidation of 1-BrP to CO2. Further, VOC accounted for ca. 72% of administered dose for the 100 mg/kg group vs. ca. 50% for the other doses in which VOC were collected through dosing. The differences in the average values of the VOC and CO2 for the high and mid dose group were significantly different (P < 0.001). The drop in CO2 and concomitant increase in VOC as dose increased to 100 mg/kg reflected a dose-dependent relationship in the route of elimination and metabolism of 1-BrP in the rat. In mice, there was an apparent moderate decrease in the percentage of radioactivity excreted in urine or exhaled as 14CO2 (P < 0.05) with a increase in dose from 20 to 100 mg/kg.
Plasma bromide data were analyzed from rat studies to determine the effect of dose level on the pathway of metabolism of 1-BrP. The micromoles of bromide released relative to micromoles released via oxidation to 14CO2 in rats are shown in Fig. 1. At a dose of 6 mg/kg, the molar ratio of bromide to 14CO2 was ca. 0.90. The proportion of 1-BrP dose metabolized via oxidation relative to other bromide releasing pathways was dose dependent, since the ratio of Br- to 14CO2 increased as dose increased. At 100 mg/kg, the relative contribution of oxidation fell significantly as the mass of exhaled 14CO2 asymptotically approached an upper limit of ∼20 μmol of CO2.

- Profile of radioactivity in urine
HPLC radiochromatographic profiles of rat urine collected 0–4 h following intravenous administration of [14C]1-BrP at nominal doses of 5, 20, and 100 mg/kg are shown in Fig. 2. In the rat, 1-BrP derived urinary radioactivity was distributed among several metabolites. Parent compound, which eluted at ca. 43 min on this system, was not detected in any rat or mouse urine sample. The major metabolite peaks at 14 and 27 min coeluted with synthetic N-acetyl-S-(2-hydroxypropyl)cysteine and N-acetyl-S-propylcysteine, respectively. These metabolites have previously been described for rats exposed to 1-BrP and are proposed to be derived from conjugation of 1-BrP with glutathione. In rats, following the 5 mg/kg dose, N-acetyl-Spropylcysteine is a relatively minor component compared with earlier eluting peaks. As dose increased from 5 to 20 and then to 100 mg/kg, the relative proportion of N-acetyl-S-(2 -hydroxypropyl)cysteine and N-acetyl-S-propylcysteine shifted such that the N-acetyl-S-propylcysteine peak predominated at 100 mg/kg, accounting for more than 80% of the urinary radioactivity. Formation of N-acetyl-S-propylcysteine results in release of a bromide ion without oxidation.
HPLC radiochromatographic profiles of mouse urine collected 8–12 h following intravenous administration of [14C]1-BrP at 4, 17, and 104 mg/kg are shown in Fig. 3. In the mouse, 1-BrP derived urinary radioactivity was distributed between a single predominant metabolite, which co-eluted with synthetic N-acetyl-S-(2-hydroxypropyl)cysteine, and several lesser metabolites. The peak co-eluting with N-acetyl-Spropylcysteine at 27 min was relatively minor (<5%) regardless of dose. Mouse urinary profiles remained similar over a dose range of 5–100 mg/kg nominal dose.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
- Metabolism studies
The proton-decoupled 13C NMR spectra of control rat urine and of urine collected 0–24 h following i.v. administration of 20 and 100 mg/kg [1,2,3-13C-1-BrP are shown in Fig. 5. The carbons of compounds in control urine give rise to signals which appear as singlets because of low incidence of adjacent 13C nuclei. The spectrum of control urine (Fig. 5a) shows intense singlets at 162.5 ppm (assigned to urea) and 70.0 ppm (vehicle alkamuls) and singlets with less intensity due to sugars, hippurate, citrate, and creatine. The 13C NMR spectrum of urine from rats administered 20 and 100 mg/kg [1,2,3-13C]1-BrP (Figs. 5b and c) contain multiplets due to 13C–13C coupling between the adjacent labeled carbons of each metabolite. Chemical shifts associated with the parent compound 1-BrP, 12.4 ppm (d; Jcc = 32), 25.9 ppm (d,d; Jcc = 32, 32) and 37.4 ppm (d; Jcc = 32), were not detected. The proton-decoupled 13C NMR spectra of urine collected 0–24 h from a rat administered a single 20 mg/kg dose of [1,2,3-13C]1 -BrP following pretreatment with ABT is shown in Fig. 5d. Following treatment with ABT, the [13C]1-BrP derived metabolite peaks were reduced in number. Multiplets remaining in the 12, 22, and 26 ppm regions showed a marked increase in intensity. There was a significant increase in the intensity of singlets in the 68–76 ppm range corresponding to sugars.

