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Toxicological information

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:
January 10, 2000-May 17, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented study conducted to GLP requirements.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2001

Materials and methods

Objective of study:
other: To compare the metabolism of acrylamide administered orally (po), dermally, intraperitoneally (ip), and by inhalation, and to measure the haemoglobin adducts produced.
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to compare the metabolism of AM administered orally (po), dermally, intraperitoneally (ip), or by inhalation, and to measure the hemoglobin adducts produced in rat exposed to 1,2,3-13C and 2,3- 14C Acrylamide
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Acrylamide
EC Number:
201-173-7
EC Name:
Acrylamide
Cas Number:
79-06-1
Molecular formula:
C3H5NO
IUPAC Name:
prop-2-enamide
Details on test material:
- Name of test material (as cited in study report): Acrylamide - Analytical purity: 99.9%
- Radiochemical purity (if radiolabelling): 99%
- Specific activity (if radiolabelling): 5 mCi/mmol
- Locations of the label (if radiolabelling): [1,2,3-13C] AM and [2,3-14C]AM
- Supplier of radiochemical: American Radiolabeled Chemical, Inc. (St.Louis, MO).
Radiolabelling:
yes
Remarks:
C13 and C14

Test animals

Species:
other: rats and mice
Strain:
other: F344 rats and B6C3F1 mice
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC)
- Age at study initiation: 9–10 weeks old
- Weight at study initiation: 212–237 g for the [14C]AM dermal study, 195–209 g for the 13C dermal study, and 197–206 g for the ip study 205 and 217 g for rats and between 26 and 34 g for mice for inhalation.
- Fasting period before study: no data
- Housing: micro-isolator cages containing Alpha Dri direct contact bedding.
- Individual metabolism cages: yes: The rats were transferred to glass metabolism cages immediately after dosing.
- Diet (e.g. ad libitum): NIH-07 diet, Ziegler Brothers
- Water (e.g. ad libitum): reverse-osmosis water
- Acclimation period: at least 13 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64–79°F
- Humidity (%): 30–70%.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-h light-dark cycle (0700–1900 h for light phase)

