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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
excretion
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study investigated the urinary excretion of acrylonitrile metabolites following inhalation, i.v. or i.p. exposure
GLP compliance:
not specified
Remarks:
: published study
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Male adult Sprague-Dawley rats
Route of administration:
other: inhalation (vapour), intravenous and intraperitoneal
Vehicle:
not specified
Details on exposure:
Rats were exposed to acrylonitrile vapour, or acrylonitrile was administered in a single dose i.v. or i.p.
Duration and frequency of treatment / exposure:
6 hours - inhalation
Remarks:
Doses / Concentrations:
Inhalation: 0, 4, 20 or 100 ppm. i.v. or i.p.: 0.6-15 mg/kg bw
No. of animals per sex per dose:
5 males per group
Control animals:
yes, concurrent no treatment
Positive control:
Not examined.
Details on study design:
The formation of urinary metabolites following exposure to acrylonitrile administered via 3 routes was determined
Details on dosing and sampling:
No further information
Statistics:
No information available
Preliminary studies:
Not applicable
Details on absorption:
Not investigated in this study.
Details on distribution in tissues:
Not investigated in this study.
Details on excretion:
Following inhalation exposure:

Thiocyanate was the major metabolite identified in urine, with levels of excreted HMA being higher than levels of CMA. CMA represented only 8% of total urinary metabolites. As exposure levels of acrylonitrile increased, excretion of thiocyanate became relatively more important, as shown by the ratio of excreted thiocyanate to the sum of CMA and HMA, rising from 0.47 at 4 ppm to 0.89 at 20 ppm, and 2.93 at 100 ppm.

Following i.v. or i.p. administration:

CMA represented 74-78% of metabolites excreted in the urine. Levels of thiocyanate excreted were low.
Metabolites identified:
yes
Details on metabolites:
Thiocyanate was the major metabolite following inhalation exposure, with levels of excreted HMA being higher than levels of CMA. In contrast, CMA represented 74-78% urinary metabolites following i.v. or i.p. administration.

Inhalation exposure

Following inhalation exposure, 15% of the inhaled acrylonitrile was excreted as thiocyanate - this was identified as the major urinary metabolite. Levels of excreted 2-hydroxyethylmercapturic acid (HMA) were higher than levels of 2-cyanoethylmercapturic acid (CMA; 8% of total urinary radioactivity). As exposure levels increased, the excretion of thiocyanate became relatively more important as shown by the ratio of excreted thiocyanate to the sum of CMA and HMA at 4 ppm (0.47), 20 ppm (0.89) and 100 ppm (2.93).

Intravenous / intraperitoneal dosing

CMA represented 74-78% of total urinary metabolites follwoing iv and ip dosing; in contrast the urinary levels of excreted thiocyanate were low.

Conclusions:
The results of this study indicate that the metabolic profile of acrylonitrile in the rat is different following inhalation exposure or parenteral administration. Findings indicate that the metabolism of acrylonitrile via conjugation with glutathione is the predominant pathway following parenteral administration, whereas cytochrome P450- oxidation (via CEO and generating thiocyanate) is the predominant pathway following inhalation exposure.
Executive summary:

The authors investigated the urinary metabolism of acrylonitrile in the rat, following exposure by different routes. Adult male Sprague-Dawley rats were exposed acutely to acrylonitrile using different routes of administration: inhalation (6 hour exposure), intravevous or intraperitoneal injection. Urinary metabolites measured at 24 hours after administration were identified as 2-cyanoethylmercapturic acid (CMA), 2 -hydroxyethylmercapturic acid (HMA) and thiocyanate. In all experiments the relationship between excretion of total urinary metabolites and the degree of exposure was reasonably linear. However, there was a marked influence of the route of administration on the pattern excretion. Following i.p. and i.v. injection, CMA was the most important metabolite while after inhalation it was thiocyanate. The results of the study indicate an important effect of the dose on the metabolism and excretion of acrylonitrile.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
excretion
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
Investigation of the urinary excretion of acrylonitrile and its metabolites in rats following inhalation exposure, influence of exposure time and exposure level
GLP compliance:
not specified
Remarks:
: published study
Radiolabelling:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Adult male Wistar rats
Route of administration:
inhalation
Vehicle:
unchanged (no vehicle)
Details on exposure:
Groups of four adult male Wistar rats were exposed by inhalation to 0, 1, 5, 10, 50 or 100 ppm acrylonitrile for 8 hours.
Duration and frequency of treatment / exposure:
8 hours
Dose / conc.:
0 ppm
Remarks:
Untreated controls
Dose / conc.:
1 ppm
Dose / conc.:
5 ppm
Dose / conc.:
10 ppm
Dose / conc.:
50 ppm
Dose / conc.:
100 ppm
No. of animals per sex per dose:
4 males per group
Control animals:
yes, concurrent no treatment
Positive control:
Not required
Details on study design:
No further details
Details on dosing and sampling:
Urine was collected during the 8 hour exposure period, and for 24 hours post exposure.
Statistics:
No information available
Preliminary studies:
Not applicable.
Details on absorption:
Not investigated in this study
Details on distribution in tissues:
Not investigated in this study
Details on excretion:
There was a dose-related increase in the excretion of unchanged acrylonitrile in urine collected during the exposure period. Mean levels at 100 ppm were 25 µmol/mL compared with 1 µmol/mL at 10 ppm. In the subsequent 24 hours, levels of unchanged acrylonitrile in urine fell to very low levels in all groups (mean 1.6 µmol/mL in the 100 ppm group). CMA was the predominant metabolite during the exposure period (mean 53.6 µmol/mL at 100 ppm), increasing with concentration and with excretion reducing in the subsequent 24 hours (22.7 µmol/ml). More HMA was excreted in the 24 hour post-exposure period than during exposure (mean 4.7 µmol/mL at 100 ppm post exposure compared with 2.7 µmol/mL during exposure). Lower levels of S-carboxymethyl cysteine (2.43 µmol/mL at 100 ppm during exposure, falling to 1.2 µmol/mL post exposure) while levels of thiodiglycolic acid increased in the post exposure period (3.2 µmol/mL at 100 ppm, compared to 2.7 µmol/mL during exposure).
Metabolites identified:
yes
Details on metabolites:
The metabolites quantified were 2-cyanoethylmercapturic acid (CMA), 2-hydroxyethylmercapturic acid (HMA), S-carboxymethyl cysteine and its further metabolite thiodiglycolic acid.

A dose-related increase in the excretion of unchanged acrylonitrile was apparent in urine collected during the 8-hour exposure period.  Mean levels at 100 ppm were 25 μmol/mL compared with 1 μmol/mL at 10 ppm. However in the subsequent 24-hour post-exposure period, the levels of unchanged urinary acrylonitrile fell to very low levels in all groups (1.6 μmol/mL at 100 ppm).  2-cyanoethylmercapturic acid (CMA) was identified as the predominant urinary metabolite during the exposure period (53.6 μmol/mL at 100 ppm); the levels of CMA increased with exposure level.  CMA excretion declined during the 24 hour post-exposure period (22.7 μmol/ml).  In contrast, a larger proportion of the metabolite 2-hydroxyethylmercapturic acid (HMA) was excreted in the post-exposure period (4.7 μmol/mL at 100 ppm) than during exposure (2.7 μmol/ml).  Lower levels of the metabolite S-carboxymethyl cysteine (2.43 μmol/mL at 100 ppm) were detected during the exposure period; levels declined post-exposure (1.2 μmol/ml), while levels of the metabolite thiodiglycolic acid increased post-exposure (3.2 μmol/mL at 100 ppm) compared with 2.7 μmol/mL during the exposure period.

Conclusions:
The dose-related increase in excretion of unchanged acrylonitrile in urine collected during the exposure period, was indicative of saturation of the metabolic processs for acrylonitrile. The authors suggested that CMA was the most sensitive indicator metabolite of acrylonitrile exposure.
Executive summary:

The authors investigated the urinary metabolites of acrylonitrile in the rat for their potential use in biological monitoring for occupational exposure. Groups of four male Wistar rats were exposed by inhalation for 8 hours to acrylonitrile at concentrations ranging of 1-100 ppm. Urine was collected during exposure and for up to 24 hours post exposure and analysed for unchanged acrylonitrile and its metabolites cyanoethylmercapturic acid (CMA), S-carboxymethyl cysteine, hydroxyethyl mercapturic acid (HMA) and thiodiglycolic acid by gas chromatography. Significant amounts of unchanged acrylonitrile was detected at exposure levels of 10 ppm and above. The presence of the metabolites HMA and S-carboxymethyl cysteine was seen following exposure to levels of 1 or 5ppm; levels of S-carboxymethyl cysteine were proportionally greater following an 8-hour exposure and levels of HMA were proportionally greater following a 24-hour exposure. Urinary CMA was higher during exposure than the post-exposure period.  Thiodiglycolic acid levels following exposure to 5ppm were significantly lower than those seen at 10 ppm, while the concentration of CMA was higher following exposure to 5 ppm than following exposure to 1 ppm acrylonitrile. At exposure levels of up to 5 ppm, combined levels of the metabolites (S-carboxymethyl cysteine, thiodiglycolic acid and HMA) accounted for approximately 20% of the dose; levels of unchanged acrylonitrile and CMA accounted for a similar proportion of the dose. At higher exposure levels, however, levels of S-carboxymethyl cysteine, thiodiglycolic acid and HMA accounted for ~10% of the dose and levels of unchanged acrylonitrile and CMA increased to ~40%. The dose related increase in excretion of unchanged acrylonitrile in urine collected during the exposure period, is indicative of saturation acrylonitrile metabolism. The authors concluded that a correlation exists between levels of exposure to acrylonitrile and its urinary excretion, and that urinary acrylonitrile, CMA and thiodiglycolic acid may be useful biomarkers for exposure monitoring.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
The distribution, elimination and covalent binding of radiolabelled acrylonitrile were investigated in the rat following gavage dosing.
GLP compliance:
not specified
Remarks:
: published study
Radiolabelling:
yes
Remarks:
[1-14C]-acrylonitrile
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Adult male Sprague-Dawley rats
Route of administration:
oral: gavage
Vehicle:
not specified
Details on exposure:
Rats were given a single oral dose of 46.5 mg/kg of [1-14C]-acrylonitrile
Duration and frequency of treatment / exposure:
Single oral (gavage) treatment
Dose / conc.:
46.5 mg/kg bw (total dose)
Remarks:
Single gavage dose
No. of animals per sex per dose:
3 male rats/group/time interval
Control animals:
no
Positive control:
Not relevant
Details on study design:
Rats were administered a single oral dose of the test substance and monitored over a 10-day period. The excretion of radiolabel was determined in urine, faeces, expired air and bile. Tissue levels of radioactivity were determined at various time intervals.
Details on dosing and sampling:
Rats were given a single oral dose. Measurements were taken at 1, 3, 6, 12, 24, 48, 72, 168 and 240 hours after dosing.
Preliminary studies:
Not applicable.
Details on absorption:
Total excretion at the end of the 10 days was approximately 75% of the initial dose, indicating a retention of approximately 25% of the dose. Excretion was largely urinary, with only 2% excreted in the faeces. The results therefore indicate almost complete absorption.
Details on distribution in tissues:
Results showed highest initial concentrations of radioactivity occurred in the gastrointestinal tract, consistent with ongoing absorption. High concentrations were found in liver, kidney and lung tissues up to 24 hours after administration. The heart, thymus, spleen, adrenals, brain and skin showed maximum concentrations at 3-6 hours after dosing, followed by a gradual decrease in radioactivity. Highest levels of radioactivity up to 72 hours were found in the gastrointestinal tract, suggesting either a secretion process of acrylonitrile metabolites into the stomach or the binding of acrylonitrile or its metabolites to the stomach mucosa. The levels of unbound radioactivity declined progressively with time in the various organs studied, although significant retention in red blood cells was noted for up to 10 days. The covalent binding of radioactivity as a proportion of the total radioactivity increased over the study period, with most of the covalently bound radiolabel located in non-cytosolic (inuclear, mitochondrial and microsomal) fractions.
Details on excretion:
40% of the radiolabel was excreted in urine, 2% in faeces, 9% in expired air and 14CO2, 0.5% as H14CN and 4.8% as unchanged acrylonitrile. 27% of the 14C was recovered in bile. Total excretion at the end of the 10 days was 75%, with 25% retention of radiolabel.
Metabolites identified:
not measured
Details on metabolites:
Metabolism was not investigated in this study.

