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Neurotoxicity

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Description of key information

The neurotoxicity of acrylonitrile has been investigated in a number of non-standard studies.

Indications of neurotoxicity have been seen in a number of animal studies with acrylonitrile.  Findings may be attributable to direct effects, the generation of cyanide, cholinergic effects or a combination of these, and are consistent with the toxicity of acrylonitrile.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
neurotoxicity: chronic oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Neurological examinations in rats following oral and inhalational exposure to acrylonitrile
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
No further information
Route of administration:
oral: unspecified
Vehicle:
not specified
Details on exposure:
Rats were exposed orally to acrylonitrile once a day, 5 days per week for 12 weeks. The results of this study prompted a second study where rats were exposed to acrylonitrile vapour for 6 hours a day, 5 days per week for 24 weeks.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available/
Duration of treatment / exposure:
Oral: 12 weeks.
Inhalation: 24 weeks.
Frequency of treatment:
Oral: once a day, 5 days per week for 12 weeks.
Inhalation: 6 hours per day, 5 days per week for 24 weeks.
Dose / conc.:
12.5 mg/kg bw/day
Remarks:
Oral gavage
Dose / conc.:
25 mg/kg bw/day
Remarks:
Oral gavage
Dose / conc.:
50 mg/kg bw/day
Remarks:
Oral gavage
Dose / conc.:
25 ppm
Remarks:
Inhalation
Dose / conc.:
50 ppm
Remarks:
Inhalation
Dose / conc.:
100 ppm
Remarks:
Inhalation
No. of animals per sex per dose:
No information
Control animals:
not specified
Details on study design:
No information available
Observations and clinical examinations performed and frequency:
Oral and inhalation exposure : Motor and sensory conduction velocities (MCV & SCV), and amplitudes of the sensory and motor action potentials (ASAP & AMAP) of the tail nerve.
Specific biochemical examinations:
No information available
Neurobehavioural examinations performed and frequency:
Oral and inhalation exposure: Neurophysiological examination
Sacrifice and (histo)pathology:
No information available
Other examinations:
No information available
Positive control:
No information available
Statistics:
No information available
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Details on results:
Mean bodyweights were below controls in the mid and high dose animals given acrylonitrile by gavage and in the high dose group exposed via inhalation. Rats given acrylonitrile orally developed behavioural sensitisation to subsequent administration.

One week after onset of treatment the rats developed salivation, locomotor hyperactivity, and moderately intense sterotypies associated with fur wetting. These effects started shortly after gavage dosing, lasted two to three hours and become more pronounces as treatment continued. High dose animals developed hindlimb weakness associated with decreases in sensory conduction velocities and amplitudes of sensory action potentials and could not rear from the 9th week on, but the weakness abated during the recovery period.

Rats exposed to acrlonitrile vapour exhibited time- and concentration-dependent decreases in motor and sensory conduction velocities and amplitudes of sensory action potentials. These changes were only partially reversible after an eight week recovery period. Related nitriles (i.e., methAN, trans-3-pentenenitrile, 3-methyl-2-butenenitrile, and 4-penetenitrile) did not cause abnormal behavioral or electrophysiological changes in spite of an obvious general toxicity.
Dose descriptor:
NOAEL
Remarks:
[gavage]
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Hindlimb weakness, changes in peripheral nerve conduction velocity and action potential
Remarks on result:
other:
Dose descriptor:
LOAEL
Remarks:
[gavage]
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Hindlimb weakness, changes in peripheral nerve conduction velocity and action potential
Remarks on result:
other:
Dose descriptor:
LOAEC
Remarks:
[inhalation]
Effect level:
25 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Changes in nerve conduction velocity and action potential.
Remarks on result:
other:
Dose descriptor:
NOAEC
Effect level:
25 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Changes in nerve conduction velocity and action potential.
Remarks on result:
not determinable
Remarks:
no NOAEC identified

Rats given acrylonitrile orally developed behavioural sensitisation characterised by salivation, locomotor hyperactivity and moderately intense stereotypies. Moreover, rats in the high dose group developed weaknesses in hindlimbs associated with decreases in SCV and ASAP. Rats exposed to acrylonitrile vapour exhibited time- and concentration-dependent decreases in MCV, SCV, and ASAP, which were partially reversible after 8 weeks of recovery. For oral exposures, this study identifies NOAEL and LOAEL values of 25 and 50 mg/kg bw/d for hindlimb weakness and changes in peripheral nerve conduction velocity and action potential. For inhalation exposures, this study identifies a LOAEL value of 25 ppm, based on changes in nerve conduction velocity and action potential.