Administration / exposure

Route of administration:
other: Oral, Dermal, IP, Inhalation
Vehicle:
water
Details on exposure:
Three separate groups of exposures were conducted:
(1) dermal, po, and ip administration of [1,2,3-13C]AM to rats,
(2) dermal administration of [2,3-14C] AM to rats,
(3) inhalation exposure of rats and mice to a mixture of [1,2,3-13C]AM and [2,3-14C] AM.
TEST SITE
- Area of exposure: 5 x 10 cm
- Type of wrap if used: Hilltop Chamber™ (2.5 cm2)
REMOVAL OF TEST SUBSTANCE
- Washing (if done):
- Time after start of exposure:
TEST MATERIAL
- Amount(s) applied: 138±1.49 mg/kg; 378±14.0 µmol [1,2,3-13C]AM or 162±2.70 mg/kg; 507±-24 µmol [14C]AM/AM
- Concentration: A 53 mg/g dosing solution of [14C]AM/AM and a 48 mg/g dosing solution of [1,2,3-13C]AM
USE OF RESTRAINERS FOR PREVENTING INGESTION: No
TYPE OF INHALATION EXPOSURE: nose only
GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: Cannon nose-only tower (Cannon et al., 1983).
- System of generating aerosols: Acrylamide vapour was generated from solid acrylamide in a glass J-tube heated to 75°C in a temperature controlled water bath. Heated nitrogen was passed through the J-tube, and the resulting acrylamide vapour passed through a column of glass beads. Heated oxygen was passed through an impinger containing water, and was mixed with the acrylamide vapour in nitrogen in a mixing tee.
- Concentration of test material in vehicle:A mixture of [1,2,3-13C]AM (90%) and [2,3-14C]AM (10%) was prepared with a specific activity of 568.65 µCi/mmol
Duration and frequency of treatment / exposure:
One for oral and IP
3 x 24h for dermal
6h for inhalation
Doses / concentrations
Remarks:
Doses / Concentrations:
Ip dose: 50 mg/kg
Oral dose: nominal of 50mg/kg, administrated dose 59.5±8.0 mg/kg
Dermal application: 150 mg/kg
Inhalation exposure: the maximum achievable nose-only concentration was 5.6 ppm for unlabeled AM and 2.9 ppm for the mixture of 13C- and 14C-labeled AM.
No. of animals per sex per dose / concentration:
4 male rats for the IP oral and dermal routes
8 male rats and 8 male mice for inhalation
Control animals:
no
Positive control reference chemical:
no
Details on study design:
- Dose selection rationale: An ip dose of 50 mg/kg was selected to facilitate direct comparison with results obtained following a 50 mg/kg gavage dose of [1,2,3-13C]AM to male F344 rats (Sumner et al.,1992). A 150 mg/kg dermal application was selected based on in vitro studies indicating that up to 30% of applied AM is absorbed (Marty, 1998). The maximum achievable nose-only inhalation exposure concentration was 5.6 ppm for unlabeled AM and 2.9 ppm for the mixture of 13C- and 14C-labeled AM. Previous studies with rats or mice administered [14C]AM (Hashimoto and Aldridge, 1970; Miller et al., 1982) or [13C]AM (Sumner et al., 1992, 1997, 1999) indicated that 40 to 70% of the dose was excreted in urine within 24 h following exposure.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Following inhalation exposure, dermal application, or ip administration, four male rats and four male mice (inhalation only) were placed in all-glass metabolism cages (1/cage). Air was drawn through the metabolism cage under negative pressure and passed through charcoal filters on exiting the cage.
Urine (over dry ice) and faeces were collected for 0–24 h. Exhaled volatiles and 14CO2 (1.0 M potassium hydroxide traps) were collected 0–2, 2–6, and 6–24 h following administration of [14C]AM. At 24 h after [14C]AM dermal application or inhalation exposure, rodents were sacrificed for the collection of blood (CO2, cardiac puncture) and lungs, abdominal fat, subcutaneous fat, thymus (dermal study only), spleen, liver, testes, epididymis, kidneys, brain, stomach, intestines, skin (site of application, dermal study only), skin (non-dose site, dermal study only), skin (inhalation study), and carcass. Blood was also collected from rats exposed to [13C]AM. The blood was centrifuged to prepare washed red blood cells for hemoglobin adduct analysis. All samples were stored at –20°C with the exception of urine which was stored at –80°C. Urine volumes (0–24 h) were 6 to 7 ml (rats, [14C]AM, dermal), 6–9 ml (rats, inhalation, [13C 14C]AM), 1 to 2 ml (mice,inhalation, [13C 14C]AM), 8–11 ml (rats, ip, [13C]AM), and 11–14 ml (rats,dermal, [13C]AM).
Distribution: Red blood cells were separated from plasma by centrifugation at 2000 x g for 20 min. Tissues, blood, plasma, red blood cells, and faeces (softened with 1% Triton X-100) were digested in tetraethyl ammonium hydroxide (TEAH), and aliquots were neutralized with concentrated HCl and decolorized with hydrogen peroxide (30% H2O2). Total radioactivity was determined using a Packard 1900 CA Tricarb LA Analyzer after addition of liquid scintillation fluid (EcoLume™, ICN, CA). Aliquots of the urine, KOH traps, and cage and nose-only tube washes were analyzed directly by scintillation counting after addition of scintillation fluid. Exhaled volatile [14C]AM equivalents were extracted from charcoal traps using N,N-dimethylformamide (DMF), and aliquots of the extracts were analyzed by scintillation counting.
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: blood (CO2, cardiac puncture) and lungs, abdominal fat, subcutaneous fat,
thymus (dermal study only), spleen, liver, testes, epididymis, kidneys, brain, stomach, intestines, skin (site of application, dermal study only), skin (non-dose site, dermal study only), skin (inhalation study), and carcass. Urine (over dry ice) and feces were collected for 0–24 h. Exhaled volatiles and 14CO2 (1.0 M potassium hydroxide traps) were collected 0–2, 2–6, and 6–24 h following administration of [14C]AM.
- Method type(s) for identification: N-(2-carbamoylethyl)valine (AAVal) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal), formed by reaction of AM and GA respectively with the N-terminal valine residue in hemoglobin, were measured by an LC-MS/MS method. Analysis was conducted using a PE Series 200 HPLC system interfaced to a PE Sciex API 3000 LC-MS with a Turbo-ionspray interface. Chromatography was conducted on a Phenomenex Luna Phenyl-Hexyl Column (50 mm x 2 mm, 3µm) eluted with 0.1% acetic acid in water and methanol at a flow rate of 350µl/min, with a gradient of 45–55% methanol in 2.1 min. The elution of adducts was monitored by Multiple Reaction Monitoring (MRM) in the negative ion mode. Quantitation of AAVal was conducted using the ratio of analyte to internal standard, with a calibration curve generated using AAVal-leu-anilide. Quantitation of GAVal was conducted using the ratio of analyte to internal standard. For samples in which rodents are administered a single dose of [1,2,3–13C]AM to track its metabolism using 13C NMR spectroscopy, the 13C AAVal and 13C GAVal can be distinguished in the negative ion mode from the natural abundance analyte and labeled internal standard since the 13C-containing adduct side chain is lost from the adduct PTH in the collision cell.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled blood (CO2, cardiac puncture) and lungs, abdominal fat, subcutaneous fat,
thymus (dermal study only), spleen, liver, testes, epididymis, kidneys, brain, stomach, intestines,
skin (site of application, dermal study only), skin (nondose site, dermal study only), skin (inhalation study), and carcass.
Urine (over dry ice) and feces were collected for 0–24 h.
Exhaled volatiles and 14CO2 (1.0 M potassium hydroxide traps) were collected 0–2, 2–6, and 6–24 h following administration of [14C]AM.

