N-(butoxymethyl)acrylamide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

January 10, 2000-May 17, 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Target record

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.

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

Radiolabelling:

yes

Remarks:

C13 and C14

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)

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

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.

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.

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.

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).

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.

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.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Target record

Objective of study:

absorption

excretion

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

Absorption and excretion of D3-acrylamide in male human volunteers after single oral and repeat dermal administration with identification of metabolites.

GLP compliance:

yes

Radiolabelling:

yes

Species:

other: Human volunteers

Strain:

other: Not applicable

Sex:

male

Details on test animals or test system and environmental conditions:

24 human volunteers between the ages of 26 and 68, weighing between 71 and 101 kg were exposed to acrylamide. They had not consumed any cafinated products within the previous 3 days or any alcohol or tobacco over the previous 7 days.

Route of administration:

other: Oral and Topical

Vehicle:

water

Details on exposure:

In the oral phase, individuals received 0,5, 1.0 or 3.0 mg/kg. In the dermal phase individuals received 3.0 mg/kg daily for three days.

Duration and frequency of treatment / exposure:

Once in the oral study and three times in the dermal study.

Remarks:

Doses / Concentrations:
0.5, 1.0 and 3.0 mg/kg in the oral phase and 9.0 mg/kg in the dermal phase.

No. of animals per sex per dose / concentration:

5 male volunteers

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

No

Details on study design:

Twenty-four volunteers participated in this study. They were all male Caucasians (with the exception of one Native American) weighing between 71 and 101 kg and between 26 and 68 years of age. All volunteers were aspermic and had not used tobacco products for the past 6 months. They passed a drug screen and had not taken prescription drugs or caffeinated products over the previous 3 days. They had not consumed alcohol-containing beverages or medications within 7 days of study entry, and for the duration of the study.
Each experimental group consisted of six individuals of which one was a placebo. There were two phases to this study: an oral phase and a dermal phase. In the oral phase, three groups of six people were administered 0.5, 1.0, or 3.0 mg/kg 13C3 AM. Individuals were presented with test substance at approximately 9:00 A.M. to initiate the study. Urine was collected at 0–2, 2–4, 4–8, 8–16, and 16–24 h. In the dermal phase, a 50% (wt/vol) solution of 13C3 acrylamide was applied directly on the skin to a clean, dry, marked off, 24 cm2 (3 cm x 8 cm) area on the volar forearm. After applying the appropriate amount of material, the liquid was evaporated to dryness using a commercial hair dryer and covered with a sterile gauze pad. After drying the AM solution, the tape which had been used to demark the area of application was removed and placed in a vial containing 20 ml of water. The water (dermal dam solution) was analysed for AM by high-performance liquid chromatography (HPLC). The site of application was covered with gauze for 24 h at which time the gauze was removed and the area was washed with 1000 ml of water. The recovered wash water was analysed by HPLC for AM. Dermal applications alternated between left and right arms, starting with the subject’s dominant arm. Blood was collected immediately prior to compound administration and 24, 48, 72, and 96 h later. Urinary metabolites of acrylamide. (immediately prior to administration of the second and third doses, after gauze removal and prior to leaving the clinic). Hormone blood samples were drawn immediately prior to compound administration, after 24 h and on day 5 when the volunteers left the clinic.
Each exposure group contained six volunteers. Of the six volunteers in each group, five received the designated amount of AM, and one received no AM. AM was applied to the skin for 24 h on one forearm, and a blood sample was collected at 24 h following the first administration. This was repeated on the following 2 days, with AM applied on alternating arms, for a total of three dermal doses of AM at 24 h intervals. A total of five blood samples was collected from each volunteer administered AM dermally, on day 1 (prior to the first dose), day 2, day 3, day 4, and day 5. The sample obtained on day 5 was at 24 h following removal of the occlusion at the site of application. Urine samples were collected at intervals of 0–2, 2–4, 4–8, 8–16, and 16–24 h following administration of AM. The volume of urine in each sample was recorded, and sample aliquots were transferred to sample vials for storage.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled :
Twenty-four volunteers participated in this study. They were all male Caucasians (with the exception of one Native American) weighing between 71 and 101 kg and between 26 and 68 years of age. All volunteers were aspermic and had not used tobacco products for the past 6 months. They passed a drug screen and had not taken prescription drugs or caffeinated products over the previous 3 days. They had not consumed alcohol-containing beverages or medications within 7 days of study entry, and for the duration of the study.
Each experimental group consisted of six individuals of which one was a placebo. There were two phases to this study: an oral phase and a dermal phase. In the oral phase, three groups of six people were administered 0.5, 1.0, or 3.0 mg/kg 13C3 AM. Individuals were presented with test substance at approximately 9:00 A.M. to initiate the study. Urine was collected at 0–2, 2–4, 4–8, 8–16, and 16–24 h.
In the dermal phase, a 50% (wt/vol) solution of 13C3 AM was applied directly on the skin to a clean, dry, marked off, 24 cm2 (3 cm x 8 cm) area on
the volar forearm. After applying the appropriate amount of material, the liquid was evaporated to dryness using a commercial hair dryer and covered with a sterile gauze pad. After drying the AM solution, the tape which had been used to demark the area of application was removed and placed in a vial containing 20 ml of water. The water (dermal dam solution) was analyzed for AM by high-performance liquid chromatography (HPLC). The site of application was covered with gauze for 24 h at which time the gauze was removed and the area was washed with 1000 ml of water. The recovered wash water was analyzed by HPLC for AM. Dermal applications alternated between left and right arms, starting with the subject’s dominant arm. Blood was collected immediately prior to compound administration and 24, 48, 72, and 96 h later. Urinary metabolites of AM. (immediately prior to administration of the second and third doses, after gauze removal and prior to leaving the clinic). Hormone blood samples were drawn immediately prior to compound administration, after 24 h and on day 5 when the volunteers left the clinic.
Each exposure group contained six volunteers. Of the six volunteers in each group, five received the designated amount of AM, and one received no AM. AM was applied to the skin for 24 h on one forearm, and a blood sample was collected at 24 h following the first administration. This was repeated on the following 2 days, with AM applied on alternating arms, for a total of three dermal doses of AM at 24 h intervals. A total of five blood samples was collected from each volunteer administered AM dermally, on day 1 (prior to the first dose), day 2, day 3, day 4, and day 5. The sample obtained on day 5 was at 24 h following removal of the occlusion at the site of application. Urine samples were collected at intervals of 0–2, 2–4, 4–8, 8–16, and 16–24 h following administration of AM. The volume of urine in each sample was recorded, and sample aliquots were transferred to sample vials for storage.