Tissue levels of radioactivity in rats given a single oral dose of 46.5 mg/kg [1 -14C]-acrylonitrile

Tissue

[1-14C]-acrylonitrile, ng equivalent/mg protein1

1 hr

3 hr

6 hr

12 hr

24 hr

48 hr

72 hr

168 hr

240 hr

Expired air2

16.9±1.8

31.4±2.9

93.9±15

112.5±17

838±87

11.2±1.1

*

*

*

Blood3

79.5±8.0

90.0±9.1

71.7±7.2

63.0±7.0

46.6±5.4

28.7±4.0

28.7±7.0

21.5±27

17.9±1.3

Stomach

513.7±71

367.6±34

332.8±36

379.9±41

362.9±31

204.6±25

118.2±12

22.2±0.9

4.2±0.5

Liver

92.1±5.7

87.5±2.2

74.9±2.0

62.0±0.2

56.8±0.8

32.3±2.6

17.7±1.7

8.6±1.3

2.9±0.2

Kidney

74.2±2.4

76.5±1.3

72.0±4.2

63.3±0.5

48.1±0.44

26.2±3.4

14.1±2.8

6.85±0.5

3.95±0.7

Lung

39.5±5.6

53.7±3.1

64.1±5.3

27.4±0.3

22.9±0.5

27.6±4.1

17.2±1.3

9.7±0.3

0.4±0.1

Heart

25.4±3.8

28.6±1.8

35.8±1.5

21.8±0.2

21.1±0.1

14.6±1.7

13.6±1.0

5.4±0.8

2.9±0.5

Thymus

17.9±2.9

25.6±2.0

27.7±2.5

20.8±0.1

21.1±0.1

14.0±0.8

7.5±2.2

3.6±0.5

1.8±0.4

Spleen

29.0±2.5

36.2±1.9

48.0±4.4

31.7±0.1

21.8±0.1

16.9±1.2

13.1±0.8

9.3±0.5

7.5±1.5

Adrenals

26.5±0.6

35.1±4.0

37.5±3.5

28.7±0.2

21.4±0

14.3±1.7

12.1±2.0

4.7±0.1

4.43±1.0

Brain

16.1±2.2

15.7±1.8

16.1±2.4

11.5±0.1

10.7±0.1

8.6±1.0

7.7±0.3

1.8±0

1.8±0.1

Skin

35.1±3.3

54.1±6.1

60.5±4.3

31.8±0.2

32.1±0.4

37.5±4.4

28.6±1.6

33.0±1.2

18.1±3.5

1 Values are means ±S.E. of 3 animals

2 Expressed as µg equivalents in total14C CO2and14C HCN

3 µg equivalents/ml

* Values less than 0.05 (loq)

Conclusions:
The study reports 25% retention of a single oral dose of acrylonitrile in the rat. Data indicate the covalent binding of acrylonitrile or its metabolites rather than true bioaccumulation. The study indicates the almost complete absorption of acrylonitrile in rats administered a single gavage dose. Data indicate the covalent binding of acrylonitrile or its metaboites rather than true bioaccumulation.
Executive summary:

The distribution, elimination and covalent binding of [1 -14C]-acrylonitrile was investigated in adult male Sprague-Dawley rats. Rats were administered a single oral dose of 46.5 mg/kg bw. Urinary excretion was found to be the major excretory route for acrylonitrile, with 40% excreted in 24 hours. Total excretion at the end of the 10 day srudy period was approximately 75% of the initial dose, indicating a retention of approximately 25%. Findings are consistent with almost complete oral absorption. The highest levels of acrylonitrile were found in the gastrointestinal tract up to 72 hours after administration, suggesting the binding of acrylonitrile (or a metabolite) within the stomach mucosa. High concentrations were also found in liver, kidney and lung tissues up to 24 hours after administration. The authors suggest that the extensive interaction noted in this study between acrylonitrile (or a metabolite) with the gastric mucosa may play a role in the development of tumours, ulcers and acrylonitrile-induced gastrointestinal bleeding.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline available
Principles of method if other than guideline:
Specific investigation of the comparative metabolism in vitro of CEO
GLP compliance:
no
Radiolabelling:
yes
Remarks:
[2,3-14C]CEO
Species:
other: In vitro study using microsomes from human, male F344 rat and male B6C3F1 mice
Details on study design:
This study investigated the role of epoxide hydrolase in the hydrolysis of CEO by HPLC analysis of the products from [2,3-14C]CEO.
Details on absorption:
Not investigated
Details on distribution in tissues:
Not investigated
Details on excretion:
Not investigated
Metabolites identified:
no
Details on metabolites:
The hydrolysis of the metabolite CEO was investigated, however investigations focussed on the rate of hydrolysis rather than the products of hydrolysis.

Incubation with hepatic microsomal or cytosolic fractions from male F344 rats or B6C3F1 mice did not enhance the rate of hydrolysis of CEO (0.69 nmol/min). Human hepatic microsomes significantly increased the rate of hydrolysis of CEO, whereas human hepatic cytosolic fraction did not. Human hepatic microsomal hydrolysis was found to be heat-sensitive and potently inhibited by 1,1,1 -trichloropropeneoxide (IC50 =23 µM), indicating that the reaction was mediated by epoxide hydrolase. The hydrolysis of CEO catalysed by hepatic microsomes from six individuals exhibited normal saturation kinetics with KM of 0.6 -3.2 mM and Vmax of 8.3 -8.8 nmol hydrolysis products/min/mg protein. Pre-treatment of rodents with phenobarbital or acetone-induced hepatic microsomal hydrolysis activity toward CEO, whereas treatment with ß-naphthoflavone, dexamethasone or acrylonitrile itself was without effect.

Conclusions:
The results of this study show that humans possess an additional detoxication pathway for CEO that is not active in rodents (but is inducible).
Executive summary:

This study investigated the role of epoxide hydrolase in the hydrolysis of CEO by HPLC analysis of the products from radiolabelled CEO. Incubation with hepatic microsomal or cytosolic fractions from male F344 rats or male B6C3F1 mice did not increase the rate of hydrolysis of CEO. Human hepatic microsomes significantly increased the rate of hydrolysis, whereas human hepatic cytosols did not. Human hepatic microsomal hydrolysis activity was heat-sensitive and potently inhibited by 1,1,1-trichloropropene oxide, indiating the involvement of epoxide hydrolase. The hydrolysis of CEO by hepaticmicrosomes from six individuals exhibited normal saturation kinetics. Pre-treatment of rodents with phenobarbital or acetone induced the hepatic microsomal hydrolysis activity of CEO, whereas treatment with ß-naphthoflavone, dexamethasone or acrylonitrile had no effect. The results of this study show that humans possess an additional detoxication pathway for CEO that is not active in rodents (but is inducible). The authors conclude that the presence of an active epoxide hydrolase hydrolysis activity toward CEO in humans should be considered in any assessment of cancer risk due to acrylonitrile/.

Endpoint:
basic toxicokinetics in vivo
Type of information:
other: expert review / secondary source
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
The EU RAR summarises and reviews the data available on the toxicokinetics of acrylonitrile. The toxicokineitcs of acrylonitrile have been investigated in a number of non-standard studies in various species using different routes of administration; the RAR provides a valuable summary and interpretation of the extensive and sometimes conflicting data.
Objective of study:
absorption
Qualifier:
no guideline followed
Principles of method if other than guideline:
A large number of studies of various designs are reviewed in the EU RAR. The findings of those studies investigating absorption are summarised.
GLP compliance:
not specified
Remarks:
: largely published studies
Radiolabelling:
yes
Remarks:
: the majority of the studies used 14C-radiolabelled acrylonitrile
Species:
rat
Strain:
other: the reviewed studies used various rat strains
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
not specified
Details on exposure:
The studies reviewed are largely oral gavage studies in the rat, with some studies also investigating inhalation. ip and iv administration.
Duration and frequency of treatment / exposure:
Single dose
Remarks:
Doses / Concentrations:
Various dose levels are used in the studies reviewed.
No. of animals per sex per dose:
No information available
Control animals:
no
Positive control:
Not required
Details on study design:
Published review: limited individual study methodolgy is reported.
Details on dosing and sampling:
Published review: limited individual study methodolgy is reported.
Statistics:
Not performed
Preliminary studies:
Not applicable
Details on absorption:
The absorption of acrylonitrile was demonstrated to be 95-98% of an administered oral dose. Peak blood levels are achieved within 3 hours of oral administration. Half lives of 61 and 70 mins in blood and liver, respectively,have been determined after oral administration. Higher levels in these tissues were achieved after i.v. and i.p. administration than after oral dosing, with a rapid decrease of initial high concentrations. Half lives of 19 minutes for blood and 15 minutes for liver were determined.
Details on distribution in tissues:
Acrylonitrile and/or its metabolites accumulated in the liver, kidney, intestinal mucosa, adrenal cortex and blood (after oral administration).
Details on excretion:
Investigations are reviewed elsewhere
Metabolites identified:
not measured
Details on metabolites:
Not applicable: metabolic investigations are reviewed elsewhere

Absorption following oral exposure

Kedderis et al (1993) demonstrated absorption of approximately 95 -98% of an administered oral dose of acrylonitrile. Ahmed et al (1982) and others indicates that peak blood levels are achieved within 3 hours of oral administration. Gut et al (1981) determined half lives of 61 and 70 minutes in blood and liver, respectively, after oral administration. Higher levels in these tissues were achieved after i.v. and i.p. administration than after oral dosing, with a rapid decrease of initial high concentrations. Half lives of 19 minutes for blood and 15 minutes for liver were determined. Sandberg & Slanina (1980) showed that acrylonitrile and/or its metabolites accumulated in the liver, kidney, intestinal mucosa, adrenal cortex and blood (after oral administration). However, it is reported that most of the radioactivity asscoiated with acrylonitrile was irreversibly bound to proteins (Peter & Bolt, 1981), making it difficult to determine whether the high levels of [14C]-radioactivity in various tissues were due to free acrylonitrile, its metabolites or to cyanoethylated proteins. Nerudova et al (1981) suggest that free acrylonitrile is relatively uniformly distributed and that the higher concentrations of radioactivity seen in some organs and erythrocytes are related to the reaction products of acrylonitrile with soluble and protein sulphydryl groups.

Absorption following dermal exposure

The data available for dermal absorption are reviewed elsewhere, but are consistent with the extensive absorption of acrylonitrile following dermal exposure.

Absorption following inhalation exposure

The available data are limited but indicate almost complete retention of acrylonitrile in the rat following inhalation exposure. Additional information suggest that absorption may be biphasic and that the rate of uptake may be increased following glutathione depletion.

Conclusions:
The data reviewed in the EU RAR suggest the rapid and almost complete absorption of acrylonitrile following oral administration in the rat.
Executive summary:

The 2004 EU RAR summarises and reviews the extensive dataset available on the toxicokinetics of acrylonitrile. The data are largely non-standard published studies but indicate rapid and extensive (almost complete) absorption of acrylonitrile following oral and inhalation exposure. The data on absorption following dermal exposure are summarised elsewhere but also indicate extensive absoprtion following dermal exposure.

Endpoint:
basic toxicokinetics in vivo
Type of information:
other: expert review / secondary source
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Objective of study:
excretion
Qualifier:
no guideline followed
Principles of method if other than guideline:
The findings of a number of non-standard studies investigating the excretion of acrylonitrile are summarised in the EU RAR (2004).
GLP compliance:
no
Remarks:
: not relevant for the EU RAR, the individual studies are from the literature and are not GLP
Radiolabelling:
yes
Remarks:
14-C vinyl
Species:
other: the studies summarised use various species, but mainly the rat
Strain:
other: various
Sex:
male
Details on test animals and environmental conditions:
Various species/strains were used, including male Sprague-Dawley rats and male Fischer 344 rats.
Route of administration:
oral: gavage
Vehicle:
other:
Details on exposure:
The studies reviewed in the EU RAR mainly used gavage dosing, however the results of studies using other exposure routes are also reported.
Duration and frequency of treatment / exposure:
Various treatment patterns are used.
Remarks:
Doses / Concentrations:
Various
No. of animals per sex per dose:
Various
Control animals:
not specified
Details on study design:
Various studies investigating the excretion of acrylonitrile
Preliminary studies:
Not applicable
Details on absorption:
Studies reviewed/summarised in the EU RAR and relevant to absorption are reported elsewhere
Details on distribution in tissues:
Studies reviewed/summarised in the EU RAR and relevant to distribution are reported elsewhere
Details on excretion:
Following oral dosing, approximately 5% of the total dose of acrylonitrile administered is estimated to be exhaled unchanged. The net amount of unchanged acrylonitrile eliminated in expired air in Sprague-Dawley rats (n=3) given a single oral dose of 46.5 mg/kg of [1-14C]-acrylonitrile reached a peak at 30 minutes after dosing. Thereafter it rapidly decreased, not being detectable 2.5 hours after treatment. Although only 2.5% of radioactivity was exhaled as trapped 14CO2 during the first 12 hours, a maximum of 9% of the dose was recovered as 14CO2, 0.5% as H14CN and 4.8% as unchanged acrylonitrile in 24 hours. In this study 40% was excreted in urine and 2% in faeces in 24 hours.

Urinary excretion is the major excretory route, with only 3-8% of a given dose being excreted in the faeces. The bulk of the urinary excretion takes places within 24 hours. In another study, 46 mg/kg of [2-14C]-acrylonitrile was administered by gavage to male F344 rats. After 24 hours 10.7+0.8% had been excreted as CO2, 2.0+0.4% as volatiles, 67.0+2.2% in the urine, 11.4+0.6% in faeces, with 9.8+0.2% remaining in blood and 4.1+0.02% in the tissues.
Metabolites identified:
not measured
Details on metabolites:
Studies reviewed/summarised in the EU RAR and relevant to metabolism are reported elsewhere

It has been demonstrated that the CO2 excreted via respiratory air is mainly from the intermediate cyanide, by varying the position of the radiolabel within the molecule.

Conclusions:
Urinary excretion is shown to be the major excretory route for acrylonitrile, administered by the oral and other routes. The bulk of the urinary excretion takes place within 24 hours. The total excretion of radiolabel after 10 days was 75%, indicating a retention of about 25% acrylonitrile either bound to macromolecules or in the form of non-excretable conjugates. Excretion in exhaled air of unchanged acrylonitrile, carbon dioxide and hydrogen cyanide is also noted.
Executive summary:

Urinary excretion is the major excretory route for acrylonitrile, administered by the oral and other routes. The bulk of the urinary excretion takes place within 24 hours. The total excretion of radiolabel after 10 days was 75%, indicating a retention of about 25% acrylonitrile either bound to macromolecules or in the form of non-excretable conjugates. The excretion in exhaled air of unchanged acrylonitrile, carbon dioxide and hydrogen cyanide is also noted.