- From how many animals: (samples pooled or not)
- Method type(s) for identification N-(2-carbamoylethyl)valine (AAVal) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal), formed by reaction of AM and GA respectively with the N-terminal valine residue in hemoglobin, were measured by an LC-MS/MS method
Analysis was conducted using a PE Series 200 HPLC system interfaced to a PE Sciex API 3000 LC-MS with a Turboionspray
interface.
Chromatography was conducted on a Phenomenex Luna Phenyl-Hexyl Column (50 mm x 2 mm, 3µm) eluted with 0.1% acetic acid in water and methanol at a flow rate of 350µl/min, with a gradient of 45–55% methanol in 2.1 min.
The elution of adducts was monitored by Multiple Reaction Monitoring (MRM) in the negative ion mode
Quantitation of AAVal was conducted using the ratio of analyte to internal standard, with a calibration curve generated using AAVal-leu-anilide.
Quantitation of GAVal was conducted using the ratio of analyte to internal standard.
For samples in which rodents are administered a single dose of [1,2,3–13C]AM to track its metabolism using 13C NMR spectroscopy,
the 13C AAVal and 13C GAVal can be distinguished in the negative ion mode from the natural abundance analyte and labeled internal standard since the 13C-containing adduct side chain is lost from the adduct PTH in the collision cell.