Conclusions:
The nervous system of the rat appears to be a target following either oral or inhalation exposures of acrylonitrile.
Executive summary:

In this study, the authors investigated motor and sensory conduction velocities (MCV and SCV) and amplitudes of the sensory and motor action potentials (ASAP and AMAP) of the tail nerve in male Sprague-Dawley rats during chronic oral treatment with five unsaturated aliphatic nitriles. Rats were given doses of 12.5, 25 and 50 mg/kg bw acrylonitrile; 50, 70 and 90 mg/kg bw methacrylonitrile; 25, 50 and 100 mg/kg bw trans-3-pentenenitrile; 50, 100 and 200 mg/kg bw 3-methyl-2-butenenitrile or 4-pentenenitrile daily on 5 days per week for 12 weeks. Neurophysiological examinations were also carried out in rats exposed by inhalation to 25, 50 and 100 ppm of acrylonitrile for 6 hours a day, 5 days per week for 24 weeks. Rats given acrylonitrile orally developed behavioural effects characterized by salivation, locomotor hyperactivity and moderately intense stereotypies. Moreover, rats in the high dose group developed weakness in hindlimbs associated with decreases in SCV and ASAP. Rats exposed to acrylonitrile by inhalation exhibited time- and concentration-dependent decreases in MCV, SCV and ASAP which were partially reversible after 8 weeks of recovery. None of the other four nitriles caused any abnormal behaviour or any changes in the electrophysiological parameters in spite of an obvious general toxicity. Based upon these results, the authors conclude that the nervous system of the rat appears to be a target of acrylonitrile following either oral or inhalation exposure.

Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The study investigated the effects on cerebral neuronal morphology and apoptosis in rats following the repeated (sub-chronic) gavage administration of acrylonitrile
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
48 Wistar rats (140-180 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. The rats were acclimatized to the animal facility for 1 week with a 12-h light/dark cycle and free access to food and water. The rats were randomly divided into four groups with 12 rats (six male and six female) in each group.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Four groups of six rats/sex were adminstered acrylonitrile by gavage (in water) at dose levels of 0, 5, 10, or 20 mg/kg bw on 5 days a week for 13 weeks.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week
Dose / conc.:
0 mg/kg bw/day
Remarks:
Controls
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
10 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
No. of animals per sex per dose:
Six
Control animals:
yes, concurrent vehicle
Details on study design:
After the 13 weeks of treatment, the brains of the rats were obtained while they were under anaesthesia with ether. The formalin-fixed, paraffin wax-embedded brain samples were sectioned at 5 μm and stained with hematoxylin-eosin. Four of the rat brains in each group were examined for neuronal apoptosis by TUNEL. Six slides of each rat brain were examined in order to determine the amount and distribution of apoptotic neurons. DNA was extracted from the rat brains and DNA ladders were examined following agarose gel electrophoresis. Bcl-2 protein, PCNA, and p53 protein were analysed immunohistochemically.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Neurones from the 5 mg/kg bw/ d appeared almost normal, with only occasional vacuolation.

Neurones from the control group brains appeared histologically normal. Neurones from the 5 mg/kg bw/d appeared almost normal, with only occasional vacuolation. Significant vacuolation and widening of the interspaces around the brain vessels were observed in neurones from the 10 mg/kg bw/d group; changes were more marked in the neurones of the 20 mg/kg bw/d group.

Electron microscopy showed that neurones from the control group were normal. Neurones from the 5 mg/kg bw/d group were also largely normal, with malformed nuclei seen only occasionally. Disordered myelin sheaths, malformed neuronal nuclei and chromatin condensation were all observed in neurones from the 10 mg/kg bw/d group; apoptotic bodies were also seen occasionally. Obviously malformed neuronal nuclei with chromatin condensation were observed in the 20 mg/kg bw/d group. The mean numbers of apoptotic cells were 1.50/slide in the 5 mg/kg bw/d group, 2.50/slide in the 10 mg/kg bw/d group and 2.34/slide in the 20 mg/kg bw/d. The numbers of apoptotic cells in these treated groups were significantly less than those seen in the control group (4.50/slide). Agarose gel electrophoresis did not reveal any DNA laddering in any group. There were more Bcl-2-positive cells in the treated groups, and the staining intensities of the apoptotic cells in the treated groups were greater than that of the control group, but there were no significant differences in the numbers of apoptotic cells and the staining intensities among the three treated groups. The stain particles were dispersed in the nuclei, and the Bcl-2-positive cells were dispersed in the rat brains. There was no significant expression of PCNA or p53 protein in the rat brains in any of the treated or control groups.