Endpoint:
basic toxicokinetics in vivo
Type of information:
other: expert review / secondary source
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The EU RAR (2004) reports the results of a large number of non-standard investigative studies investigating the toxicology and toxicokinetics of acrylonitrile. The findings of metabolism studies are summarised here.
GLP compliance:
no
Radiolabelling:
yes
Species:
other: Rats, mice and rabbits
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
Various studies are reviewed using different species.
Route of administration:
oral: gavage
Vehicle:
not specified
Duration and frequency of treatment / exposure:
Various treatment patterns were used in the summarised studies.
Remarks:
Doses / Concentrations:
Various dose levels were used in the summarised studies.
No. of animals per sex per dose:
Various
Control animals:
not specified
Positive control:
Not required
Details on study design:
The individual studies are of various designs
Details on dosing and sampling:
The individual studies are of various designs
Statistics:
Not reported
Preliminary studies:
Not applicable
Details on absorption:
Studies focusing on the absorption of acrylonitrile and summarised in the EU RAR are reported separately in this dossier
Details on distribution in tissues:
Studies focusing on the distribution of acrylonitrile and summarised in the EU RAR are reported separately in this dossier
Details on excretion:
Studies focusing on the excretion of acrylonitrile and summarised in the EU RAR are reported separately in this dossier
Metabolites identified:
yes
Details on metabolites:
Pathway 1

A number of studies in various experimental animal species have identified that the major metabolite of acrylonitrile (following oral administration) is N-acetyl-S-(2-cyanoethyl)cysteine. This metabolite results from the conjugation of acrylonitrile with glutathione, the initial step of pathway [1]. Further metabolism via this pathway results in a number of metabolites in the rat urine. Major metabolites include thiocyanate, N-acetyl-S-2-(2-cyanoethyl)cysteine and 4-acetyl-5-cyanotetrahydro-1,4-2H-thiazine-3-carboxylic acid. The chemical structures of the remaining metabolites were not identified but none contained the -CN group of acrylonitrile.

Pathway 2

The cytochrome P450-dependent pathway of acrylonitrile biotransformation [2] includes a number of consecutive enzyme-catalysed or spontaneous reactions. Studies indicate that the initial metabolic step in the pathway is the cytochrome P450-mediated epoxidation of the vinyl bond in acrylonitrile to form the reactive epoxide metabolite 2-cyanoethylene oxide (CEO). CEO subsequently reacts with cellular glutathione at either the 2- or 3- position. The 2-position CEO conjugate is further metabolised to N-acetyl-S-(1-cyano-2-hydoxyethyl)cysteine; the 3-position CEO conjugate is metabolised to cyanide and, following the rhodanese-mediated reaction with endogenous thiosulphate, to thiocyanate. Studies using oral administration to the rat report approximately 20-25% metabolism of acrylonitrile to urinary thiocyanate. The relative proportions of the 2- and 3- position glutathione conjugates of CEO formed during the initial step of pathway [2] determine the amount of cyanide liberated; the metabolism of CEO to cyanide has been shown by some investigators to be more extensive in mice than in rats and it is postulated that this difference may be responsible for the sensitivity of mice to the acute toxicity of acrylonitrile.

The formation of carbon dioxide is also important in the metabolism of acrylonitrile, accounting for 2.5-17% of the administered dose.

Metabolism following inhalation exposure

The metabolism of acrylonitrile following inhalation exposure is qualitatively similar to that following oral administration, however some quantitative differences have been noted. Thiocyanate is reported to be the major metabolite of acrylonitrile following inhalation exposure; the proportion of acrylonitrile metabolised to urinary thiocyanate by pathway [2] is reported to be 15-16%. Other urinary metabolites were identified as N-acetyl-S-(2-cyanoethyl)cysteine (2-cyanoethylmercapturic acid; CMA) resulting from pathway [1]and N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine (2-hydroxyethylmercapturic acid; HMA), formed by pathway [2]; the relative proportion of thiocyanate (compared to CMA and HMA) increases at higher levels of exposure. Other workers report a non-linear increase in the excretion of the pathway [1] metabolites CMA and -S-(2-cyanoethyl)thioacetic acid with increasing dose. Tardif et al suggest that the saturation of the cytochrome P-450 pathway [2] occurs at relatively higher exposure levels in the rat that the generation of CEO via pathway [2] is the major metabolic route for inhaled acrylonitrile up to 100 ppm. Other workers report a dose-related increase in the urinary excretion of unchanged acrylonitrile following inhalation exposure. Additional pathway [2] metabolites following inhalation exposure have been identified as S-carboxymethyl cysteine and thiodiglycolic acid

Metabolism following other exposure routes

Tardif et al report that CMA is the most important urinary metabolite (74-78% of total), with lower levels of urinary thiocyanate compared to other exposure routes.

Metabolic pathways

On the basis of the studies reviewed, the EU RAR concludes that acrylonitrile is metabolised by two pathways.

Pathway 1: direct conjugation of acrylonitrile with glutathione (GSH), either with or without catalysis by glutathione transferases, representing a detoxification step.

Pathway 2: the cytochrome P450-mediated oxidation of acrylonitrile to cyanoethylene oxide (CEO), considered to be an activation step.

The initial step of Pathway 1 can either occur non-enzymatically via a Michael reaction or via catalysis by GSH-transferase. This reaction has been proposed to be responsible for the observed depletion of GSH from various tissues (brain, lung, liver, kidney, stomach, red blood cells) after acrylonitrile administration. The binding of acrylonitrile and CEO to GSH and other protein sulphydryl groups may be responsible for the inhibition of various thiol-dependent enzymes observed following acrylonitrile administration. The prolonged failure to maintain adequate levels of intracellular glutathione can result in the impairment of cellular redox processes the increased binding of acrylonitrile and CEO to cellular macromolecules. Consistent with this is the observation that exogenous thiols such as cysteine and N-acetylcysteine have been shown to afford some protection against the toxicity of acrylonitrile in experimental studies.

A number of studies have shown that the major metabolite of acrylonitrile in the rat, rabbit and other experimental animals is N-acetyl-S-(2 -cyanoethyl)cysteine formed following the conjugation of acrylonitrile with GSH (Pathway 1). Fennell et al (1991) and Kedderis et al (1993) determined the urinary metabolite profile of radiolabelled acrylonitrile over 72 hours following the gavage administration of single doses (0.09 -28.8 mg/kg bw) to male rats and reported that metabolites arising from the conjugation of acrylonitrile with GSH (Pathway 1) represented approximately 85% of all urinary metabolites. The excretion of the Pathway 1 metabolites N-acetyl-S-(2-cyanoethyl)cysteine and -S-(2-cyanoethyl)thioacetic acid has been shown to increase in a non-linear fashion with increasing dose, suggesting the existence of a competing and saturable pathway. This pathway is considered likely to be the cytochrome P450-dependent Pathway 2.

The relative contributions of the two pathways to the overall biotransformation of acrylonitrile metabolism are clearly influenced by dose level, with saturation of the cytochrome-P450 dependent Pathway 2 reported by various authors following gavage, iv and ip administration and metabolism of acrylonitrile predominantly to N-acetyl-S-(2- cyanoethyl)cysteine by Pathway 1. A different pattern is seen following the low dose administration of acrylonitrile in the drinking water, diet or by inhalation, where metabolism to CEO by Pathway 2 appears to predominate. The work of Pilon et al (1988a,b) shows that experimental depletion of glutathione shifts the metabolism of acrylonitrile from Pathway 1 to Pathway 2.

Conclusions:
Data from studies in the rat indicate the metabolism of acrylonitrile by two pathways. The oxidation of acrylonitrile to CEO can be considered an activation step, while conjugation of acrylonitrile or CEO with GSH can be considered as a detoxification step. The EU RAR concludes that the comparative data indicate that the rate of conjugation of ACN or CEO with GSH is lower in humans than in either rats or mice, but that the hydrolysis of CEO by epoxide hydrolase is very high in humans, while this detoxification pathway is apparently absent in rodents. The data therefore indicate that CEO is detoxified by GSH in rats or mice, but predominantly by epoxide hydrolase in humans. The metabolite resulting from the human epoxide hydrolase pathway (glycolaldehyde cyanohydrin) is rapidly converted to hydoxyacetaldehyde and hydrogen cyanide. The oxidation of acrylonitrile to CEO is considered to be an activation step, while the conjugation of acrylonitrile or CEO with GSH can be considered as a detoxification step. The balance between these two processes may play a role in the carcinogenic susceptibility of different animal species. The predominant biotransformation pathway appears to be dependent on the systemic dose.
Executive summary:

The metabolism of acrylonitrile in experimental animals has been extensively investigated and appears to proceed via two pathways. The metabolism of acrylonitrile has been shown to be influenced by species, dose level and route of administration.

Pathway 1 represents a detoxification step and involves the conjugation of acrylonitrile with glutathione. The terminal rat metabolite in this pathway is 2 -cyanomethylmercapturic acid (CMA). CMA is a relatively minor metabolite (8%) following inhalation exposure but is seen at much higher levels (~75%) following intraperitoneal or intravenous exposure. Metabolism via Pathway 1 also appears to predominate following gavage dosing but is less important following low-level exposure in the diet, drinking water or by inhalation. Data suggest that this pathway is saturable and is less important in the mouse than the rat.

Pathway 2 represents an activation step and involves the cytochrome P450 -mediated formation of the epoxide metabolite 2 -cyanoethyleneoxide (CEO). CEO can be conjugated with glutathione either at the 2 -position to form (via an intermediate) the metabolite 2-hydroxyethylmercapturic acid (HMA) or alternatievly can be conjugated with glutathione at the 3 -position to form (via intermediate metabolites) N-acetyl-S-(carboxymethyl)cysteine or (via thiodiglycolic acid) thionyldiacetic acid. The further metabolism of the 3 -position glutathione conjugate results in the liberation of cyanide, which is converted by rhodanese to thiocyanate following reaction with endogenous thiosulphate. The relative proportions of the 2 -and 3- position conjugates influence the amount of cyanide liberated, and may potentially be responsible for species differences in the acute toxicity of acrylonitrile. Pathway 2 is quantitatively more important following low-level exposure to acrylonitrile in the diet, drinking water or by inhalation and also becomes more important following the saturation of Pathway 1 at high levels of exposure. Investigators have also demonstrated that the generation of carbon dioxide from radiolabelled acrylonitrile originates from the cyano-group on the metabolite.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
Comparison of the toxicokinetics of acrylonitrile in normal and glutathione-depleted rats
GLP compliance:
not specified
Remarks:
: published study
Radiolabelling:
yes
Remarks:
[2,3-14C]-acrylonitrile
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Adult male Fischer 344 rats; control and GSH-depleted
Route of administration:
oral: gavage
Vehicle:
not specified
Details on exposure:
Adult rats were gavaged with a single dose of acrylonitrile at a dose level of 4 mg/kg bw
Duration and frequency of treatment / exposure:
Single dose
Dose / conc.:
4 mg/kg bw (total dose)
Remarks:
Single gavage dose
Control animals:
no
Positive control:
Not relevant
Details on study design:
[2,3-14C]-acrylonitrile was administered to rats that were glutathione depleted (and rats that were non-glutathione depleted), rats were monitored for 24 hours.
Details on dosing and sampling:
A single oral dose of radiolabelled acrylonitrile was administered to rats. Uptake into target organs was determined over a 24 hour period.
Statistics:
No information available.
Preliminary studies:
Not applicable.
Details on absorption:
Glutathione depletion resulted in greater uptake into target organs (brain, stomach, liver, kidney and blood) compared to non-depleted rats
Details on distribution in tissues:
Glutathione depletion was reported to increase the uptake of radioactivity into tissues.
Details on excretion:
Urinary excretion of the metabolite thiocyanate was increased by 300% in glutathione-depleted rats.
Metabolites identified:
yes
Details on metabolites:
Metabolism to 2-cyanoethylene (CEO) was increased by 300% in glutathione-depleted rats.

In addition to increasing the uptake of radioactivity into the tissues, glutathione depletion caused an increase in covalently bound tissue radioactivity at 6 and 24 hours after dosing.