The proton-decoupled 13C NMR spectra of control rat urine and of urine collected 0–24 h following gas uptake exposure to [1,2,3-13C]1-BrP (800 ppm) are shown in Figs. 6a and b, respectively. The 13C NMR spectra of urine collected following inhalation exposure were similar to that of urine following i.v. administration with the exception that signal to noise was improved significantly due to a higher concentration of metabolites. Additional multiplets in the 70 ppm range, obscured by signals from components of the dose vehicle, were revealed in spectra of urine from inhalation exposure. The proton-decoupled 13C NMR spectra of control mouse urine and of urine collected 0–24 h following gas uptake exposure to [1,2,3-13C]1-BrP (800 ppm) are shown in Figs. 6c and d, respectively. Doublets present in rat urine are present in mouse urine. However, three doublets at ca. 66.5, 39.8, and 21 ppm are the predominant signals.

INADEQUATE spectroscopy was used to correlate carbon signals in the complex mixture, allowing determination of connectivity between carbon signals that arise from each [1,2,3 -13C]1-BrP derived metabolites. Inspection of the INADEQUATE spectrum of rat urine collected following inhalation exposure suggests at least six major 3-carbon systems (Fig. 7a), while that of mouse urine suggested at least three 3-carbon systems (Fig. 7b). This information and the multiplicity and Jcc coupling constants from the 1 -dimensional spectra suggested seven structural types (Table 3) for metabolites derived from [1,2,3-13C]1-BrP. Where possible, metabolites were further characterized based on comparison of LC–MS/MS data from urine samples with those obtained for synthetic standards of biochemically feasible metabolites.

Quantitative comparisons were made using HPLC radiochromrograms. To aid in assignment of peaks associated within the radiochromatogram, urine from [1,2,3-13C]1-BrP treated rats was mixed with urine collected from animals dosed with 20 mg/kg [14C]1-BrP (5:1, v:v) and concentrated five-fold under a gentle stream of nitrogen. Aliquots of the urine containing 14C and 13C labeled 1-BrP metabolites were injected onto the HPLC system and eluent fractions collected, pooled, and concentrated, and then analyzed with 13C NMR and MS/MS. Plots of radioactivity collected vs. time following fraction collection contained seven major metabolite fractions. Five of the seven metabolite types determined by NMR could be definitively associated with individual metabolite fractions.

- Metabolites of structure R-S–CH2CH2CH3
The INADEQUATE spectra of rat and mouse urine following inhalation exposure (Figs. 7a and b, respectively) have carbon peaks aligned along the same double quantum frequency (y-axis) between 33 ppm and 22 ppm and between 22 ppm and 12 ppm. The 1D spectrum of urine for all 13C-1 -BrP exposures (Table 3, Figs. 5 and 6) contain two doublets at 33.4 ppm (Jcc = 34 Hz) and 12 ppm (Jcc = 34 Hz), and a doublet of doublets is at 22.9 ppm (Jcc = 34, 34 Hz). The chemical shifts and coupling constant of 34 Hz are consistent with 3 contiguous sp3 carbons. These shifts are consistent with those calculated for the structure type R–S–CH2CH2CH3, where R is a functional group such as a cysteine, cysteine-glycine, or N-acetylcysteine. 13C NMR studies of the labeled compounds afford detection of the chemical shift of the enriched carbons which can be used to discern the nature of the conjugate based on known substituent effects. Further delineation of the RS substituent was obtained through isolation and MS identification.

LC–MS/MS spectra in the positive and negative polarity modes showed peaks with m/z ratios of 209 and 207. Based on molecular weight, fragmentation pattern, and comparison with a synthetic standard, a R-S-CH2CH2CH3 structure was definitively assigned as N-acetyl-S-propylcysteine, where R is N-acetylcysteine. The 13C NMR spectrum obtained for the urine fractions eluting at 32–34 min indicated the presence of this Type A metabolite, and MS/MS analysis of this fraction supported this assignment.