Conclusions:
The authors suggest that acrylonitrile may induce serious morphological change in rat brain neurones and also that acrylonitrile may inhibit neuronal apoptosis.
Executive summary:

The study aimed to evaluate the effects of acrylonitrile on cerebral neuronal morphology and apoptosis in rats. Wistar rats were gavaged with acrylonitrile at dose levels of 0, 5, 10 or 20 mg/kg bw/d on five days a week for 13 weeks. Neuronal morphology and the presence of apoptosis was examined by light and electron microscope, DNA electrophoresis, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated (dUTP) nick-end labelling. Significant vacuolation and the widening of the interspaces around blood vessels were observed in the groups that received the highest dose level of acrylonitrile. Disordered myelin sheaths, malformed neuronal nuclei and chromatin condensation at the periphery of the nucleus were also observed in the treated rats. The number of apoptotic neurons was significantly decreased in the treated groups compared to the control group. The number of Bcl-2-positive neurons and the levels of staining were increased in the treated rats compared to those of the control group. The authors suggest that acrylonitrile may induce serious morphological changes in rat neurons and inhibit neuronal apoptosis. The conclusion of the authors on a role for the inhibition of neuronal apoptosis in the aetiology of brain tumours in the rat is questionable as these tumours are demonstrated to be of microglial rather than neuronal origin.

Endpoint:
neurotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessment of the effects of acrylonitrile administration on acetylcholinesterase activity in mice in vivo, following a single intraperitoneal dose
GLP compliance:
no
Limit test:
no
Species:
mouse
Strain:
other: Kunming
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Laboratory Animal Center of Jiangsu University
- Age at study initiation: Not reported
- Weight at study initiation: 19-21 g
- Fasting period before study: Not applicable
- Housing: Not reported
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
physiological saline
Details on exposure:
Groups of mice were administered a single dose of acrylonitrile in physiological saline by intraperitoneal injection at dose levels of 0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5.0, 10, or 20 mg/kg bw. Additional groups were pre-treated with ethanol (5%) in the drinking water for one week prior to administration of acrylonitrile.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Single intraperitoneal injection with or without ethanol pre-treatment
Frequency of treatment:
Single intraperitoneal injection with or without ethanol pre-treatment (1 week, drinking water)
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
Vehicle
Dose / conc.:
0.156 mg/kg bw (total dose)
Dose / conc.:
0.312 mg/kg bw (total dose)
Dose / conc.:
0.625 mg/kg bw (total dose)
Dose / conc.:
1.25 mg/kg bw (total dose)
Dose / conc.:
2.5 mg/kg bw (total dose)
Dose / conc.:
5 mg/kg bw (total dose)
Dose / conc.:
10 mg/kg bw (total dose)
No. of animals per sex per dose:
8 males
Control animals:
yes, concurrent vehicle
Observations and clinical examinations performed and frequency:
Behaviour was assessed immediately following injection
Specific biochemical examinations:
Mice were terminated 24 hours following administration. Acetylcholinesterase activity in whole blood and homogenised brain samples was measured using the Ellman method. Liver CYP2E1 activity was measured based on the oxidation of p-nitrophenol in vitro by hepatic microsomes.
Positive control:
No