Statistics:

The kinetics of AM elimination in urine following oral administration of AM were analyzed using WinNonlin version 4.0.1 from Pharsight (Cary, NC). Noncompartmental analysis using Model 210 for extravascular input and urine data was used to calculate the elimination rate constant and half-life.

Preliminary studies:

None

Details on absorption:

1. Dermal absorption, on occluded human skin, in vivo, is 6.5% over a 96 hour period and only 4.5 % was recoverable in urine.
2. Approximaely 50 % of the admionistered dose was recoverd in the urine over the time period involved.

Details on excretion:

Pharmacokinetic Analysis of AM Elimination in Urine
Following Oral Administration
0.5 mg/kg 1.0 mg/kg 3 mg/kg
Half-life (h) 3.13 ± 1.33 3.25 ± 0.43 3.49 ± 0.78
Elimination rate 0.261 ± 0.128 0.216 ± 0.030 0.206 ± 0.042
constant (h1)


Values represent mean ± SD (n ¼ 5 subjects).

Test no.:

#1

Toxicokinetic parameters:

half-life 1st: acrylamide in urine= 3.1-3.5 hours

Metabolites identified:

yes

Details on metabolites:

Urinary Metabolites: Cysteine-S-propionamide, N-acetyl cysteine-S-propionamide, N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)cysteine, N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine and N-acetyl cysteine-S-propionamide sulfoxide.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
This study demonstrates that acrylamide is rapidly distributed throughout the body where it is readily metabolized prior to excretion. The biotransformation of acrylamide was mainly mediated through glutathione conjugation followed by excretion in the urine of the mercapturic acid, N-acetyl-S-(3-amino-3oxypropyl)cysteine. Other metabolites are also excreted but their significance is unknown. The half-life of parent acrylamide in the body is extremely short; however, a small percentage of radiolabel remains in tissues for several weeks. Finally, neural tissues do not selectively concentrate parent acrylamide or metabolites when compared to non-neural tissues. The data suggest that the mechanism by which acrylamide induces neuropathy does not involve accumulation of parent acrylamide. Whether or not accumulation of biotransformation products are responsible for this toxicity remains to be established.

Reference 3

Endpoint:

dermal absorption in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

See attached Analogue Approach Report.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Methods are described in 7.1.1 Basic Toxicokinetics.