Based on integration of peaks in the NMR spectrum acquired so that the integrated areas of the 13C resonance are quantitative (30-s delay and decoupling gated off during the delay) of 0–24 urine following 800 ppm inhalation exposure to rats, this metabolite-type contributes to approximately 37% of the total excreted metabolites in rat.

- Metabolites of structure R-S(O)-CH2CH2CH3
Another 3-carbon system has chemical shifts with connectivity between 53.2 ppm and 15.4 ppm and between 15.4 and 12.0 ppm. Inspection of the 1D spectra shows evidence of connectivity with doublets present at 53.2 ppm (Jcc = 34 Hz) and 12.0 ppm (Jcc = 35 Hz) and a doublet of doublets at 15.5 ppm (Jcc = 35, 34 Hz). These coupling constants are consistent with a 3 contiguous carbon sp3 system. Comparisons between the measured chemical shifts and those calculated for plausible metabolites suggest the structure R–S(O)–CH2CH2CH3, where R is N-acetylcysteine. Additional signals are present near 12, 15, and 52 ppm with approximately half the intensity of the signals from metabolite B. These signals are designated B' and are indicative of the presence of an additional metabolite with a similar structure to that of type B. This is suggestive of a system containing two chiral centers (diastereomers) or could be derived from variations in the “RS” substituent (e.g., plausible differences between cysteine, Nacetylcysteine, and cysteine). This metabolite is formed following 100 mg/kg i.v. and inhalation exposure but is relatively minor at the lower i.v. dose (20 mg/kg).

LC–MS/MS analysis of urine in the positive and negative polarity modes showed peaks with m/z ratios of 225.0 and 222.8, respectively. Further inspection of the LC–MS spectrum showed a fragment with m/z ratio of 162.9 for the N-acetyl cysteine fragment. Based on molecular weight, fragmentation pattern, a R-S(O)-CH2CH2CH3 structure was definitively assigned as N-acetyl-3-[(propylsulfinyl)alanine, where R is Nacetylcysteine. The 13C NMR spectrum of the urine fraction eluting at 14–17 min contained signals consistent with this metabolite, and MS/MS data for this fraction further confirmed the assignment.

Based on integration of peaks in the NMR spectrum of 0–24 urine following 800 ppm inhalation exposure to rats, this metabolite-type contributes to approximately 5% of the total excreted metabolites in rat.

- Metabolites of structure X-CH2CH(OH)CH3
The third 3-carbon system has chemical shifts with connectivity between 39.8 ppm and 66.5 ppm and between 66.5 and 21.0 ppm. The doublets at 39.8 ppm (Jcc = 39 Hz) and 21.0 ppm with long-range coupling (Jcc = 39, 6.9 Hz) and the doublet of doublets at 66.5 ppm (Jcc = 39, 39 Hz) observed within the 1D spectra further support their connectivity. The coupling constants are consistent with three contiguous sp3 carbons containing a secondary alcohol and are assigned to metabolite of the structure X-CH2CH(OH)CH3 based on comparisons to calculated chemical shift values. Calculated chemical shifts of the structures N-acetyl cysteine-S-CH2CH(OH)CH3 and Br-CH2CH(OH)CH3 are similar; both being consistent with the observed shifts. Additional signals are present near 66, 40, and 12 ppm with approximately half the intensity of the CH2 signal from metabolite type C and are designated C′. These signals are suggestive of a system containing two chiral centers (diastereomers) such as R-S-CH2CH(OH)CH3. However, it is also plausible that both the Br- and RS-moities exist requiring further isolation and MS identification.

LC–MS spectrum in the positive polarity modes showed a peak with m/z ratio of 225. Based on molecular weight, fragmentation pattern, and comparison with a synthetic standard the X-CH2CH(OH)CH3 structure was definitively assigned as N-acetyl-S-(2-hydroxypropyl)cysteine, where X is N-acetylcysteine. Signals consistent with this metabolite were located in the NMR data obtained for the fraction collected at 19–21 min.

Based on integration of peaks in the NMR spectrum of 0–24 urine following 800 ppm inhalation exposure to rats, this metabolite-type contributes to approximately 16% of the total excreted metabolites in rat.