Two mice administered acrylonitrile at 20 mg/kg bw (one with and one without ethanol pre-treatment) died. All acrylonitrile-treated mice exhibited salivation and reddish ears, noses and paws. Treatment with doses greater than 2.5 mg/kg bw resulted in a general 'wet' appearance, attributed by the authors to excessive sweating. Hyperactivity was reported within 5 minutes of administration and was followed 30 minutes later by general hypoactivity. No changes in behaviour were apparent in the ethanol pretreatment control mice; behavioural effects in acrylonitrile-treated mice were not affected by ethanol pre-treatment. Pre-treatment with ethanol increased CPYP2E1 activity by approximately 70%. A slight (14-16%) but statistically significant increase in blood acetylcholinesterase activity was seen in mice treated with acrylonitrile at dose levels of 0.156 -1.25 mg/kg bw, but not at higher dose levels of 2.5 -20 mg/kg bw. A broadly comparable response was seen in mice pre-treated with ethanol. A more marked depression (down to approximately 55% of the control value) in brain acetylcholinesterase activity was seen in mice treated with acrylonitrile at dose levels of 0.156 -5 mg/kg bw; the largest reduction in activity was seen at 0.313 mg/kg bw. No significant changes were seen at 10 or 20 mg/kg bw. Pre-treatment with ethanol altered the shape of the dose-response curve for the inhibition of acetylcholinesterase inhibition by acrylonitrile. Maximal inhibition (-25%) was seen at a dose level of 0.313 mg/kg bw acrylonitrile and a significant reduction was also seen at 0.625 mg/kg bw. A significant increase (+25%) in brain acetylcholinesterase activity was seen at 5 mg/kg bw; however activity at 10 and 20 mg/kg bw was comparable to controls.

Conclusions:
The authors note the non-monotonic dose-response relationship for the inhibition of acetylcholineste rase activity by acrylonitrile. The response is postulated to be due to the inhibition of acetylcholinesterase due to an interaction between acrylonitrile and cysteine residues on the enzyme. It is also suggested that the increase in acetylcholinesterase activity is due a stimulation of acetylcholine release. The authors further suggest that the biphasic response of brain acetylcholinesterase activity is hormetic; i.e. exposure to a low dose of acrylonitrile initiates a compensatory reaction that leads to a temporary increase in acetylcholinesterase activity. The influence of ethanol pre-treatment on response suggests an effect of acrylonitrile rather than a metabolite.
Executive summary:

This study was designed to investigate the effects of acrylonitrile on blood and brain acetylcholinesterase activity in mice, and also assess the influence of pre-treatment with ethanol. Groups of 8 male Kunming mice were administered a single intraperitoneal dose of acrylonitrile in physiological saline at dose levels of 0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5.0, 10, or 20 mg/kg bw. Additional groups were pre-treated with ethanol (5%) in the drinking water for one week prior to administration of acrylonitrile. Mice were terminated 24 hours following administration. Acetylcholinesterase activity in whole blood and homogenised brain samples was measured using the Ellman method. Liver CYP2E1 activity was measured based on the oxidation of p-nitrophenol in vitro by hepatic microsomes. Two mice administered acrylonitrile at 20 mg/kg bw (one with and one without ethanol pre-treatment) died. All acrylonitrile-treated mice exhibited salivation and reddish ears, noses and paws. Treatment with doses greater than 2.5 mg/kg bw resulted in a general 'wet' appearance, attributed by the authors to excessive sweating. Hyperactivity was reported within 5 minutes of administration and was followed 30 minutes later by general hypoactivity. No changes in behaviour were apparent in the ethanol pre-treatment control mice; behavioural effects in acrylonitrile-treated mice were not affected by ethanol pre-treatment.

Endpoint:
neurotoxicity
Remarks:
other: review
Type of information:
other: expert review / secondary source
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The author provides a descriptive, interpretive and critical appraisal of animal and human studies, and addresses these within the context of human health risk assessment.
Qualifier:
no guideline required
Principles of method if other than guideline:
Literature review
GLP compliance:
no
Remarks:
: not relevant for a literature review
Limit test:
no
Species:
other: various
Strain:
other: various
Sex:
male/female
Details on test animals and environmental conditions:
The author reviews the findings of a number of studies with specific focus on neurotoxicity and neuropathology
Route of administration:
other: various routes used in the individual studies
Duration of treatment / exposure:
The author reviews the results of a number of studies using different exposure routes and dosing schedules
Frequency of treatment:
The author reviews the results of a number of studies using different exposure routes and dosing schedules
Remarks:
Doses / Concentrations:
various
Basis:

Details on results:
Potentially relevant findings in single dose rodent studies include laboured breathing and convulsions, as well as cholinergic signs such as salivation, diarrhoea, vasodilation and gastric secretion; however no convulsions have been reported in some studies at gavage doses of up to 40 mg/kg bw in rats. Signs of intoxication (salivation, lacrimation, diarrhoea, polyuria and vasodilation) are reported in rats after gavage or subcutaneous dose levels of 20, 40 and 80 mg/kg bw in a dose-dependent manner shortly after treatment; a few hours later, dose-dependent respiratory depression and convulsions were observed at 40 and 80 mg/kg bw groups).