Conclusions:
The findings of this study indicate that glutathione has a key role in the metabolism and toxicity of acrylonitrile.
Executive summary:

The authors investigated the role of glutathione in the metabolism of acrylonitrile using a glutathione-depleted rat model. A single oral dose of 4 mg/kg bw [2,3 -14C]-acrylonitrile was administered to male Fischer 344 rats. Rats were either glutathione depleted or non-depleted controls. Glutathione depletion resulted in greater uptake into target organs (brain, stomach, liver, kidney, and blood) compared with non-depleted rats. Metabolism to CEO and the urinary excretion of thiocyanate were both increased by 300% by glutathione depletion. In addition to increasing the uptake of radioactivity into the tissues, glutathione depletion caused an increase in covalently bound radioactivity between 6 and 24 hours after dosing. The authors suggested that glutathione could play a role in the extent of metabolism of acrylonitrile to the key metabolite CEO by the cytochrome P450 system and hence the distribution of acrylonitrile-derived species to tissue macromolecules and nucleic acids.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
Toxicokinetics of acrylonitrile in the rat following exposure by inhalation and the influence of glutathione
GLP compliance:
not specified
Remarks:
: published study
Radiolabelling:
yes
Remarks:
[2,3-14C]-acrylonitrile
Species:
rat
Strain:
Fischer 344
Sex:
male
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
Rats were exposed to radiolabelled acrylonitrile vapour in an inhalation chamber.
Duration and frequency of treatment / exposure:
240 minutes
Remarks:
Doses / Concentrations:
25-750 ppm
Control animals:
not specified
Details on study design:
Rats were exposed to acrylonitrile vapour to determined the rate of uptake of [2,3-14C]-acrylonitrile over 240 minutes exposure. GSH depleted rats were also exposed to 100 ppm.
Details on dosing and sampling:
Uptake was assessed by measurement of the concentration in the exposure chamber over a 240 minute period. Radioactivity was measured in various tissues following exposure.
Statistics:
No further information
Preliminary studies:
Not applicable
Details on absorption:
Biphasic uptake of [2,3-14C]-acrylonitrile was seen, characterised by a dosage-dependent rapid phase lasting for approximately 60 minutes and a subsequent slow phase which lasted from 60 minutes to the end of exposure. The rate of uptake for both phases was linearly related to the initial concentration of acrylonitrile in the chamber. Using the rate of uptake curve for the rapid phase, the authors estimated a rate of 4.82 mg/kg/hr uptake at an exposure level of 100 ppm.
Details on distribution in tissues:
The depletion of GSH resulted in a decrease in total radioactivity recovered in the brain, stomach, liver, kidney and blood and a concomitant decrease in the acrylonitrile-derived nondialysable radioactivity in these organs. In non-GSH depleted rats, accumulation of radiolabel was greatest in brain RNA, but no radioactivity was detected in DNA of any organ examined. In GSH-depleted rats, the radiolabel concentration was higher in the brain RNA than in liver or stomach RNA, but was also 50% lower than that observed in brain RNA of non-GSH-depleted rats.
Details on excretion:
Urinary excretion of thiocyanate, derived from the epoxide pathway of acrylonitrile metabolism, was doubled in GSH-depleted rats compared with non-depleted rats.
Metabolites identified:
yes
Details on metabolites:
Thiocyanate was excreted in the urine.

Glutathione depletion increased the uptake of acrylonitrile, the covalent binding of acrylonitrile metabolites, and also increased the excretion of thiocyanate.

Conclusions:
Glutathione depletion increased the uptake of acrylonitrile, the covalent binding of acrylonitrile metabolites, and also increased the excretion of thiocyanate.
Executive summary:

The toxicokinetics of acrylonitrile following inhalation were investigated in male F344 rats (control and glutathione-depleted) exposed for 4 hours to concentrations of 25 -750 ppm. The study demonstrates biphasic uptake of radiolabelled acrylonitrile in male F344 rats following inhalation exposure to all concentrations. Findings were characterised by a concentration-independent rapid phase lasting for approximately 60 minutes and a subsequent slow phase. The rate of uptake for both phases was linearly related to the initial concentration of acrylonitrile. Using the rate of the uptake for the rapid phase, the authors estimate a rate of 4.82 mg/kg/hr uptake at an exposure level of 100 ppm. The study also shows that glutathione depletion results in an increase in the rate of acrylonitrile uptake via inhalation in both phases. In common with the oral exposure study reported by the same authors, depletion of GSH was shown to result in a decrease in total radioactivity recovered in the brain, stomach, liver, kidney and blood and a concomitant decrease in the non-dialysable radioactivity in these organs. In control rats, the accumulation of radiolabel was greatest in brain RNA, but no radioactivity was detected in the DNA of any organ examined.  In GSH-depleted rats, the radiolabel concentration was higher in the brain RNA than in liver or stomach RNA, but was also 50% lower than that observed in brain RNA of control rats. The urinary excretion of thiocyanate (derived from the epoxide pathway of acrylonitrile metabolism) in GSH-depleted rats was twice that of controls.

Endpoint:
basic toxicokinetics
Type of information:
other: expert review / secondary source
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Independently peer-reviewed review of the available data on the toxicokinetics of acrylonitrile.
Objective of study:
other: review of available toxicokinetics data
Qualifier:
no guideline followed
Principles of method if other than guideline:
This comprehensive review of the toxicology of acrylonitrile includes an assessment of the toxicokinetics, based on a summary and review of the literature.
GLP compliance:
no
Remarks:
: not relevant to the review, individual studies are not GLP
Radiolabelling:
other: some of the reviewed studies used radiolabelled material.
Species:
other: The reviewed studies were performed mainly in the rat, with some data available from other species including the mouse and human
Strain:
other: various
Sex:
male/female
Details on test animals and environmental conditions:
Various
Route of administration:
other: the reviewed studies used various routes of administration, but predominantly oral (gavage) and inhalation
Vehicle:
other: various
Duration and frequency of treatment / exposure:
The reviewed studies used a number of treatment schedules
Remarks:
Doses / Concentrations:
The reviewed studies used various different doses / exposure concentrations
No. of animals per sex per dose:
various
Control animals:
other: control animals were included in some of the reviewed studies
Positive control:
Not relevant
Details on study design:
Various designs are used in the reviewed studies
Statistics:
Not performed
Preliminary studies:
Not applicable
Details on absorption:
Acrylonitrile is a small, water soluble molecule. Absorption occurs by passive diffusion of the acrylonitrile molecule through the gastrointestinal tract, respiratory tract, or skin. Absorption is rapid and extensive following ingestion or inhalation.

Ingestion

In rats exposed to a single oral dose of 46.5 mg/kg bw, peak tissue concentrations were generally achieved within three hours of exposure, indicating rapid oral absorption(Ahmed et al., 1982, 1983). Following oral exposure of rats to 0.09 to 28.8 mg/kg bw and of mice to 0.09 to 10 mg/kg bw, the absorption also appears to be complete (Kedderis et al., 1993). Based upon the amount of radiolabel excreted in the urine and faeces up to 72 hours after dosing, absorption ranged from 82-100% in the rat, and from 85-100% in the mouse. There is no evidence of any dose-dependency on the extent of absorption. Results from an unpublished study in rats also indicate that acrylonitrile is well absorbed, ranging from 90-98% (Young et al., 1977).

Inhalation

High retention of AN (91.5%) was reported in rats exposed to initial air concentrations of 1800 ppm (3,900 mg/m3) acrylonitrile, which became depleted in a biphasic manner reaching approximately 10-20 ppm after five hours of exposure in a closed chamber system. Based upon an analysis of time-course data, the amount of acrylonitrile exhaled by the rats was estimated to be 8.5% (Peter and Bolt, 1984). Absorption was also essentially complete in Rhesus monkeys exposed to approximately 30 ppm AN for six hours (Peter and Bolt, 1984).
Details on distribution in tissues:
Following absorption, acrylonitrile and its metabolites are readily distributed throughout the body. Available data regarding distribution of summarised below via route of exposure.

Ingestion

Following oral or intravenous exposure to radiolabelled acrylonitrile, most of the radiolabel accumulated in the liver, kidney, stomach mucosa, lung, and adrenal cortex and blood of rats and monkeys (Sandberg & Salina, 1980). Gut et al (1981) determined half-lives of 61 and 70 minutes in blood and liver, respectively, in exposed Wistar rats. Elimination rates were similar for intravenous, intraperitoneal, and subcutaneous exposures, although the metabolite composition differed for oral exposures. In adult male Sprague-Dawley rats receiving a single oral dose of 46.5 mg/kg bw, the highest initial concentrations of radiolabel were observed in the stomach and intestines (Ahmed et al, 1982, 1983). The red blood cells retained significant amounts of radioactivity for more than ten days after treatment, whereas the radiolabel declined sharply in plasma. Initially, the highest levels of radioactivity were found in the stomach and stomach content followed by the intestine. In liver, kidney, brain, spleen, adrenal, lung, and heart tissues the radioactivity of the acid soluble fractions declined while covalent binding to macromolecules remained unchanged. In subcellular fractions of liver, kidney, spleen, brain, lung, and heart, 20-40% of the total radioactivity was bound to nuclear, mitochondrial, and microsomal fractions whereas in cytosolic fractions only 6-14% was bound over a period of six hours.

The position of the radiolabel shows some differences, as when compared to [1-14C]-acrylonitrile administered to animals, the percentage of covalent binding of [2,3-14C]-acrylonitrile was significantly higher even 72 hours after dosing. The irreversible binding of [2,3-14C]-acrylonitrile to proteins, RNA, and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg bw was studied (Farooqui & Ahmed, 1983b). Binding of acrylonitrile to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach, and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain, and stomach than that in the liver. Covalent binding indices for the liver, stomach, and brain at 24 hours after dosing were, 5.9, 51.9, and 65.3, respectively. More recent studies have failed to confirm the binding observed to DNA (Pilon et al, 1988a,b; Whysner et al, 1998). It has been hypothesised that the label binding observed in the early study may be attributable to the contamination of DNA with protein-bound radiolabel. Irreversible binding to tissue macromolecules was assessed in control and glutathione-depleted F-344 rats treated with an oral dose of 4 mg/kg bw [2,3-14C]acrylonitrile (Pilon et al, 1988a). Glutathione was depleted in rat tissues by the administration of a combined intraperitoneal phorone/buthionine sulfoximine treatment. The amount of total radioactivity recovered from brain, stomach (target organs), liver, kidney, lung, and blood (non-target organs) was similar between control and glutathione-depleted rats. However, stomach, lung, blood, and liver showed an increase in total radioactivity content after glutathione depletion. Glutathione depletion also caused an increase in acrylonitrile-derived non-dialysable radioactivity in liver, lung, kidney, stomach, blood, and brain macromolecules between six and 24 hours after the dose. There was no organ-specific accumulation of radiolabel in RNA in control rats. However, an increase in the radiolabel associated with RNA in the target organs but not in the non-target organs was measured in glutathione-depleted rats. The tissue distribution of CEO was investigated in F344 rats and B6C3F1 mice following a single oral dose of 3 mg/kg (Kedderis et al, 1993). Radioactivity from [2,3-14C]-CEO was widely distributed in the major organs by two hours and decreased by 71-90% within 24 hours, demonstrating that there was no preferential tissue uptake or retention of CEO. CEO was detected in rodent blood and brain 5-10 min after an oral dose of 10 mg/kg acrylonitrile, demonstrating that this metabolite rapidly circulates to extrahepatic target organs following exposure. Acrylonitrile and CEO were detected in the blood of male Fisher-344 rats after administration of 3,10, or 30 mg acrylonitrile/kg bw by gavage. Acrylonitrile and CEO were also measured in the liver and brain for the 10 mg/kg dose (Kedderis et al, 1996). Peak tissue concentrations for acrylonitrile and CEO were achieved within 0.2 hours. Although acrylonitrile concentrations were comparable for both tissues (1-10 mg/L), CEO concentrations were considerably lower (~0.01-0.02 mg/L) in the brain compared to liver (0.05-0.08 mg/L).

Inhalation

The effect of glutathione depletion on acrylonitrile distribution was investigated in male F344 rats (Pilon et al., 1988b) given a 4 mg/kg bw dose of [2,3-14C]-acrylonitrile by inhalation. Tissue uptake of acrylonitrile into brain, stomach, liver, kidney, and blood was enhanced by glutathione depletion in exposed male Fischer 344 rats. However, GSH depletion caused a decrease in total radioactivity recovered in brain, stomach, liver, kidney, and blood and a concomitant decrease in the acrylonitrile-derived non-dialysable radioactivity in these organs. I n control rats, accumulation of radiolabel was greatest in brain RNA, but no radioactivity was detected in DNA of any organ examined. In GSH-depleted rats, the radiolabel concentration was higher in brain RNA than in the liver or stomach RNA, but was also 50% lower than that observed in brain RNA of control rats. Following a three hour exposure of male Fisher-344 rats to 186, 254, or 291 ppm acrylonitrile, acrylonitrile and CEO concentrations were detected in blood, brain, and liver (Kedderis et al, 1996). Concentrations of acrylonitrile and CEO were generally higher in brain than in liver, and decreased rapidly in both tissues following cessation of exposure.