- Metabolites with X-CH2C(O)CH3 structure
An additional 3-carbon system is observed with connectivity between chemical shifts at 41.9 ppm and 209.3 ppm and between 209.3 ppm and 27.7 ppm. The doublets at 41.9 ppm (Jcc = 40 Hz) and 209.3 ppm (Jcc = 41 Hz) and the doublet of doublets at 209.3 ppm (Jcc = 39, 39 Hz) observed within the 1D spectra further support their connectivity. Coupling constants of 39 and 40 Hz are consistent with connected sp3 and sp2 carbons. The chemical shift of the central carbon 209.3 ppm is distinctive of a ketone. Shift calculations fit the base structure of XCH2C(O)CH3 where X can be Br or a RS group. Additional signals present near 42 ppm, 209 ppm, and 28 ppm with approximately equal intensity of the signals from metabolite type D (designated D') suggest the presence of additional metabolites that have slightly different shielding environments for the carbons similar to that of metabolite type D. Since the halogenated 2-oxopropyl metabolite could not yield diastereomers, the assignment is most likely attributed to a variation in the R group. LC–MS/MS analysis of urine in the positive polarity mode showed peak with m/z ratio 223.2 and fragmentation pattern consistent with the assignment of N acetyl-S-(2-oxopropyl)cysteine. Signals associated with metabolite D were located in the urine fraction eluting at 19–21 min. This fraction 5 also contained signals for metabolite C, where the ratio of C/D metabolites was ∼1.5:1. The LC\\MS/MS data did not indicate the presence of bromine.

Based on integration of peaks in the NMR spectrum acquired from 0 to 24 urine following 800 ppm inhalation exposure to rats, this metabolite-type contributes to approximately 12% of the total excreted metabolites.

- Metabolites of structure X-CH2CH(OR)CH3
The fifth 3-carbon system has chemical shifts with connectivity between 36.5 ppm and 74.5 ppm and between 74.5 ppm and 19.2 ppm. The doublets at 36.5 ppm (Jcc = 41 Hz) and 19.2 ppm (Jcc = 40 Hz) and the doublet of doublets at 74.5 ppm (Jcc = 40, 40 Hz) observed within the 1D spectra further support their connectivity. The coupling constants are consistent with a contiguous system of sp2 and sp3 carbons or three sp3 carbons containing electron-withdrawing substituents such as a hydroxyl group. The chemical shift of the center carbons is more consistent with the latter and is assigned to metabolite of the structure X-CH2CH(OR)CH3 based on comparisons to calculated chemical shifts (R = alkyl). Calculated shifts for both R-S-CH2CH(OR)CH3 and Br–CH2CH(OR)CH3 are consistent with NMR shift values LC–MS/MS analysis of urine in the positive polarity modes showed equally abundant peaks with m/z ratios of 315.8 and 318.0, a molecular weight consistent with a glucuronide conjugate of Br-CH2CH(OH)CH3 and also a fragment with m/z ratio of 235.9, consistent with loss of bromine. These data are consistent with the assignment of 1-bromo-2-propanol-glucuronide. 13C NMR signals for this metabolite are associated with the fraction eluting at 13–15 min, overlapping with metabolite type B.

Based on integration of peaks in the NMR spectrum acquired so that integration is quantitative of 0–24 urine following 800 ppm inhalation exposure to rats, this metabolite-type contributes to approximately 9% of the total excreted metabolites.