GLP compliance:

yes

Radiolabelling:

yes

Species:

other: Human volunteers

Strain:

other: Not applicable

Sex:

male

Details on test animals or test system and environmental conditions:

Details are given in 7.1.1.

Type of coverage:

occlusive

Vehicle:

water

Duration of exposure:

3 periods of 24 hours

Doses:

3.0 mg/kg

No. of animals per group:

5 human volunteers

Control animals:

yes

Remarks:

Human volunteers

Details on study design:

A 50% (wt/vol) solution of 13C3 AM was applied directly on the skin to a clean, dry, marked off, 24 cm2 (3 cm 3 8 cm) area on the volar forearm. After applying the appropriate amount of material, the liquid was evaporated to dryness using a commercial hair dryer and covered with a sterile gauze pad. After drying the AM solution, the tape which had been used to demark the area of application was removed and placed in a vial containing 20 ml of water. The water (dermal dam solution) was analyzed for AM by high-performance liquid chromatography (HPLC). The site of application was covered with gauze for 24 h at which time the gauze was removed and the area was washed with 1000 ml of water. The recovered wash water was analyzed by HPLC for AM. Dermal applications alternated between left and right arms, starting with the subject’s dominant arm. Blood was collected immediately prior to compound administration and 24, 48, 72 and 96 hours later (immediately prior to administration of the second and third doses, after gauze removal and prior to leaving the clinic). Hormone blood samples were drawn immediately prior to compound administration, after 24 hours and on day 5 when the volunteers left the clinic.

Signs and symptoms of toxicity:

yes

Remarks:

One individual demonstrated dermal hypersensitivity to acrylamide.

Dermal irritation:

no effects

Absorption in different matrices:

None used.

Total recovery:

83.94%

Dose:

3.0 mg/kg

Parameter:

percentage

Absorption:

6 %

Remarks on result:

other: 24 hours

Conversion factor human vs. animal skin:

No coversion factor since this was a human study.

Conclusions:

In this well conducted study, designed to maximise absorption, the recovery of the labelled material was very satisfactory at 83.9%. The study demonstrated that dermal absorption in humans is slow. Only 4.5% of the applied dose was absorbed from an occluded patch on the forearm over a 24 hour period which equates to less than 0.2% absorption per hour.

Description of key information

Based on the available data, an oral absorption of 100 % is considered as a worst-case to address the oral absorption of NBMA.

Based on the results of the dermal absorption study in human volunteers (Fennell et al. 2006)and the physico-chemical properties of both AD and NBMA the dermal absorption of NBMA cannot be any greater than the soluble species investigated (AD). A dermal absorption value of 6 % proposed for AD is considered sufficiently precautionary to address the dermal absorption of NBMA.

An inhalation absorption of 100% has been proposed.

 

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

6

Absorption rate - inhalation (%):

100

Additional information

Introduction

Read-across from Acrylamide (AD) to NBMA is considered valid for the evaluation of the toxicokinetic properties. The acrylamide moiety present in both substances represents the most relevant structure for mammalian toxicity. Furthermore, it is known that these molecules inter-convert. There exists an adequate body of studies available on AD, whereas the target substance has limited toxicological data available. Where data is available on both substances, the chemical and biological characteristics of these compounds are very similar. Where differences between these two compounds have been detected, the AD results are potentially more problematic than those identified for NBMA. The use of data from AD to substitute for missing data for NBMA provides a good but conservative estimate for the evaluation of toxicokinetic endpoints for which read-across is proposed.

 

Systemic availability of NBMA depends on its ability to be absorbed across body surfaces. Factors that affect this process include water solubility, lipophilicity (measured by the partition coefficient, Kow), and molecular size. The substance has a molecular weight of 157.21 g/mol, a solubility in water of 35.2 mg/L and a log Kow of 1.182. None of these values would indicate that uptake of NBMA by the body would be impeded. In addition, given values noted for AD and NBMA, it is considered justified to adopt an analogue approach, and consider the use of published pharmacokinetic studies on acrylamide as reasonable surrogates for use in the evaluation of the toxicokinetics of NBMA.