Other Routes

Radiolabel tends to be irreversibly bound to tissue proteins (Peter and Bolt, 1981). Within cells, radioactivity levels were higher in nuclear, mitochondrial, and microsomal fractions compared to cytosolic fraction. Binding was not dependent on NADPH, and occurred in heat-inactivated microsomes, indicating that metabolism was not required. Binding of the radiolabel was inhibited by the addition of thiols (glutathione, cysteine, mercaptoethanol). The distribution and accumulation of acrylonitrile after the single intraperitoneal injection of [2,3-14C]acrylonitrile were examined by whole-body autoradiography and by the determination of radioactivity in several tissues and subcellular fractions after a whole-body perfusion (Sato et al, 1982). The radiolabel was seen strongly in blood, particularly in red blood cells, and in several tissues including lung, liver, and kidney. Longer retention of radioactivity in brain and muscle was
observed. At the sub-cellular level, a relatively high specific radioactivity was seen in cytosolic fractions of the brain, liver, and kidney. Following iv injection of [1-14C]-acrylonitrile in the rat, high concentrations of total radiolabel were found in blood, liver, duodenum, kidney, and the adrenal glands (Silver et al., 1987). Except for blood, there was a time-dependent decrease in total radiolabel in these tissues. Compared with other major organ systems, the levels of covalently bound radiolabel were lower in the adrenal glands. An in vitro study was conducted to determine partition coefficients for acrylonitrile and CEO (Teo et al 1994). Active uptake of acrylonitrile was observed in rat blood due to reaction with blood sulphydryl groups, while CEO reacted with all tissues examined (rat blood, muscle, fat, liver, and brain). The active uptake processes were first order as evidenced by a linear decrease in the log of the vial headspace concentrations over time. Linear extrapolation of the log of the apparent partition coefficient to zero time, where the contribution of the active uptake process is zero, yielded an estimated partition coefficient of 487 for acrylonitrile in blood. Equilibrium was achieved with acrylonitrile after treatment of blood with diethyl maleate to modify blood sulphydryl groups, with a partition coefficient of 512. The directly measured acrylonitrile blood:air PC was 437, which compared well with the estimated values. Treatment of tissues with diethyl maleate or 2,4-dinitrofluorobenzene did not abolish the active uptake of CEO. However, pre-treatment of tissues with CEO itself abolished subsequent CEO uptake. The CEO blood:air PC estimates obtained from zero time extrapolation of four CEO concentrations (1672 +/- 139) and from CEO pre-treatment (1658 +/- 137, n = 8) were in good agreement. The time course of acrylonitrile and CEO in the blood of male Fischer-344 rats was measured for iv doses of 3.4, 47, 55, and 84 mg/kg acrylonitrile (Gargas et al., 1995). Peak acrylonitrile levels (exceeding 100 mg/L at the highest dose) were achieved within 0.1 hours of exposure, whereas peak CEO levels (up to 0.1 mg/L) were achieved more slowly (~0.4 hours). A dose-dependent formation of haemoglobin adducts was observed, corresponding to approximately 2-5% of the administered dose. The dose dependence of acrylonitrile covalent binding to tissue protein, following a single acute exposure over a broad range of doses (administered subcutaneously), was investigated (Benz et al. 1997b). Covalent binding was a linear function of acrylonitrile dose in the lower dose range (0.02-0.95 mmol/kg). The slopes of the dose-response curves indicated that tissues varied by nearly 10-fold in their reactivity with acrylonitrile. The relative order of covalent binding was as follows: blood > > kidney = liver > forestomach = brain > glandular stomach > > muscle. Similar dose-response behaviour was observed for globin total covalent binding and for globin N-(2-cyanoethyl) valine (CEValine) adduct formation. The latter adduct was found to represent only a small percentage (0.2%) of the total acrylonitrile adduction to globin. Regression of tissue protein binding versus globin total covalent binding or globin CEValine adduct indicated that both globin biomarkers could be used as surrogates to estimate the amount of acrylonitrile bound to tissue protein. At higher acrylonitrile doses, above approximately 1 mmol/kg, a sharp break in the covalent binding dose-response curve was observed, and is explained by the nearly complete depletion of liver glutathione and the resultant termination of acrylonitrile detoxification. Following subcutaneous injection of 115 mg/kg [2,3-(14)C]-acrylonitrile in male Sprague-Dawley rats, protein binding in the liver was reported (Nerland et al., 2001). One set of bound proteins was identified as glutathione-S-transferase, predominantly at a reactive cysteine site on one particular subunit (rGSTM1) of the enzyme. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit. In vitro studies have shown that acrylonitrile can bind to reactive cysteine sites in other proteins as well, including the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Campian et al., 2002). acrylonitrile irreversibly inhibits GAPDH in a temperature-dependent manner with second-order rate constants of 3.7 and 9.2 M-1s-1 measured at 25 and 37 degrees C, respectively. acrylonitrile was found to inactivate GAPDH by covalently binding to cysteine 149 in the active site of the enzyme.
Details on excretion:
The excretion of acrylonitrile and its metabolites occurs predominantly via the urine, with smaller amounts excreted in either the faeces or exhaled breath.

Ingestion

When a single dose of [1-14C]-acrylonitrile was given orally to rats, approximately 27% of the administered dose was excreted in bile in six hours (Ghanayem & Ahmed, 1982). This amount was increased in rats fasted overnight or pre-treated with cobalt chloride. Pre-treatment of rats with phenobarbital produced no change, while diethyl maleate pre-treatment significantly decreased the proportion of the dose excreted in bile in six hours. In rats, following exposure to [1,2-14C] acrylonitrile via oral or ip injection, the majority of the radiolabel (82-93%) was excreted in the urine (Sapota, 1982). A smaller percentage of the dose (3-7%) was exhaled unchanged in 24 hours. Ahmed et al (1982, 1983) report that rats given an oral dose of 46.5 mg/kg radiolabelled acrylonitrile excreted approximately 40% of the radiolabel in urine, 2% in faeces, 9% in expired air as carbon dioxide, 0.5% as HCN and 4.8% as unchanged acrylonitrile in 24 hours. Bile flow increased three times after the administration of acrylonitrile and over a period of six hours, 27% of the radiolabel was recovered in bile. After gavage administration of equimolar doses of [2-14C]-methacrylonitrile (MAN) or [2-14C]-acrylonitrile to male F344 rats, substantial differences were observed in the excretion of these two chemicals (Burka et al, 1994). Approximately 39% of the administered methyl-acrylonitrile dose was eliminated as carbon dioxide within 24 hours of dosing, compared to 11% of an equimolar dose of acrylonitrile. In addition, 31% of the methyl-acrylonitrile dose was exhaled as organic volatiles in 24 hours compared to less than 2% of acrylonitrile. HPLC analysis showed that acrylonitrile was the only organic volatile exhaled by acrylonitrile-treated rats. Urinary excretion of methyl-acrylonitrile was 22% compared to 67% acrylonitrile. The major urinary metabolite of acrylonitrile results from direct conjugation with glutathione, whereas the major urinary methyl-acrylonitrile metabolite results from conjugation of the epoxide with glutathione.

Male F344 rats and B6C3F1 mice were co-administered [1,2,3-13C] acrylonitrile (16-17 mg/kg bw) and [1,2,3-13C]-acrylamide (21-22 mg/kg bw) after 0 or 4 days of administration of unlabeled acrylonitrile or acrylamide (Sumner et al, 1997). Rats and mice excreted metabolites derived from glutathione conjugation with acrylonitrile or acrylamide or derived from glutathione conjugation of the respective the epoxides CEO or glycidamide. In mice, increased urinary excretion of total acrylonitrile- and acrylamide-derived metabolites following repeated co-administration suggested a possible increase in metabolism via oxidation. In addition, mice showed an increased proportion of dose excreted as metabolites derived from glutathione conjugation after five exposures as compared with one exposure that may be related to a significant increase in the synthesis of gluathione or an increase in glutathione transferase activity. No differences between one and five exposures for the rat were reported for AN.

Inhalation

Quantitative analysis of the dose-dependent urinary excretion of acrylonitrile and its metabolites was carried out in male Wistar rats following inhalation exposure of the animals to 1, 5, 10, 50 and 100 ppm for eight hours (Muller et al 1987). The excretion pattern of the compound and its metabolites was dependent on the exposure level; it is concluded that urinary determination of unchanged acrylonitrile and two of its metabolites (cyanoethylmercapturic acid and thioglycolic acid) may be useful for biological monitoring of industrial exposure. Adult male Sprague-Dawley rats were exposed acutely to 0, 4, 20, or 100 ppm via inhalation for six hours (Tardif et al, 1987). Urinary metabolites measured 24 hours after administration were 2-cyanoethylmercapturic acid, 2-hydroxyethylmercapturic acid, and thiocyanate. The relationship between excretion of total urinary metabolites and the degree of exposure was reasonably linear.

Other exposure routes

The excretion of acrylonitrile and its metabolites was investigated in male Sprague-Dawley rats following acute exposure to 0, 0.6, 3.0, or 15 mg/kg bw via iv, or ip dosing (Tardif et al., 1987). Urinary metabolites measured 24 hours after administration were 2-cyanoethylmercapturic acid, 2-hydroxyethylmercapturic acid, and thiocyanate. The relationship between excretion of total urinary metabolites and the degree of exposure was reasonably linear.
Metabolites identified:
yes
Details on metabolites:
The metabolism of acrylonitrile is an important determinant of toxicity, and has direct implications on the mode of action.

Acrylonitrile is initially metabolised by two pathways: (1) conjugation with glutathione, either through catalysis with a cytosolic glutathione-S-transferase (GST) or non-enzymatically; and (2) epoxidation by microsomal cytochrome P4502E1 forming 2-cyanoethylene oxide (CEO). The primary metabolites from both pathways are subject to further metabolism. The glutathione conjugate of acrylonitrile can be converted to a mercapturic acid and excreted in urine. CEO, on the other hand, is metabolised by two pathways: (2a) conjugation with glutathione, either through catalysis with cytosolic GST or non-enzymatically, forming conjugates on the second or third carbon; and (2b) hydrolysis by microsomal epoxide hydrolase. The secondary metabolites of CEO can undergo further metabolism/decomposition. Of potential toxicological importance, cyanide can be released from the CEO metabolite generated by the epoxide hydrolase pathway and from the GSH conjugate formed on the third carbon. Cyanide is detoxified by the mitochondrial enzyme, rhodanese, which uses thiosulphate to form thiocyanate. Thiocyanate has been measured in the blood and brain of Sprague-Dawley rats exposed to 20, 50, 80, or 115 mg/kg bw acrylonitrile by gavage and peak blood cyanide concentrations were attained one hour after dosing in mice and three hours after dosing in rats. HCN has also been detected in exhaled breath of rats exposed to acrylonitrile by the oral route; accounting for 0.5% of a 46.5 mg/kg bw dose rats (Ahmed et al, 1982). The release of cyanide appears to require CYP2E1 activity. While the primary site of acrylonitrile metabolism is the liver, other tissues have the capacity to metabolise acrylonitrile. In rats, metabolism has been demonstrated in vitro in microsomal fractions from a number of tissue sites, including testes, kidney, lung, nasal tissue, small intestines and brain. Human lung lipoxygenase has also demonstrated an appreciable activity for metabolising acrylonitrile to cyanide in vitro, suggesting that there may be additional enzymatic pathways leading to the formation of CEO and the release of cyanide.

Influence of the route of exposure

Based on urinary metabolite data, conjugation with glutathione represents the predominant metabolic pathway for acrylonitrile in rats exposed by gavage or iv or ip injection. The primary metabolite observed in rats and other test species is N-acetyl-S-(2-cyanoethyl)cysteine. Other major metabolites include thiocyanate and 4-acetyl-5-cyanotetrahydro-1,4-2H-thiazine-3-carboxylic acid. However, the metabolism of acrylonitrile exhibits a strong first-pass effect, and as such, the relative importance of each pathway depends upon the route of exposure. In rats, oxidation of acrylonitrile, as indicated by urinary excretion of thiocyanate was greater following oral exposure (23%) when compared to intraperitoneal (4%), subcutaneous (4.6%) and intravenous (1.2%) exposure. Similarly, a marked influence of the route of administration on the pattern of metabolic excretion was reported in rats exposed via inhalation, ip, or iv injection (Tardif et al., 1987). After ip and iv injection, 2-cyanoethylmercapturic acid was the most important metabolite, whereas after inhalation exposure thiocyanate was the primary metabolite.

Species Differences

Mice appear to form CEO at a greater rate than rats. The hydrolysis of CEO by epoxide hydrolase is virtually absent in mice and rats but can be induced in both species.

Influence of dose

Several metabolic factors contribute to non-linear kinetics for acrylonitrile, including the presence of a saturable metabolic pathway and the depletion of cofactors required for metabolism. The urinary excretion of N-acetyl-S-(2-cyanoethyl)cysteine and S-(2-cyanoethyl)thioacetic acid across a wide range of oral doses (0.09-28.8 mg/kg) bw was increased in a non-linear manner. Glutathione depletion has been observed in a number of tissues (brain, lung, liver, kidney, stomach, adrenal gland, erythrocytes) in rats exposed to acrylonitrile and results in an increase in the proportion of acrylonitrile metabolised via the oxidative pathway. The metabolism of acrylonitrile to CEO following oral or inhalation exposures (as indicated by urinary thiocyanate) was enhanced approximately two- to three-fold in glutathione-depleted F344 rats (Pilon et al., 1988).

The authors of the review have considered the data available on the toxicokinetic behaviour of acrylonitrile in various species. It is concluded that a consideration of the factors affecting metabolism is important to the dose-response assessments for acrylonitrile, to ensure that efforts taken to perform both interspecies extrapolation and high-to-low dose extrapolation are appropriate.

Conclusions:
No bioaccumulation of acrylonitrile is predicted due to its rapid metabolism and excretion. However several studies have shown the binding of a reactive metabolite to cellular macromolecules.
Executive summary:

This comprehensive review of the toxicology of acrylonitrile includes an assessment of the toxicokinetics, based on a summary and review of the literature.

Acrylonitrile is rapidly and extensively absorbed following oral or inhalation exposure and is rapidly distributed. Metabolism is rapid and extensive and proceeds via two major pathways, detoxification by glutathione or activation to the epoxide metabolite 2 -cyanoethylene oxide (2 -CEO) by cytochrome P4502E1. The glutathione conjugate of acrylonitrile is further metabolised to a mercapturic acid and is excreted in urine. CEO may be further metabolised by conjugation with glutathione or (via an inducible pathway) hydrolysis by epoxide hydrolase. Cyanide is released from the CEO metabolite generated by the epoxide hydrolase pathway and may be of toxicological importance; cyanide is detoxified by rhodanese to form thiocyanate which is excreted in urine.

The mode of action of acrylonitrile toxicity indicates that many of the adverse effects of are attributable to the formation of one or more metabolites (2-cyanoethylene oxide (CEO) or cyanide). Therefore, the metabolism of acrylonitrile is an important determinant of toxicity, which can vary across individuals and species. Although there may be small differences in the metabolism of acrylonitrile in human populations due to genetic polymorphisms, the impact of these differences on susceptibility is likely to be small.  More importantly, there do not appear to be important differences between males and females, and children are not expected to be at greater risk than adults based on kinetic factors determining the metabolic activation of acrylonitrile. The metabolism of acrylonitrile is complicated by a number of sources of nonlinear kinetics and important species differences are identified with respect to metabolism. Furthermore, the metabolism of is influenced by the route of exposure, species to which it is administered, the magnitude of the exposure, and the vehicle in which it is administered.