- Metabolites of structure R-S(O)-CH=C(OH)CH3
The final 3-carbon system has chemical shifts with connectivity between 20.6 ppm and 82.8 ppm and between 82.8 ppm and 151.5. Connectivity is further confirmed by inspection of the 1D spectra which show doublets at 82.8 ppm (Jcc = 71 Hz) and 20.6 ppm (Jcc = 43 Hz) and the doublet of doublets at 151.5 ppm (Jcc = 72, 43 Hz). The distinctive vinylic chemical shifts and the coupling constants of 43 and 71 Hz suggest a sp2–sp2–sp3 system. The spectrum is consistent with a secondary propeneol resulting from the enolization of a methyl ketone. Calculated chemical shifts (154 ppm, 81 ppm and 23 ppm) for the enol structure are consistent with the observed chemical shifts. Reported 13C NMR spectral data of phosphoenol structures give similar chemical shift patterns. The additional signals present near 21 ppm, 151 ppm, and 82 ppm with approximately equal intensity of the signals from metabolite type F (designated F') suggest the presence of an additional metabolite that has a slightly different shielding environment for the carbons similar to that of metabolite type F. This could suggest a compound containing 2 chiral centers (diastereomers). This would be expected in the case of S-oxide. Overall, the data could be interpreted to support assignment of the metabolite F structure as R-S(O)-CH=C(OH)CH3, the enol form of N-acetyl-3-[(2-oxopropyl)sulfinyl]alanine. 13C NMR signals consistent with this metabolite were detected in the fraction eluting at 8–10 min, together with additional [1,2,3-13C]1-BrP derived signals that were not detected directly in the urine. It is possible that the method of fractionation and concentration resulted in some structural modifications.

- Other possible metabolites
Inspection of the 1D spectra show several small doublets that are suggestive of additional 1-BrP metabolites but do not appear in the INADEQUATE spectra due to low signal to noise ratio. In 13C spectra generated from urine following inhalation exposure are a doublet of doublets at 68 ppm (Jcc = 40, 40 Hz) matched in intensity to a doublet at 31 ppm (Jcc = 40 Hz) and a doublet at 21 ppm (Jcc = 40 Hz). These chemical shifts are consistent with the structure HSCH2CH(OH)CH3 and are supported by calculated chemical shifts for this structure, and these resonances are tentatively assigned to this structure. Within the liver homogenate spectrum is a doublet of doublets at 74 ppm matched in intensity to a doublet at 35.3 ppm (Jcc = 37 Hz) and a doublet at 64.3 ppm (Jcc = 37 Hz) (data not shown). These chemical shifts are consistent with the structure BrCH2CH(OH)CH2OH are supported by calculated chemical shifts for this structure, and these resonances are tentatively assigned to this structure.

- Contribution of cytochrome P450 and glutathione on BrP metabolism
ABT pretreatment significantly changed the proportion of radioactivity appearing in urine, VOC, and CO2 (Table 1), indicating that reduction in P450 content had a significant impact on 1-BrP metabolism. However, BSO pretreatment did not significantly affect 1-BrP disposition. ABT pretreatment resulted in an ∼80% reduction in 14CO2 excretion and a ∼30% reduction in urine radioactivity while exhaled volatile organics increased ∼52%. The profiles of [14C]1-BrP-derived radioactivity in urine following pretreatment with BSO or ABT are shown in Fig. 4. Following treatment with ABT, urine metabolites were reduced in number to a single major metabolite, N-acetyl-S-propylcysteine, which accounted for >90% of the total radioactivity, and one other metabolite. The urinary metabolite profile of rats pretreated with BSO did not show the marked changes seen in the ABT group, though the level of N-acetyl-S-propylcysteine was lowered moderately with a concomitant increase in metabolites shown to be eliminated by ABT pretreatment.

Any other information on results incl. tables

Table 1 Disposition of [1-14C] 1-BrP derived radioactivity following intravenous administration of 1-BrP to rats and mice

Species/Dose (mg/kg)