Statistics:
Statistical analysis was conducted using Instat 2.01 (Graphpad Software, San Diego, CA). Evaluation of species differences in the extent of GA derived metabolites, AAVal, and GAVal, was conducted with Student’s t-test. For evaluation of differences resulting from route of exposure, comparisons of AAVal and GAVal were only made where the dose administered was the same, i.e., with ip and gavage administration of 50 mg acrylamide/kg, with inhalation exposure at 0 and 24 h after exposure, and with 2 dermal administration studies. Differences in GAVal:AAVal ratio were compared across all treatments with ANOVA using a Tukey Kramer multiple comparisons test for all pairwise comparisons.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Following dermal application of 162 mg/kg 14C[AM], the amount of the applied dose that was absorbed was 14, 15, 27, and 30% for the four rats.
Details on distribution in tissues:
DERMAL EXPOSURE: Following the 24-h dermal application of [14C]AM, blood cells had the highest relative level (1 µmol/g tissue) of radioactivity (excluding skin at the dose site) compared with all other tissues. The skin (non-dose site, 0.4 µmol/g tissue) and liver, spleen, testes, and kidney (0.3 µmol/g tissue) had nearly the same levels. Radioactivity (0.2 µmol/g tissue) was also recovered in the lungs, thymus, brain, and epididymis. Low levels (0.05 µmol/g tissue) of radioactivity were recovered in fat.
Inhalation: The total [14C]AM equivalents recovered from male rats (89±8.9 µmol/kg body weight) were at least 2.8 times lower than [14C]AM equivalents recovered from male mice (245±45 and 401±102µmol/kg body weight at 0 and 24 h following exposure, respectively). For rats, the radioactivity (µmol) recovered immediately following exposure was not markedly different from that recovered 24 h following exposure. For mice, the average µmol recovered immediately following exposure (8±1.1 µmol) was not significantly different from the average µmol recovered 24 h following exposure (11±2.7 µmol). The total recovered AM-equivalents 24 h following a 2.9 ppm inhalation exposure to AM (19µmol) was 6 times lower that the radioactivity recovered following a 162 mg/kg (112 µmol) dermal application of AM.
INHALATION EXPOSURE:
Rats : Immediately or 24 h following the 6-h inhalation exposure to 2.9 ppm AM, blood cells of rats had the highest relative level (0.1 µmol/g tissue) of radioactivity compared with all other tissues. Plasma levels were higher immediately following exposure termination (0.03 µmol/g tissue) and reduced 24 h later (0.004 µmol/g tissue). The rank order of relative (µg/g tissue) radioactivity immediately following exposure was blood > testes > skin > liver > kidneys > brain > spleen > lung > epididymis. After 24 h, the rank order of radioactivity was blood > skin > spleen > lung > liver > kidney > brain > testes > epididymis > fat. Lowest radioactivity levels were observed for fat at either time point.
Mice: the rank order of relative radioactivity immediately after exposure was testes, skin, liver, kidney, epididymis, brain, lung, blood, and fat. After 24 h, the rank order was skin, subcutaneous fat, testes, blood, epididymis, liver, lung, spleen, brain, abdominal fat, and kidney.
Transfer into organsopen allclose all
Test no.:
#1
Transfer type:
other: dermal
Observation:
distinct transfer
Test no.:
#2
Transfer type:
other: inhalation
Observation:
distinct transfer
Details on excretion:
DERMAL EXPOSURE: The major portion of the dose was excreted in 0–24 h urine (8% of the applied dose or 36% of total absorbed dose) or remained in the body (53% of the absorbed dose) following the 24-h AM-dermal application. A minor portion of the absorbed dose was recovered in faeces (< 1%) or eliminated as organic volatiles (1%) or 14CO2 (2%).
INHALATION EXPOSURE: For rats, the major portion of the inhaled dose was excreted in urine (31% of total absorbed dose) or remained in the body (56%) by 24 h following exposure termination. A minor portion of the absorbed dose was recovered in faeces (3%) or eliminated as organic volatiles and 14CO2 (2%). A similar distribution of the inhaled dose was determined for mice with 27% in urine, 46% in tissues, 5% in faeces, 2% as organic volatiles, and 1% as 14CO2.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Metabolites of [1,2,3-13C]AM After gavage: Metabolites derived from direct conjugation of AM with glutathione (AM-GSH) included N-acetyl-S-(3-amino-3-oxopropyl)cysteine (metabolite1) and S-(3-amino-3 oxopropyl)cysteine (metabolite 1').Diastereomers of N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl) cysteine (metabolite 2,2') and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)cysteine (metabolite 3, 3') were detected (GSHGA) and are derived from GSH conjugation with GA (GA,metabolite 4). 2,3-Dihydroxypropionamide and its acid (metabolite5,5') were definitively assigned only in samples from rats administered [1,2,3-13C]acrylamide by gavage.
Metabolites of [1,2,3-13C]AM After IP injection: N-acetyl-S-(3-amino-3-oxopropyl)cysteine (metabolite1) and S-(3-amino-3 oxopropyl)cysteine (metabolite 1').Diastereomers of N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl) cysteine (metabolite 2,2') and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)cysteine (metabolite 3, 3') were detected (GSHGA) and are derived from GSH conjugation with GA (GA,metabolite 4).