The authors of the review have considered the data available on the toxicokinetic behaviour of acrylonitrile in various species. It is concluded that a consideration of the factors affecting metabolism is important to the dose-response assessments for acrylonitrile, to ensure that efforts taken to perform both interspecies extrapolation and high-to-low dose extrapolation are appropriate.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23rd June-17th August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
[14C]acrylonitrile
Species:
other: rat and human skin, in vitro
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Duration of exposure:
1 hour
Doses:
The application volume was 10 µL/cm2
Details on in vitro test system (if applicable):
Charles River UK supplied five samples of full-thickness skin obtained from male CD® rats (Rat/IGS (Crl: CD®(SD) (IGS BR)) aged 6-8 weeks old, weighing 200 to 250 g.

Five samples of full-thickness human skin (2 breast, 1 abdomen, 1 abdomen/breast and 1 abdomen/arms/back) were obtained from donors aged 23 to 53 years old. Samples were cleaned of subcutaneous fat and connective tissue using a scalpel blade. The skin samples were washed in cold running water and dried using tissue paper. The skin samples were then cut into smaller pieces (where appropriate), wrapped in aluminium foil, placed into self sealing plastic bags and stored in a freezer set to maintain a temperature of 20°C until they were used in the study.

Human and rat skin samples were removed from storage and allowed to thaw at ambient temperature. The fur of the rat skin was trimmed using electric clippers. The thickness of the full thickness skin membranes were measured using a micrometer. Split thickness membranes were prepared by pinning the full thickness skin, stratum corneum uppermost, onto a raised cork board and cutting at a setting equivalent to 200 400 µm depth using a dermatome.

An automated flow through diffusion cell apparatus was used. The flow through diffusion cells were placed in a steel manifold heated via a circulating water bath to maintain the skin surface temperature at 32C. The cells were connected to multi-channel peristaltic pumps from their afferent ports with the receptor fluid effluent dropping via fine bore tubing into scintillation vials on a fraction collector. The surface area of exposed skin within the cells was 0.64 cm2. The receptor chamber volume was 0.25 mL. The peristaltic pumps were adjusted to maintain a flow-rate of 1.5 mL/h ± 0.15 mL/h.
Phosphate buffered saline (PBS) was used as the receptor fluid. The receptor fluid was degassed by sonication prior to use. Acrylonitrile has a water solubility of 7.3% (w/w) at 20ºC in water. For an application of 10 µL/cm2 over a 0.64 cm2 application area, this would be 5.16 mg/0.64 cm2 of Acrylonitrile (density = 806 kg/m3). If 100% of Acrylonitrile was absorbed in 1 h (1.5 mL), then 5.16 mg/1.5 mL is 3.44 g/L. Therefore, this receptor fluid was not considered to be rate limiting for solubility and was accepted for use.

Sections of split thickness skin membrane 1.5 x 1.5 cm were cut out, positioned on the receptor chamber of the diffusion cell, which contained a magnetic flea and the donor chamber was tightened into place. The prepared cells were then placed in the heated manifold and connected to the peristaltic pump. The magnetic stirrer was switched on to mix the contents of the receptor chamber. An equilibration period of 15 minutes was allowed while receptor fluid was pumped through the receptor chambers at 1.5 mL/h ± 0.15 mL/h. The effluent was then collected for 30 minutes and retained as blank samples for use in the tritiated water barrier integrity assessment.

Tritiated water (250 µL, 100,000 dpm) was applied to the surface of each skin sample and the donor chamber occluded. Penetration of tritiated water was assessed by collecting receptor fluid for 1 hour and analysing the sample by liquid scintillation counting. The mean dpm applied for the tritiated water was calculated from the seven mock tritiated water samples taken at the time of dosing. Absorption was then calculated for each skin sample from the receptor fluid sample collected. Any human and rat skin sample exhibiting greater than 0.6% absorption was excluded from subsequent absorption measurements. At the end of the 1 hour period, residual tritiated water was removed from the skin surface by rinsing with water (1 2 mL). The skin was then dried with tissue paper. An equilibration period of up to 3 hours was allowed prior to collection of the pre dose sample which was collected for 0.5 hours.

Signs and symptoms of toxicity:
not specified
Remarks:
: not applicable
Dermal irritation:
not specified
Remarks:
: not applicable
Parameter:
percentage
Absorption:
1.3 %
Remarks on result:
other: 24 hours
Remarks:
Human skin
Parameter:
percentage
Absorption:
0.76 %
Remarks on result:
other: 24 hours
Remarks:
Rat skin

Distribution of radioactivity

Species:

Human

Rat

Application rate:

8015.89 µg equiv./cm2 (10 µL/cm2)

8026.48 µg equiv./cm2 (10 µL/cm2)

Distribution

(% Applied Dose)

(µg equiv./cm2)

(% Applied Dose)

(µg equiv./cm2)

Dislodgeable Dose 1 h:

27.10

2160.19

47.98

3824.99

Total Dislodgeable Dose:

27.35

2180.01

48.30

3850.48

Unabsorbed Dose:

27.74

2210.86

48.55

3870.84

Absorbed Dose:

1.05

83.88

0.55

43.59

Dermal Delivery:

1.30

103.47

0.76

60.32

Mass Balance:

29.04

2314.33

49.31

3931.15

Volatile loss after 1 h post dose:

26.80

2136.28

47.78

3809.76

Total volatile loss:

26.97

2149.68

48.07

3832.69

Conclusions:
Following the topical application of [14C]-acrylonitrile to human and rat skin membranes in vitro, the primary route of loss was via volatilisation. This factor is reflected in the low mass balance for both rat and human skin. The absorbed dose was 1.05% (83.88 µg equiv./cm2) and 0.55% (43.59 µg equiv./cm2) in human and rat skin membranes, respectively. Dermal delivery was 1.30% (103.47 µg equiv./cm2) and 0.76% (60.32 µg equiv./cm2), respectively.

The mass balance for human and rat skin membranes was 29.04% and 49.31%, respectively. The low mass balances were attributed to the volatility of acrylonitrile; findings suggest that nearly all of the lost acrylonitrile had volatilised within the first hour after application and that the filter and trap were not effective at collecting all of this volatilised material. Given the high and variable rates of volatile loss, absorption rates could not be reliably compared between the species. However the results indicate that 1.30% in human and 0.76% in rats of the applied dose was either retained in the skin or had been absorbed. The results of the study indicate that systemic absorption resulting from occupational dermal exposure to acrylonitrile is likley to be be limited due to the volatile nature of the substance.
Executive summary:

The dermal absorption of 14C-radiolabeled acrylonitrile was investigated in vitro in human and rat skin membranes. Split‑thickness human and rat skin membranes were mounted into flow‑through diffusion cells using phosphate- buffered saline as receptor fluid. The skin surface temperature was maintained at 32°C ± 1°C throughout the experiment.  A tritiated water barrier integrity test was performed prior to the main study to ensure the integrity of the skin membranes.

14C-acrylonitrile was applied to the topical surface of the skin membranes at an application volume of 10 µL/cm2. Immediately after application, the donor chamber of the cells was occluded with a trap containing an activated charcoal filter intended to collect any volatile fraction. Percutaneous absorption was assessed by collecting receptor fluid in hourly fractions from 0-8 hours post‑application and in 2‑hourly fractions from 8-24 hours post‑application.  At 1 hour post application, the volatile trap was removed and exposure was terminated by washing the skin surface with water and drying with tissue swabs.  A new trap was then used to occlude the cells. At 24 hours post application, the underside of the skin membranes and the receptor chamber were rinsed with receptor fluid. The trap was collected, the skin was removed from the diffusion cells, dried and the stratum corneum was removed with 20 successive tape strips.  The remaining skin was divided into exposed and unexposed skin and solubilised. All samples were analysed by liquid scintillation counting.

The mass balance for human and rat skin membranes was 29.04% and 49.31%, respectively. The low mass balances were attributed to the volatility of acrylonitrile; findings suggest that nearly all of the lost acrylonitrile had volatilised within the first hour after application and that the filter and trap were not effective at collecting all of this volatilised material. Given the high and variable rates of volatile loss, absorption rates could not be reliably compared between the species. However the results indicate that 1.30% in human and 0.76% in rats of the applied dose was either retained in the skin or had been absorbed. The results of the study indicate that systemic absorption resulting from occupational dermal exposure to acrylonitrile is likley to be limited by the volatile nature of the substance.

Endpoint:
basic toxicokinetics, other
Remarks:
Review of metabolism data
Type of information:
other: expert review / secondary source
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
secondary literature
Objective of study:
metabolism
Principles of method if other than guideline:
The authors provide an overview of the metabolism of acrylonitrile from published and unpublished data, with a particular emphasis on aspects relevant to genotoxicity and carcinogenicity.
GLP compliance:
no
Specific details on test material used for the study:
Not relevant
Radiolabelling:
yes
Details on species / strain selection:
The review includes data on various species and strains, as well as data from studies in vitro.
Details on test animals and environmental conditions:
The review includes data on various species and strains, as well as data from studies in vitro.
Route of administration:
other: The review includes data on various routes of administration, as well as data from studies in vitro.
Duration and frequency of treatment / exposure:
The review includes data from various studies, as well as data from studies in vitro.
No. of animals per sex per dose:
The review includes data from various studies, as well as data from studies in vitro.
Details on study design:
The review includes data from various studies, as well as data from studies in vitro.
Metabolites identified:
yes
Details on metabolites:
A proposed pathway for the metabolism of acrylonitrile is attached.

The authors propose a metabolic pathway for acrylonitrile and note the potential influence of other factors on the metabolism and potentially therefore the toxicity of acrylonitrile. Species differences in metabolism, non-linear kinetics due to sulphydryl depletion, non-linear kinetics due to enzyme induction and inhibition and local tissue metabolism may all play a part in influencing the toxicity of acrylonitrile.

Conclusions:
The authors propose a metabolic pathway for acrylonitrile and note the potential influence of other factors on the metabolism and potentially therefore the toxicity of acrylonitrile. Species differences in metabolism, non-linear kinetics due to sulphydryl depletion, non-linear kinetics due to enzyme induction and inhibition and local tissue metabolism may all play a part in influencing the toxiicty of acrylonitrile.
Executive summary:

As part of a broader review, the authors consider the metabolism data available for acrylonitrile. The emphasis of the review is on the role palyed by metabolism in the genotoxicity and carcinogenicity of acrylontrile.

Acrylonitrile and some of its metabolites are reactive molecules, capable of interacting with cellular macromolecules to varying degrees. For this reason, metabolism is considered to be important determinant of the genotoxicity and carcinogenicity of acrylonitrile. An overview is provided of the pathways involved in acrylonitrile metabolism, including a brief discussion of complicating factors (species differences, sources of nonlinear toxicokinetics, local tissue metabolism) that should be considered when interpreting studies on the genotoxicity of acrylonitrile and their implications to human health risk assessment.

 

Acrylonitrile is metabolised via two pathways (see figure attached). The first pathway involves conjugation with glutathione (GSH), either through catalysis by the cytosolic enzyme, glutathione-S-transferase (GST), or nonenzymatically. The second pathway proceeds via and oxidation by the microsomal enzyme, cytochrome P450 CYP2E1, initially forming 2 -cyanoethylene oxide (CNEO).  This oxidative pathway can result in the release of cyanide (CN-), which has been reported to require CYP2E1 activity.  Other enzyme systems are also postulated to play a role in the oxidative metabolism of acrylonitrile. For example, cytochrome C peroxidase isolated from S. cerevisiae was found to catalyse the oxidation of acrylonitrile, as indicated by cyanide release at a rate similar to rat liver microsomal P450.  Lactoperoxidase has also shown activity for the oxidation of acrylonitrile in vitro.  Partially purified human lung lipoxygenase has also demonstrated appreciable activity, oxidising acrylonitrile to release cyanide in vitro.  These findings are also supported by studies conducted using structurally similar chemicals which suggest that other enzyme systems and pathways may be involved in the oxidation of nitriles, including the myeloperoxidase-mediated oxidation of chloroacetonitrile, the xanthine oxidase-mediated oxidation of dibromoacetonitrile and the non-enzymatic oxidation of dichloroacetonitrile in the presence of reactive oxygen species (peroxides) in vitro. 

 

The metabolites of acrylonitrile from both the oxidative and GSH-conjugation pathways are subject to further metabolim.  The acrylonitrile-GSH conjugate is converted to a mercapturic acid, which is subsequently excreted in urine.  The oxidative metabolite CNEO is metabolised by two pathways; conjugation with glutathione either through catalysis by GST or nonenzymatically, forming conjugates on the second or third carbon; and hydrolysis by epoxide hydrolase.  The secondary metabolites of CNEO can undergo further metabolism or decomposition.  Of particular toxicological importance is the fact that cyanide can be released from the CNEO metabolite generated by the epoxide hydrolase pathway and also from the GSH conjugate formed on the third carbon of CNEO.  Cyanide is relatively short-lived in the body and is rapidly metabolised, primarily by the mitochondrial enzyme rhodanese, using thiosulphate as a cofactor to form thiocyanate.  In this regard it is notable that thiocyanate has been detected in the blood and urine of volunteers following short-term inhalation exposure to acrylonitrile, in the urine of workers exposed occupationally to acrylonitrile and has also been measured in the blood and brain of rats exposed to acrylonitrile by oral gavage.  A minor metabolic pathway for cyanide involves its reaction with cystine to form 2 -aminothiazoline-4 -carboxylic acid (ATCA) which is excreted in the urine.