Carcass

Urine

Feces

CO2

VOC

% Dose recovered

Rata

3.4

6.1 ± 1.5

17.1 ± 1.6

1.9 ± 1.1

27.6 ± 3.7

25.2 ± 2.0

82.7 ± 8.6

15.7

5.9 ± 1.4

19.2 ± 3.1

2.1 ± 0.9

31.1 ± 4.5

31.6 ± 5.1

95.0 ± 11.7

94.1

1.9 ± 1.8

13.3 ± 0.8

0.4 ± 0.1

9.7 ± 0.4

71.0 ± 1.7b

96.2 ± 1.3

Ratc

5.9

5.4 ± 0.6

16.9 ± 0.8

0.8 ± 0.2

27.7 ± 2.3

52.3 ± 2.1

103 ± 4.6

24

3.3 ± 0.2

14.0 ± 1.1

0.6 ± 0.2

19.2 ± 1.1

49.9 ± 3.8

87 ± 0.9

Moused

3.7

3.7 ± 1.2

23.4 ± 6.4

2.9 ± 1.1

22.1 ± 12.0

44.7 ± 9.6

96.9 ± 20.9

17.4

1.9 ± 0.52

18.9 ± 6.2

3.7 ± 1.7

25.6 ± 4.8

39.3 ± 10.1

94.1 ± 9.8

10.4

3.9 ± 2.5

14.3 ± 6.6

3.4 ± 2.6

19.3 ± 4.1

48.2 ± 5.4

89.0 ± 13.8

Rat

ABT pretreatment + 22.0

0.8 ± 0.1

9.8 ± 0.9

0.2 ± 0.1

4.0 ± 0.3

7.6 ± 4.1

90.5 ± 5.2

BSO pretreatment + 24.2

2.0 ± 0.2

13.8 ± 0.8

0.4 ± 0.1

18 ± 0.3

51.7 ± 1.7

86.2 ± 2.2

aDoses delivered via tail vein.

bDoses delivered via tail vein while expired air collected. VOC trapped during iv administration represent 82.3 ± 2.9% of total volatiles.

cDoses delivered via jugular cannula while animal enclosed in metabolism chamber.

dDoses delivered via tail vein while expired are collected.

 

Table 2 Residual radioactivity retained in liver following administration of 1-BrP to rats and mice

Species/Dose (mg/kg)

1-BrP ng eq per gram tissue

Rata

3.4

1100 ± 626

15.7

2801 ± 1243

100

7003 ± 1065

Ratb

5.9

1575 ± 557

24

4072 ± 1107

Mousea

3.7

164 ± 25.9

17.4

592 ± 245

10.4

2161 ± 448

Ratb

ABT pretreatment + 22.0

461 ± 30

BSO pretreatment + 24.2

2380 ± 171

aDoses delivered via tail vein

bDoses delivered via jugular cannula while animal enclosed in metabolism chamber

 

Table 313C NMR chemical shifts, carbon-carbon coupling constants, carbon-carbon correlation for signals derived from 1-bromopropane in rats and mice administered [1, 2, 3-13C] 1-bromopropane

Matrix

Metabolite typea

Multiplicityb

Jccb

Measured ppm

Predictedcppm

Inadequate Connectivityd

Assignment

Urine

A1

d

33

3.9

34.5

N-Acetyl-S-propylcysteine

A2

d, d

34, 34,

21.94

22.9

A3

A3

d

35

12.33

14

A2

Urine

B1

d

34

52.85

53

B2

N-Acetyl-S-propylcysteine-S-oxide

B2

d, d

34, 34

15.42

2

B1

B3

d

35

12.02

16.3

Urine

B1'

d

34

52.89

14

B2'

d, d

34, 34

15.52

B3'

d

35

12

Urine

C1

d

39

39.77

35.2

C2

N-acetyl-S-(2-hydroxypropyl)cyteine

C2

d, d

39, 39

66.5

64.4

C3, C1

C3

d

39

20.95

22.8

C2

Urine

C1'

d

39

39.75

C2'

d, d

39, 39

66.25

C3'

d

39

20.89

Urine

D1

d

40

41.98

41.9

D2

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

D2

d, d

39, 39

209.3

206

D3, D1

D3

d

41

27.77

29

D2

Urine

D1'

d

40

41.86

D2'

d, d

39, 39

209.36

D3'

d

41

27.65

Urine

E1

d

41

36.46

36

1-bromo-2-hydroxypropane-glucuronide

E2

d, d

40, 40

74.46

72

E3

E3

d

40

19.15

21

E2

Urine

F1

d (lr)

71 (5)

82.84

82

F2

N-acetyl-3-[(2-oxopropyl)sulfinyl]alanine

F2

d, d (lr)

72, 43 (5)

151.5

160

F1, F3

F3

d

43 (5)

20.61

21

F2

Urine

F1'

d (lr)

71 (5)

82.87

F2'

d, d (lr)

72, 45 (5)

151.7

F3'

d

43 (5)