Any other information on results incl. tables

IP Administration: Rats excreted 62±12% of the dose in the 0 to 24-h urine. The percentage of excreted dose varied 1.5-fold among the four rats (range, 53 to 79%). The GSH-AM derived metabolites (1,1’) accounted for approximately two-thirds of the excreted dose (69% of total excreted metabolites) following ip administration (Table 3). GA (metabolite 4; 7%) and GSH-GA-derived metabolites (metabolites 2,2 and 3,3; 24%) accounted for the remainder.

Oral administration: Data similar to those generated by ip administration were found. The GSH-AM derived metabolites (1,1’) accounted for approximately two thirds of the excreted dose (71% of total excreted metabolites) following ip administration . GA (metabolite 4; 7%), GSH-GA-derived metabolites (metabolites 2,2' and 3,3';20%), and GA hydrolysis (metabolite 5, 1.7%) accounted for the remainder.

Dermal Application: Following dermal application of 138 mg/kg [1,2,3- 13C]AM, rats excreted 1.5% (range, 0.43 to 2.8%) of the applied dose in the 0 to 24-h urine. Metabolites 1–4 were quantitated for the two rats with the highest percentage of the dermal dose excreted in urine (1.6 and 2.8%). Only metabolite 1,1’ and GA could be detected and quantitated for the two additional rats. For the two rats in which quantitative values were obtained for metabolites 1–4, the AM-GSH-derived metabolites accounted for 46 to 58% of the total excreted metabolites . For these two rats, GA accounted for 14 to 20% of the total excreted metabolites, and the GSH-GA-derived metabolites accounted for 28 to 34% of the total excreted metabolites.

Inhalation Exposure: A significant portion of the metabolites detected in urine from rats exposed to [13C]AM-vapour were derived from AMGSH (1,1', 64% of the excreted metabolites. Metabolites derived from GA-GSH (2,2', 3,3') accounted for 36% of the excreted metabolite, while GA was not detected in rats exposed via inhalation. In rats, the extent of oxidation via glycidamide (metabolites 2, 3, 4, and 5) was slightly higher on

inhalation exposure when compared with po or ip administration. In contrast to rats, mice exposed to AM vapor had a similar percentage of metabolites attributed to GA (31%) and GSH-AM (27%), while GSH-GA accounted for 42% of the excreted metabolites . In mice, approximately twothirds of the urinary metabolites arise from oxidation of AM to GA.

Hemoglobin Adducts of AM and GA

For quantitation of AAVal and GAVal, the AAVal and GAVal PTH derivatives were analyzed by LC-MS/MS in the negative ion mode with a turboionspray interface. The major ion formed was the parent ion (M-H–), and the major daughter ions resulted from loss of the AM or GA side chain. This provided the capability to distinguish among the adduct ions derived from AM, from [1,2,3-13C]AM, and from the internal standard labelled with valine-13C5, since the loss of the AM andGA side chains results in three distinct reactions that can be monitored to detect each form of the adduct (Fennellet al.,2003). For globin samples from rats and mice administered [1,2,3-13C]AM or administered [1,2,3-13C]AM in combination with [2,3-14C] AM, analyses were conducted for the adducts formed by both the 13C-enriched and natural abundance forms of AA and GA. Three separate chromatograms were obtained for two forms of the analyte and the internal standard. A similar set of chromatograms was obtained for GAVal in the same animal. For quantitation, the two peaks for the isomers of GAVal-PTH were integrated together.