 

In acute exposure scenarios, the formation of thiocyanate from cyanide from acrylonitrile metabolism has historically been viewed as a detoxification step.  However, this may not be the case for some tissues or for long-term exposures. As a pseuodo-halide, the pharmacokinetics of thiocyanate are driven by active transport and metabolic processes reserved for halides rather than by tissue partitioning.  For this reason, plasma levels of thiocyanate persist considerably longer than either acrylonitrile, CNEO or cyanide (half-life ~1 -6 days in humans.  Long-term exposures to thiocyanate are known to produce goitre due to competition with iodine for uptake by the sodium-iodine symporter into the thyroid. Additionally, thiocyanate is actively transported to external surfaces of the body where antimicrobial activity is required, including the oral cavity, gastrointestinal tract and respiratory tract surface where thiocyanate levels are generally higher than plasma levels. Following an intravenous dose of radiolabeled potassium cyanide administered to rats, approximately 19% of the radiolabel was transported to the gastrointestinal lumen within 6 hours, presumably in the form of thiocyanate.  Data therefore indicate that tissue doses of thiocyanate may vary significantly, depending on the presence and activity of halide symporters, and may not be readily predicted by blood concentrations.  Five minutes after rats received a radiolabelled dose of acrylonitrile via intavenous injection, the tissues with the highest concentration of radiolabel were the lung, liver, small intestines contents and spleen, a distribution pattern that cannot be explained by simple partitioning.  Following transport, thiocyanate serves as a substrate for peroxidases (e.g. myeloperoxidase and lactoperoxidase) which yields hypothiocyanite, an important endogenous antimicrobial agent analogous to hypohalous acids (HOCl, HOBr). Unlike the hypohalous acids, which react indiscriminately with cellular macromolecules, the antimicrobial activity of hypothiocyanite is attributable to its ability to react almost exclusively with sulphydryls, a reaction that is largely reversible. Also unlike hypohalous acids, thiocyanate is capable of diffusing across bi-lipid membranes where it can react with intracellular sulphydryl groups. As a sulphydryl reactive agent, hypothiocyanite can deplete levels of GSH, and inhibit enzyme activities including microtubule polymerization.  While initially considered to be a mild oxidant, there is an increasing body of evidence that the toxicological consequences of hypothiocyanite formation can be significant. 

 

The metabolism of acrylonitrile is also affected by a number of important factors that should be considered when interpreting toxicity studies, as summarised briefly below:

Species Differences

Species differences in the metabolic pathways of acrylonitrile have been reported.  Clear species differences have been reported for the oxidation of acrylonitrile by cytochrome P450; studies using liver microsomes indicate thatmice and rats appear to form CNEO at a greater rate (4x and 1.5x, respectively) compared to humans.  The hydrolysis of CNEO by epoxide hydrolase is significant in humans, virtually absent in naive mice and rats but can be induced in both species.  With respect to the clearance of acrylonitrile, GSH conjugates account for approximately 36-43% of urinary metabolites in rats and 20-28% of urinary metabolites in mice. Despite having a higher rate of CNEO formation than rats, mice exhibited circulating levels of CNEO that were notably lower than the levels detected in rats, suggesting that differences exist between rats and mice with respect to CNEO clearance (e.g. by GSH conjugation).  The conjugation of CNEO with GSH occurs faster in humans (~1.5 -fold) than in either mice or rats.  With respect to thiocyanate metabolism, peroxidase activity has been detected in the mouse Harderian gland (a target for acrylonitrile carcinogenicity) but was not detected in rat Harderian gland (not a target). Species differences in metabolism can also be assessed by examining the excretion of urinary metabolites and their ratios.  At high dose levels (~10 mg/kg bw), the relative contribution of metabolites from the oxidative pathway [N-acetyl-S-(1-cyano-2-hydroxyethyl)-L-cysteine (CHEMA)] is less than that from the direct conjugation pathway [N-acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA)], resulting in CHEMA:CEMA ratios of 0.3-0.4 in rats and 0.4-0.9 in mice. Other data for the excretion of urinary metabolites in rat and mice exposed to acrylonitrile show that the ratio of CHEMA:CEMA is dose-dependent. At low dose levels (<0.5 mg/kg bw), the ratio of CHEMA:CEMA excreted in urine was greater than 3.5 in rats and greater than 1.5 in mice, suggesting that the oxidative pathway predominates at low dose levels of acrylonitrile. In comparison, urinary excretion of metabolites in humans exposed to low doses of acrylonitrile showed CHEMA:CEMA ratios of 0.26 and 0.16 for non-smokers and smokers, respectively. The dose of acrylonitrile received by smokers was not specified by the study authors but can be estimated to be less than 0.0075 mg/kg bw/d. Taken together, these data suggest that the oxidative pathway plays a much larger relative role in acrylonitrile metabolism in rodents than it does in humans. 

Nonlinear Toxicokinetics Due to Sulphydryl Depletion

An important source of nonlinear toxicokinetics for acrylonitrile includes the depletion of cellular sulphydryls such as GSH, which is likely to contribute to oxidative stress (Puppelet al., 2015). Acrylonitrile and CNEO both react with GSH and together are capable of depleting cellular GSH levels. Acrylonitrile has been shown to be a more effective depletor of tissue GSH than several acrylates (Vodickaet al., 1990). When administered at oral doses corresponding to the LD50, acrylonitrile was more effective than several other nitrile compounds in depleting GSH in rat liver, kidney and brain at 1 hpurr post exposure (Ahmedet al., 1982). GSH depletion has been observed in a number of tissues (brain, lung, liver, kidney, stomach, adrenal gland, erythrocytes) in rats exposed to acrylonitrile (Coteet al., 1984; Gutet al., 1985, Benzet al., 1997a, Vodickaet al., 1990, Silver & Szabo, 1982). Benzet al. (1997) reported significant GSH depletion in rat tissues following acute doses of approximately 20 -50 mg/kg bw. For tissues and cells that have significant peroxidase activity, the formation of hypothiocyanite from thiocyanate creates an additional stressor on GSH levels. In human erythrocytes, GSH was significantly depleted at low concentrations (10 uM), and was completely depleted at 100 uM hypothiocyanite (Arlandsonet al., 2001), which are physiologically relevant concentrations in some tissue and fluids. For example, mean thiocyanate and hypothiocyanite concentrations in saliva young adults (with no exposure to acrylonitrile) were reported to be 1.5 mM and 31 uM, respectively (Jalil, 1994).  Fromthe metabolic pathways for acrylonitrile, it is clear that there are multiple steps which are dependent upon maintenance of cysteine levels to support GSH (conjugation reactions with acruylonitrile, CNEO, and hypothiocyanite), thiosulphate and cystine. For thisreason, it is important to consider the magnitude of the acrylonitrile exposures used in toxicity studies and the potential role of sulphydryl depletion as a causative role in producing oxidative stress.

 

Nonlinear Toxicokineticsdue to Enzyme Induction or Inhibition

The induction of cytochrome P4502E1 by acrylonitrile does not appear to be important factor at toxicologically relevant doses.  However, enzyme activity for other oxidative pathways is induced by acrylonitrile exposure including stomach myeloperoxidase and xanthine oxidase. Data therefore suggest that, for some tissues, the oxidative metabolism of acrylonitrile may be increased following a single high oral doses of 25 -30 mg/kg bw. With respect to enzyme inhibition, in human erythrocytes exposed to hypothiocyanite, glutathione transferase activity was found to be completely inhibited by a physiologically relevant concentration of acrylonitrile. For tissues and cells with significant peroxidase activity, the formation of hypothiocyanite from thiocyanate could inhibit the conjugation pathways important for acrylonitrile and CNEO clearance.  Hypothiocyanate has also been shown to reversibly inactivate several enzymes with active site thiol residues; this effect of hypothiocyanite may therefore extend to multiple enzyme systems.

Local Tissue Metabolism

Studies of the metabolism of acrylonitrile have focused upon the liver as the primary site of metabolism, particularly with respect to CYP2E1 and GST activity.  The role of local tissue metabolism, particularly for other enzyme systems (e.g., peroxidases) has not been evaluated. Rodent target tissues for tumour formation (positive species indicated in parentheses) for lifetime exposures to acrylonitrile are listed below.

Brain/microglial (rat)

Zymbal's gland (rat)

Forestomach (rat, mouse)

Mammary gland (rat)

Tongue (rat)

Intestines (rat)

Nasal turbinate (rat)

Harderian gland (mouse)

When the list of target tissues is considered within the context of tissues where myeloperoxidase and lactoperoxidase activities are required to support antimicrobial action, there is considerable overlap. At these tissue sites, the formation of hypothiocyanite is likely to serve as an additional stressor to GSH/sulphydryl levels (in addition to systemic stressors contributed by acrylonitrile and CNEO, which in turn may contribute to localised oxidative stress.

Description of key information

The toxicokinetics of acrylonitrile have been investigated in a large number of studies, the most relevant of which are summarised in this dossier. The data have also been comprehensively summarised and reviewed in the EU RAR (2004); the conclusions of the EU RAR are also summarised. A more recent review of the metabolism of acrylonitrile and its potential role in genotoxicity and carcinogenicity is also provided (Albertini et al., 2016).

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
100
Absorption rate - inhalation (%):
100

Additional information

Overview of toxicokinetics

The toxicokinetics of acrylonitrile have been well characterised in animal (predominantly rat) studies using inhalation or oral administration.  Additional investigations have been performed in studies using intravenous and intraperitoneal administration.  Acrylonitrile is shown to be extensively absorbed and distributed following administration by all routes investigated; however absorption following dermal exposure is likley to be limited by volatilisation.  The metabolism of acrylonitrile procedes initially via direct conjugation with glutathione (a detoxification step) or, alternatively, via cytochrome P450-mediated oxidation (an activation step), followed by extensive further metabolism and the excretion of a range of metabolites in urine and exhaled air.  Mechanistic data indicate the covalent binding of acrylonitrile and its metabolite cyanoethylene oxide (CEO).

 

Absorption

The most relevant information on the oral absorption of acrylonitrile has been derived from gavage studies in the rat, with comparatively little information available for other species.  Findings show that acrylonitrile is rapidly and extensively (almost quantitatively) absorbed following oral administration to the rat; absorption levels of 95-98% are reported, with Cmax attained within 3-6 hours of dosing. Following inhalation exposure of rats, the absorption of acrylonitrile is also rapid and extensive.  Levels of retention in excess of 90% are reported.  The inhalation absorption of acrylonitrile may be biphasic and has also been reported to be enhanced in GSH-depleted rats.  It is notable, however, that the level of retention of acrylonitrile in the respiratory tract in human volunteers (~50%) is reported to be lower than that seen in the rat.  An inhalation absorption level of 100% (i.e. a level equivalent to that of oral absorption) is assumed for the purposes of risk assessment. The results of a dermal absorption study in rat and human skin membranes in vitro indicate very limited dermal absorption (~1%) and show that the large majority of the applied test material was lost though volatilisation. However a worst case dermal absorption of 100% (i.e. a level equivalent to that of oral absorption is assumed for the purposes of risk assessment.

 

Distribution 

Following gavage administration, acrylonitrile and/or its metabolites are shown to accumulate in the liver, kidney, intestinal mucosa, adrenal cortex, and blood, with most of the radioactivity irreversibly bound to proteins.  Free acrylonitrile may be relatively uniformly distributed; higher concentrations of radioactivity seen in some organs and erythrocytes are due to the interaction of acrylonitrile metabolites with soluble and protein sulphydryl groups.  Experimental glutathione depletion results in greater uptake of a single oral dose of acrylonitrile into the brain, stomach, liver, kidney and blood of rats and also results in an increase in the levels of covalently bound radioactivity at 6-24 hours after dosing.

Metabolism

Acrylonitrile and some of its metabolites are reactive molecules, capable of interacting with cellular macromolecules to varying degrees. For this reason, metabolism is considered to be important determinant of the genotoxicity and carcinogenicity of acrylonitrile. An overview is provided of the pathways involved in acrylonitrile metabolism, including a brief discussion of complicating factors (species differences, sources of nonlinear toxicokinetics, local tissue metabolism) that should be considered when interpreting studies on the genotoxicity of acrylonitrile and their implications to human health risk assessment. Acrylonitrile is metabolised via two pathways (see figure attached). The first pathway involves conjugation with glutathione (GSH), either through catalysis by the cytosolic enzyme, glutathione-S-transferase (GST), or non-enzymatically. The second pathway proceeds via and oxidation by the microsomal enzyme, cytochrome P450 CYP2E1, initially forming 2 -cyanoethylene oxide (CNEO). This oxidative pathway can result in the release of cyanide (CN-), which has been reported to require CYP2E1 activity.  Other enzyme systems are also postulated to play a role in the oxidative metabolism of acrylonitrile.For example, cytochrome C peroxidase isolated from S. cerevisiae was found to catalyse the oxidation of acrylonitrile, as indicated by cyanide release at a rate similar to rat liver microsomal P450. Lactoperoxidase has also shown activity for the oxidation of acrylonitrile in vitro. Partially purified human lung lipoxygenase has also demonstrated appreciable activity, oxidising acrylonitrile to release cyanide in vitro. These findings are also supported by studies conducted using structurally similar chemicals which suggest that other enzyme systems and pathways may be involved in the oxidation of nitriles, including the myeloperoxidase-mediated oxidation of chloroacetonitrile, the xanthine oxidase-mediated oxidation of dibromoacetonitrile and the non-enzymatic oxidation of dichloroacetonitrile in the presence of reactive oxygen species (peroxides) in vitro.  The metabolites of acrylonitrile from both the oxidative and GSH-conjugation pathways are subject to further metabolim. The acrylonitrile-GSH conjugate is converted to a mercapturic acid, which is subsequently excreted in urine.  The oxidative metabolite CNEO is metabolised by two pathways; conjugation with glutathione either through catalysis by GST or nonenzymatically, forming conjugates on the second or third carbon; and hydrolysis by epoxide hydrolase. The secondary metabolites of CNEO can undergo further metabolism or decomposition. Of particular toxicological importance is the fact that cyanide can be released from the CNEO metabolite generated by the epoxide hydrolase pathway and also from the GSH conjugate formed on the third carbon of CNEO. Cyanide is relatively short-lived in the body and is rapidly metabolised, primarily by the mitochondrial enzyme rhodanese, using thiosulphate as a cofactor to form thiocyanate.  In this regard it is notable that thiocyanate has been detected in the blood and urine of volunteers following short-term inhalation exposure to acrylonitrile, in the urine of workers exposed occupationally to acrylonitrile and has also been measured in the blood and brain of rats exposed to acrylonitrile by oral gavage. A minor metabolic pathway for cyanide involves its reaction with cystine to form 2 -aminothiazoline-4 -carboxylic acid (ATCA) which is excreted in the urine.