20.58

Urine

G1

d

40

31

32.5

G2

d, d

40, 40

68

68.6

G3

d

40

21

21.4

Liver

H1

d

37

35.3

35.2

H2

d, d

37, 37

74

71.5

H3

d

37

64.33

65.6

aMetabolite structure type A: R-S-CH2CH2CH3B: R-S(O)-CH2CH2CH3C: R-S-CH2CHOHCH3D: R-S-CH2C(O)CH3E: Br-CH2CH(Ogluc)CH3F: R-S(O)-CH=CHOHCH3G: HS-CH2CHOHCH3H: Br-CH2CHOHCH2OH. Structures A-H are proposed from the connectivity, multiplicity and chemical shift data. Assignments were based on these data and comparison with calculated and chemical shift values for biochemically feasible metabolites.

bMultiplicities and carbon-carbon coupling constants were measured from the ID spectrum

cPredicted chemical shifts were determined with ACD/CNMR Predictor, ACD/Labs. ACD/CNMR Predictor version 7.03, Advanced Chemistry Development Inc., Toronto ON, Canada www.acdlabs.com 2003

dCarbon connectivities were obtained using INADEQUATE spectroscopy. The carbon atom is assigned a letter corresponding to the metabolite and a carbon position number that designates its derivation from 1-bromopropane.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
The metabolism and disposition of 1-BrP was investigated in rats and mice. In rats, but not mice, the routes of disposition and pathways metabolism of 14C-1-BrP were found to differ at high (100 mg/kg) vs. low doses (20 mg/kg and below). The metabolites present in the urine of rats and mice were profiled by 13C NMR and mass spectra. The introduction of inhibitors demonstrated that oxidation of 1-BrP via cytochrome P450 at the C2 position is preferred relative to the C1 and C3 carbons. The data suggest that the principal metabolite of 1-BrP, 1-bromo-2-hydroxypropanol is conjugated with glutathione or glucuronic acid or further metabolized.
Executive summary:

In this study, the factors influencing the disposition and biotransformation of 1-BrP were examined in male F344 rats and B6C3F1 mice following inhalation exposure (800 ppm) or intravenous administration (5, 20, and 100 mg/kg). [1,2,3-13C]1-BrP and [1-14C]1-BrP were administered to enable characterization of urinary metabolites using NMR spectroscopy, LC–MS/MS, and HPLC coupled radiochromatography. Exhaled breath volatile organic chemicals (VOC), exhaled CO2, urine, feces, and tissues were collected for up to 48 h post-administration for determination of radioactivity distribution. Rats and mice exhaled a majority of the administered dose as either VOC (40–72%) or 14CO2 (10–30%). For rats, but not mice, the percentage of the dose exhaled as VOC increased between the mid (∼50%) and high (∼71%) dose groups; while the percentage of the dose exhaled as 14CO2 decreased (19 to 10%). The molar ratio of exhaled 14CO2 to total released bromide, which decreased as dose increased, demonstrated that the proportion of 1-BrP metabolized via oxidation relative to pathways dependent on glutathione conjugation is inversely proportional to dose in the rat. [14C]1-BrP equivalents were recovered in urine (13–17%, rats; 14–23% mice), feces (<2%), or retained in the tissues and carcass (<6%) of rats and mice administered i.v. 5 to 100 mg/kg [14C]1-BrP. Metabolites characterized in urine of rats and mice include N-acetyl-S-propylcysteine, N-acetyl-3-(propylsulfinyl)alanine, N-acetyl-S-(2-hydroxypropyl)cysteine, 1-bromo-2-hydroxypropane-O-glucuronide, N-acetyl-S-(2-oxopropyl)cysteine, and N-acetyl-3-[(2-oxopropyl)sulfinyl]alanine. These metabolites may be formed following oxidation of 1 - bromopropane to 1-bromo-2-propanol and bromoacetone and following subsequent glutathione conjugation with either of these compounds. Rats pretreated with 1-aminobenzotriazole (ABT), a potent inhibitor of P450 excreted less in urine (↓30%), exhaled as 14CO2 (↓80%), or retained in liver (↓90%), with a concomitant increase in radioactivity expired as VOC (↑52%). Following ABT pretreatment, rat urinary metabolites were reduced in number from 10 to 1, N-acetyl-S-propylcysteine, which accounted for >90% of the total urinary radioactivity in ABT pretreated rats.

Together, these data demonstrate a role for cytochrome P450 and glutathione in the dose-dependent metabolism and disposition of 1-BrP in the rat.