The dermal administration of 150 mg/kg [13C]AM resulted in 13C-AAVal adduct levels that were approximately 10-fold lower than those observed following ip administration of 50 mg/kg [13C]AM in male rats (Table 5). 13C-GAVal levels were also lower (approximately 4-fold) on dermal administration compared with the ip administration. Adjusting for the difference in dose administered and comparing AAVal would suggest that approximately 3.6% of the administered dose is absorbed on dermal application of [13C]AM. A second analysis of dermal administration was conducted using a mixture of natural abundance AM, and [14C]AM. In this study, the levels of AAVal detected in globin were approximately 5-fold higher than observed in the study using the [13C]AM. (Table 5). Likewise the GAVal adduct levels were also approximately 4.5-fold higher than in the study using [13C]AM. Comparing the AAVal levels observed in the second dermal application with the ip administration suggests that approximately 16.5% of the applied dose was taken up. This calculation does not account for any differences in the conversion of acrylamide to glycidamide with dose route.

On inhalation exposure of rats and mice, the amount of 13C-AAVal was similar in rats and mice and increased slightly between collection of blood immediately following exposure and at 24 h following exposure. The levels of 13C-GAVal also increased between the two time points. The amount of 13C-GAVal observed in the mouse was 3.6- and 3.8-fold that of the amount observed in the rat at the 0 and 24 h time points, respectively.

Compared with gavage administration, ip administration produced lower AAVal but higher GAVal levels. With dermal administration, the amount of AAVal and GAVal calculated using the administered doses were lower than the other routes of exposure. However, when recalculated for the dose of AM that was recovered in excreta, carcass, and tissues (representing the amount of AM absorbed), the amounts of AAVal formed approached that found with po and ip administration, and the amount of GAVal formed with dermal administration was

highest. With inhalation exposure in the rat, the amount of AAVal formed normalized to the dose taken up was lower than that formed with ip and gavage administration, but higher than that formed with dermal exposure.

GAVal formed in the rat was similar to that formed with dermal and oral administration.

In the mouse, which had the highest levels of AAVal and GAVal , correction for the amount of AM taken in resulted in a considerably lower AAVal per mmol AM administered that found with the rat with inhalation, ip or po administration. This reflects a higher intake of acrylamide per kg body weight in the mouse, and indicates a more rapid metabolism of AM in the mouse. The amount of GAVal normalized per mmol of AM/kg body weight was similar between the rat and mouse.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: Uptake due to dermal and inhalation exposure is lower than for ip.
The objective of this study was to compare the metabolism of AM administered orally (po), dermally, intraperitoneally (ip), or by inhalation, and to measure the haemoglobin adducts produced. Rats and mice were exposed to 2.9 ppm [1,2,3-13C] and [2,3- 14C]AM for 6 h. [2,3-14C]AM (162 mg/kg) or [1,2,3-13C]AM (138mg/kg) in water was administered dermally to rats for 24 h, and [1,2,3-13C]AM was administered ip (47 mg/kg). Urine and faeces were collected for 24 h. Urine was the major elimination route in rats (ip, 62% and po, 53% of the dose; dermal, 44% of the absorbed dose; inhalation, 31% of the recovered radioactivity) and mice (inhalation, 27% of the recovered radioactivity). Signals in the 13C-NMR spectra of urine were assigned to previously identified metabolites derived from AM glutathione conjugation (AM-GSH) and conversion to glycidamide (GA). AM-GSH was a major met­abolic route in rats accounting for 69% (ip), 71% (po), 52% (der­mal), and 64% (inhalation). In mice, AM-GSH accounted for only 27% (inhalation) of the total urinary metabolites. The remaining urinary metabolites were derived from GA. Valine haemoglobin adducts of AM and GA were characterized using liquid chroma­tography-mass spectrometry. The ratio of AM to GA adducts paralleled the flux through pathways based on urinary metabo­lites. This study demonstrates marked species differences in the metabolism and internal dose (Hb-adducts) of AM following inhalation exposure and marked differences in uptake comparing dermal with po and ip administration.