 

Results of toxicology studies on AD are consistent with the conclusion that the compounds are similar but toxicity decreases with chain length. AD is more toxic than NBMA. The acute oral toxicity LD50 value for AD in rats was 100-200mg/kg, whereas for NBMA a value of > 1000 mg/kg was found. Similarly, the acute dermal toxicity LD50 value in rabbits for AD was from 1141 mg/kg, while the value for NBMA was > 2000 mg/kg. Unlike AD, NBMA is not classified as acutely toxic via the oral or dermal routes and is not a skin or eye irritant. Like AD, NBMA is highly soluble, readily biodegradable and with a log Kow of 1.181 it is also unlikely to bioaccumulate. Data on the substance of interest for toxicokinetic read-across to AD will therefore be precautionary for NBMA.

 

 

Oral absorption

Following a single administration of NBMA by the oral route (> 1000 mg/kg) (Gilotti, 2006), deaths and clinical signs of systemic toxicity are indication of systemic absorption of the NBMA: Three of the five animals survived. In those that survived instances of lethargy, wetness and soiling of anogenital area, wetness and red staining of nose/mouth area, unkempt appearance and few faeces were noted early in the study, but they appeared normal from Day 4 through Day 7. Following a single administration of AD by the oral route (177 mg/kg) two of the five animals were found dead on the morning of day 2 and two were found dead on the afternoon of day 2. The surviving animal showed sedation or hypoactivity, tremors and piloerection up to day 5; rhinorrhoea was also noted on day 2, and dyspnoea on day 8. Recovery was complete on day 9 for this animal. Based on these observed effects, oral absorption does occur upon ingestion of both AD and NBMA in animals.

 

Oral pharmacokinetic studies for AD (Sumner et al. 2003), resulted in data similar to those generated by i.p. administration (Sumner et al. 2003). Sumner (2003) demonstrated that following i.p. 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 %). Following oral administration at least 53 % of the dose was present in the urine (Sumner et al. 2003). The oral absorption of NBMA, which by comparison is practically insoluble, is unlikely to be any greater than the soluble species investigated. However, considering the uncertainty regarding other routes of excision a worst-case oral absorption of 100 % has been considered.

 

Based on the available data, an oral absorption of 100 % is considered as a worst-case to address the oral absorption of NBMA.

 

 

Dermal absorption

Following a single administration of NBMA by the dermal route (at the limit does of 2000 mg/kg (Gilotti, 2006)), no relevant systemic clinical sign or changes in body weight were observed. Following a single administration of AD by the dermal route (1141 mg/kg) At the two highest exposure levels tremors and incoordination of hindlimbs were noted, and, in addition, the surviving female at 1,612 mg/kg was in poor condition and lost weight.

 

Following dermal exposure to AD in rats and mice, the major portion of the absorbed dose was excreted by 24 h in urine (8 % of the applied dose or 36 % of total absorbed dose) or remained in the body (53 % of the absorbed dose). A minor portion of the absorbed dose was recovered in faeces (< 1 %) or eliminated as organic volatiles (1 %) or 14CO2 (2 %) (Sumner et al. 2003).

 

In a well conducted dermal study in human volunteers (Fennell et al. 2006), designed to maximise absorption, the recovery of the labelled material from the volunteers was 83.9 %. The study demonstrated that dermal absorption in humans is slow. Only 4.5 % of the applied dose was absorbed from an occluded patch on the forearm over a 24 hour period which equates to less than 0.2 % absorption per hour.

 

Based on the results of the dermal absorption study in human volunteers (Fennellet al.2006)and the physico-chemical properties of both AD and NBMA the dermal absorption of NBMA cannot be any greater than the soluble species investigated (AD). A dermal absorption value of 6 % proposed for AD is considered sufficiently precautionary to address the dermal absorption of NBMA.

 

 

Inhalation absorption

Following inhalation exposure, 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. A minor portion of the absorbed dose was recovered in faeces (3 %) or eliminated as organic volatiles and¹⁴CO₂(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 % as¹⁴CO₂(Sumner S.C. 2003).

 

Based on these data an inhalation absorption of 100% has been proposed.

 

 

Distribution

A toxicokinetics assessment with radiolabelled AD (Sumner et al. 2001) is considered appropriate for the toxicokinetic assessment of NBMA. It indicated that following dermal exposure, excluding the skin at the dose site, blood cells had the highest level of radioactivity (1 µmol/g tissue). The skin, liver, spleen, testes, and kidney had nearly the same levels (0.3 µmol/g tissue). Radioactivity was also recovered in the lungs, thymus, brain, and epididymis (0.2 µmol/g tissue). Low levels of radioactivity (0.05 µmol/g tissue) were recovered in fat, which is consistent with the reported Log Kow value. 