 

In acute exposure scenarios, the formation of thiocyanate from cyanide from acrylonitrile metabolism has historically been viewed as a detoxification step. However, this may not be the case for some tissues or for long-term exposures. As a pseuodo-halide, the pharmacokinetics of thiocyanate are driven by active transport and metabolic processes reserved for halides rather than by tissue partitioning. For this reason, plasma levels of thiocyanate persist considerably longer than either acrylonitrile, CNEO or cyanide (half-life ~1 -6 days in humans. Long-term exposures to thiocyanate are known to produce goitre due to competition with iodine for uptake by the sodium-iodine symporter into the thyroid. Additionally, thiocyanate is actively transported to external surfaces of the body where antimicrobial activity is required, including the oral cavity, gastrointestinal tract and respiratory tract surface where thiocyanate levels are generally higher than plasma levels. Following an intravenous dose of radiolabeled potassium cyanide administered to rats, approximately 19% of the radiolabel was transported to the gastrointestinal lumen within 6 hours, presumably in the form of thiocyanate.  Data therefore indicate that tissue doses of thiocyanate may vary significantly, depending on the presence and activity of halide symporters, and may not be readily predicted by blood concentrations. Five minutes after rats received a radiolabelled dose of acrylonitrile via intavenous injection, the tissues with the highest concentration of radiolabel were the lung, liver, small intestines contents and spleen, a distribution pattern that cannot be explained by simple partitioning. Following transport, thiocyanate serves as a substrate for peroxidases (e.g. myeloperoxidase and lactoperoxidase) which yields hypothiocyanite, an important endogenous antimicrobial agent analogous to hypohalous acids (HOCl, HOBr). Unlike the hypohalous acids, which react indiscriminately with cellular macromolecules, the antimicrobial activity of hypothiocyanite is attributable to its ability to react almost exclusively with sulphydryls, a reaction that is largely reversible. Also unlike hypohalous acids, thiocyanate is capable of diffusing across bi-lipid membranes where it can react with intracellular sulphydryl groups. As a sulphydryl reactive agent, hypothiocyanite can deplete levels of GSH, and inhibit enzyme activities including microtubule polymerization. While initially considered to be a mild oxidant, there is an increasing body of evidence that the toxicological consequences of hypothiocyanite formation can be significant. 

 

The metabolism of acrylonitrile is also affected by a number of important factors that should be considered when interpreting toxicity studies, as summarised briefly below:

Species Differences

Species differences in the metabolic pathways of acrylonitrile have been reported.  Clear species differences have been reported for the oxidation of acrylonitrile by cytochrome P450; studies using liver microsomes indicate thatmice and rats appear to form CNEO at a greater rate (4x and 1.5x, respectively) compared to humans.  The hydrolysis of CNEO by epoxide hydrolase is significant in humans, virtually absent in naive mice and rats but can be induced in both species. With respect to the clearance of acrylonitrile, GSH conjugates account for approximately 36-43% of urinary metabolites in rats and 20-28% of urinary metabolites in mice. Despite having a higher rate of CNEO formation than rats, mice exhibited circulating levels of CNEO that were notably lower than the levels detected in rats, suggesting that differences exist between rats and mice with respect to CNEO clearance (e.g. by GSH conjugation).  The conjugation of CNEO with GSH occurs faster in humans (~1.5 -fold) than in either mice or rats. With respect to thiocyanate metabolism, peroxidase activity has been detected in the mouse Harderian gland (a target for acrylonitrile carcinogenicity) but was not detected in rat Harderian gland (not a target). Species differences in metabolism can also be assessed by examining the excretion of urinary metabolites and their ratios. At high dose levels (~10 mg/kg bw), the relative contribution of metabolites from the oxidative pathway [N-acetyl-S-(1-cyano-2-hydroxyethyl)-L-cysteine (CHEMA)] is less than that from the direct conjugation pathway [N-acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA)], resulting in CHEMA:CEMA ratios of 0.3-0.4 in rats and 0.4-0.9 in mice. Otherdata for the excretion of urinary metabolites in rat and mice exposed to acrylonitrile show that the ratio of CHEMA:CEMA is dose-dependent. At low dose levels (<0.5 mg/kg bw), the ratio of CHEMA:CEMA excreted in urine was greater than 3.5 in rats and greater than 1.5 in mice, suggesting that the oxidative pathway predominates at low dose levels of acrylonitrile. In comparison, urinary excretion of metabolites in humans exposed to low doses of acrylonitrile showed CHEMA:CEMA ratios of 0.26 and 0.16 for non-smokers and smokers, respectively. The dose of acrylonitrile received by smokers was not specified by the study authors but can be estimated to be less than 0.0075 mg/kg bw/d. Taken together, these data suggest that the oxidative pathway plays a much larger relative role in acrylonitrile metabolism in rodents than it does in humans. 

Nonlinear Toxicokinetics Due to Sulphydryl Depletion

An important source of nonlinear toxicokinetics for acrylonitrile includes the depletion of cellular sulphydryls such as GSH, which is likely to contribute to oxidative stress (Puppel et al., 2015). Acrylonitrile and CNEO both react with GSH and together are capable of depleting cellular GSH levels. Acrylonitrile has been shown to be a more effective depletor of tissue GSH than several acrylates (Vodicka et al., 1990). When administered at oral doses corresponding to the LD50, acrylonitrile was more effective than several other nitrile compounds in depleting GSH in rat liver, kidney and brain at 1 hour post exposure (Ahmed et al., 1982). GSH depletion has been observed in a number of tissues (brain, lung, liver, kidney, stomach, adrenal gland, erythrocytes) in rats exposed to acrylonitrile (Cote et al., 1984; Gut et al., 1985, Benz et al., 1997a, Vodicka et al., 1990, Silver & Szabo, 1982). Benz et al. (1997) reported significant GSH depletion in rat tissues following acute doses of approximately 20 -50 mg/kg bw. For tissues and cells that have significant peroxidase activity, the formation of hypothiocyanite from thiocyanate creates an additional stressor on GSH levels. In human erythrocytes, GSH was significantly depleted at low concentrations (10 uM), and was completely depleted at 100 uM hypothiocyanite (Arlandson et al., 2001), which are physiologically relevant concentrations in some tissue and fluids. For example, mean thiocyanate and hypothiocyanite concentrations in saliva young adults (with no exposure to acrylonitrile) were reported to be 1.5 mM and 31 uM, respectively (Jalil, 1994).  From the metabolic pathways for acrylonitrile, it is clear that there are multiple steps which are dependent upon maintenance of cysteine levels to support GSH (conjugation reactions with acruylonitrile, CNEO, and hypothiocyanite), thiosulphate and cystine. For thisreason, it is important to consider the magnitude of the acrylonitrile exposures used in toxicity studies and the potential role of sulphydryl depletion as a causative role in producing oxidative stress.

 

Nonlinear Toxicokinetics due to Enzyme Induction or Inhibition

The induction of cytochrome P4502E1 by acrylonitrile does not appear to be important factor at toxicologically relevant doses. However, enzyme activity for other oxidative pathways is induced by acrylonitrile exposure including stomach myeloperoxidase and xanthine oxidase. Data therefore suggest that, for some tissues, the oxidative metabolism of acrylonitrile may be increased following a single high oral doses of 25 -30 mg/kg bw. With respect to enzyme inhibition, in human erythrocytes exposed to hypothiocyanite, glutathione transferase activity was found to be completely inhibited by a physiologically relevant concentration of acrylonitrile. For tissues and cells with significant peroxidase activity, the formation of hypothiocyanite from thiocyanate could inhibit the conjugation pathways important for acrylonitrile and CNEO clearance. Hypothiocyanate has also been shown to reversibly inactivate several enzymes with active site thiol residues; this effect of hypothiocyanite may therefore extend to multiple enzyme systems.

Local Tissue Metabolism

Studies of the metabolism of acrylonitrile have focused upon the liver as the primary site of metabolism, particularly with respect to CYP2E1 and GST activity. The role of local tissue metabolism, particularly for other enzyme systems (e.g., peroxidases) has not been evaluated. Rodent target tissues for tumour formation (positive species indicated in parentheses) for lifetime exposures to acrylonitrile are listed below.

Brain/microglial (rat)

Zymbal's gland (rat)

Forestomach (rat, mouse)

Mammary gland (rat)

Tongue (rat)

Intestines (rat)

Nasal turbinate (rat)

Harderian gland (mouse)

When the list of target tissues is considered within the context of tissues where myeloperoxidase and lactoperoxidase activities are required to support antimicrobial action, there is considerable overlap. At these tissue sites, the formation of hypothiocyanite is likely to serve as an additional stressor to GSH/sulphydryl levels (in addition to systemic stressors contributed by acrylonitrile and CNEO, which in turn may contribute to localised oxidative stress.

Excretion

In rats administered acrylonitrile by gavage dosing, approximately 5% of the dose is excreted in exhaled air, with peak excretion in rats reported at 30 minutes after dosing.  Acrylonitrile is also excreted as other volatile metabolites including hydrogen cyanide (0.5%) and carbon dioxide (9%). It has been demonstrated that the exhaled carbon dioxide is derived from the –CN group on the acrylonitrile molecule.  The predominant route of excretion is urinary excretion; a small proportion (3-8%) of administered acrylonitrile is excreted in the faeces.  Excretion is rapid and occurs mainly within 24 hours, although approximately 25% of the administered radioactivity is retained in the body beyond 10 days, possibly due to the binding of metabolites to cellular macromolecules.

Toxicokinetics in humans

Limited human toxicokinetic data indicate metabolism via cyanoethylene oxide.  Blood thiocyanate levels in volunteers exposed to acrylonitrile concentrations below 22 ppm for 30 minutes returned to normal within 24 hours, while elevated levels were still present 12 hours after exposure to 50 ppm for 30 minutes. Urinary excretion of N-acetyl-S-(2-cyanoethyl)cysteine (CMA), derived from the glutathione conjugate of acrylonitrile has also been reported in workers exposed to airborne acrylonitrile concentrations of 3-10 ppm. These data therefore indicate that the metabolic pathways observed in experimental animals are also operative in humans. Another study confirmed high epoxide hydrolase activity and low glutathione transferase activity in humans compared with rodents, suggesting that humans possess an additional detoxification pathway for CEO. Species differences in 2-cyanoethylene oxide disposition pathways suggest that rodent data may not be useful for directly predicting the human disposition of this epoxide in humans.

Discussion on absorption rate

Theoretical assessment of the potential for dermal absorption

Acrylonitrile molecule is of comparatively low molecular weight (53.06) and is highly lipid soluble (log Pow 0.0165); these physicochemical properties of the substance favour dermal absorption. The Sapphire Group report (2004) states that acrylonitrile is 'well absorbed' through the skin. The report cites a study in human volunteers in which dermal absorption is reported to be 0.6 mg/cm2/h and a study using human skin in vitro which reports a higher penetration rate of 3.6 mg/cm2/h. The greater penetration rate in vitro is consistent with data for other chemicals.  Simple comparison of acute oral and dermal LD50 values does not take into account other kinetic factors which clearly have the potential to influence acute toxicity, however this comparison can give some indication of the extent of dermal absorption. for the rat, the EU RAR reports oral LD50 values of 72 -186 mg/kg bw and dermal LD50 values of 148 -282 mg/kg bw in the rat. A comparison of these values indicates that the dermal absorption of acrylonitrile in the rat is extensive. Further comparison of the dermal LD50 value with the subcutaneous LD50 value further indicates that the skin is not an effective barrier to acrylonitrile absorption in this species. A comparison of the oral and dermal LD50 values in the rabbit also supports this conclusion. Rao et al (2013) also report identical acute LD50 values for acrylonitrile of 95.1 (81.7 -110.6) mg/kg bw in female Wistar rats. The key acute dermal toxicity study (SNF, 2005) reports an LD50 value of >200 mg/kg bw, with 10% mortality at the single dose level investigated.

A dermal absorption study performed in vitro with acrylonitrile in human and rat skin membranes reports very limited (~1%) dermal absorption, with the large majority of applied material lost through volatilisation. The EU RAR reports case studies in which dermal exposure to acrylonitrile has resulted in systemic toxicity; in some cases symptoms of delayed toxicity are noted, indicating delayed or prolonged dermal absorption. The overall dataset therefore indicates that the dermal absorption of acrylonitrile is potenitally extensive, particularly when applied under occlusive conditions. A worst-case assumption of 100% dermal absorption can be used for purposes of risk assessment; this is likely to be an overestimate but does recognise the toxic potential of acrylonitrile. In practice, the volatility of the substance is likely to significantly reduce dermal absorption due to evaporation from the site of contact.