 

Immediately or 24 h following the 6-h inhalation exposure to AD, blood cells of rats had the highest relative level (0.1 µmol/g tissue) of radioactivity compared with all other tissues. Radioactivity levels in plasma 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 and epididymis. After 24 h, the rank order of radioactivity was blood, skin, spleen, lung, liver, kidney, brain, testes, epididymis and fat. The lowest radioactivity levels were observed for fat at either time point.

 

Following dermal exposure to 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.

 

Neural tissues did not selectively concentrate parent acrylamide or metabolites when compared to non-neural tissues (Fennell, 2005)

 

 

Metabolism

As with the Sumner (2001) study the Fennell (2006) study is considered appropriate for the toxicokinetic assessment of NBMA and provides appropriate metabolism data to substitute for missing data for NBMA for which read-across is proposed (see Introduction above). The metabolic fate of NBMA is inferred from that of AD. The ultimate metabolic fate of NMBA is likely to be similar, once the butoxymethyl group is cleaved.

 

The biotransformation of AD was mainly mediated through GSH-conjugation followed by excretion in the urine of the mercapturic acid, N-acetyl-S-(3-amino-3oxypropyl)cysteine (Fennell et al. 2006).

 

Following inhalation exposure rats and mice exposed to AD vapour (Sumner et al. 2001) had a similar percentage of metabolites attributed to glycidamide (GA) (31 %) and AD glutathione conjugation (AD-GSH) (27 %), while GSH-GA accounted for 42 % of the excreted metabolites. In mice, approximately two thirds of the urinary metabolites arise from oxidation of AD to GA.

 

The GHS-AD derived metabolites accounted for approximately two-thirds of the excreted dose (69 % of total excreted metabolites) following i.p. administration. Glycidamide (GA) (7 %) and GSH-GA-derived metabolites (24 %) accounted for the remainder. Whilst, following oral administration GSH-AD derived metabolites accounted for approximately two thirds of the excreted dose (71 % of total excreted metabolites). GA (7 %), GSH-GA-derived metabolites (20 %), and GA hydrolysis (1.7 %) accounted for the remainder (Sumner et al. 2001).

 

Valine haemoglobin adducts of AD and GA (ADVal and GAVal, respectively) where quantified and compared with gavage administration (Sumner et al. 2003), i.p. administration produced lower ADVal but higher GAVal levels. With dermal administration, the amount of ADVal and GAVal were lower than the other routes of exposure. However, when recalculated for the dose of AD that was recovered in excreta, carcass, and tissues (representing the amount of AD absorbed), the amounts of ADVal formed approached that found with p.o. and i.p. administration, and the amount of GAVal formed with dermal administration was highest. With inhalation exposure in the rat, the amount of ADVal formed normalized to the dose taken up was lower than that formed with i.p. 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 (Sumner et al. 2003).

 

In the mouse, which had the highest levels of ADVal and GAVal, correction for the amount of AD taken into account resulted in a considerably lower ADVal per mmol AD administered than that found with the rat with inhalation, i.p. or p.o. administration (Sumner et al. 2003). This reflects a higher intake of acrylamide per kg body weight in the mouse, and indicates a more rapid metabolism of AD in the mouse. The amount of GAVal normalised per mmol of AD/kg body weight was similar between the rat and mouse (Sumner et al. 2003).

 

 

Elimination

As with the Sumner (2001) study the Fennell (2006) study is considered appropriate for the toxicokinetic assessment of NBMA and provides appropriate excretion data to substitute for missing data for NBMA for which read-across is proposed (see Introduction above). The fate of NBMA is inferred from that of AD. 

 

From the same ADME study from Sumner et al (2003) conducted on AD, urine was the major elimination route in rats (i.p., 62 % and p.o., 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 GSH-AD and conversion to GA. GSH-AD was a major metabolic route in rats accounting for 69 % (i.p.), 71 % (p.o.), 52 % (dermal), and 64 % (inhalation). In mice, GSH-AD accounted for only 27 % (inhalation) of the total urinary metabolites. The remaining urinary metabolites were derived from GA. The ratio of AD to GA adducts paralleled the flux through pathways based on urinary metabolites (Sumner et al. 2003). 

 

A study using human volunteers (Fennell et al. 2006) demonstrated that acrylamide is rapidly distributed throughout the body where it is readily metabolized prior to excretion. The half-life of parent acrylamide following oral administration in the body is extremely short (approx. 3-4 hrs); however, a small percentage of radiolabel remains in tissues for several weeks.