Following a review of the available data, the author concludes that convulsions and laboured breathing are seen at acrylonitrile doses of 40 mg/kg bw or greater, and that signs of intoxication are reversible at dose levels of below 25-50 mg/kg bw/d. Following inhalation exposure to acrylonitrile, signs of irritation have been reported at 90 ppm (6h/day exposure). Cholinergic signs have been consistently reported in several studies at lower dose levels of 15 mg/kg bw/d and above. Qualitative effects (fur soiling, decreased home cage activity, circling, twitching and backwards walking) have been reported at doses as low as 4.3 mg/kg/ bw/d (50 ppm in drinking water), however other studies do not report similar effects at much higher dose levels. The available evidence also indicates varying sensitivity to the neurotoxic potential of acrylonitrile in different species.

The effects of acrylonitrile on the motor system have also been investigated in a number of studies. The author notes that no differences either in the amplitude of the motor action potential or in the motor nerve conduction velocity were seen after gavage doses up to 50 mg/kg bw/d for 12 weeks. No differences in the amplitude of the motor action potential were observed after inhalation, with the exception of animals exposed to 100 ppm 24 weeks (estimated to be 60 mg/kg bw/d); a potential effect on motor nerve conduction velocity was also noted at this exposure level. No convincing evidence of an effect in open field was seen at dose levels of up to 200 ppm in drinking water for 12 weeks (equivalent to 13.5 mg/kg bw/d), however decreased coordination was reported in another study at 13.5 mg/kg bw/d (Rongzhu et al., 2007).

The author concludes that sensory nerve conduction velocity and action potential amplitude were 'probably affected' at the gavage dose level of 50 mg/kg bw/d and at 100 ppm by inhalation (Gagnaire et al., 1998). It is also noted that lesions of the nasal epithelium were observed in rats at inhalation exposure levels of 5-45 ppm (6h/d, 7 d/wk for 10 weeks) in the study of Nemec et al. (2008). Additional studies show that acute exposure to acrylonitrile may reversibly decrease auditory sensitivity following a subcutaneous dose of 50 mg/kg bw. It is also noted that studies report that acrylonitrile can potentiate the effects of noise at a dose level of 50 mg/kg bw/d for 5 days) and, when combined with noise, can decreases auditory sensitivity with partial recovery after 4 weeks. However acrylonitrile does not appear to cause behavioural signs of vestibular dysfunction.

The results of studies on learning/memory are also reviewed; the author considers that the data do not support a conclusion that acrylonitrile has effects on cognitive function in one paper in the absence of a dose-response relationship and inadequate statistical analyses. While Rongzhu et al.(2007) state that exposure to acrylonitrile has an effect on cognitive function, no data is presented to support this.

It is documented elsewhere that chronic exposure to acrylonitrile results in an increased incidence of glial cell tumours in rats in a dose-related manner. Acrylonitrile is also shown to reversibly inhibit gap junction intracellular communication in rat glial cells. Data also indicate that glutathione depletion is an important mechanism in the induction of oxidative stress.
Dose descriptor:
NOAEL
Basis for effect level:
other: This review of neurotoxicity is not specifically designed to identify an overall effect level
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Conclusions:
The author reviews the available data on the neurotoxicity of acrylonitrile and concludes that the mechanism of neurotoxicity is still unknown and most likely multifaceted.
Executive summary:

The author concludes that, while the central nervous system has been identified as a target organ for acrylontitirle in a number of animal studies, closer examination of a number of papers does not confirm the reported effects.  A number of conclusions are drawn.  Signs of irritation have been consistently reported in animal studies and are also noted following human exposure.  Other signs and symptoms such as headache, fatigue and weakness have been documented in humans. Motor effects including decreased coordination and motor nerve conduction velocity have also been observed in studies in rats. In the sensory domain, nerve conduction velocity and action potential amplitude were probably affected at 50 mg/kg bw/d by gavage dosing and following inhalation of 100 ppm.  Acutely, acrylonitrile can reversibly decrease auditory sensitivity at 50 mg/kg bw and can potentiate the effects of noise in animals at a probably relevant noise level for humans, but at a dose level (50 mg/kg bw) and dose route (subcutaneous) without human relevance.  The documented synergy with noise, though, may be of relevance to humans and should not be overlooked.  Olfactory function can be reversibly affected in workers exposed to ABS products for several years.  Although not much can be concluded about acrylonitrile per se, it should be reminded that studies have identified nasal lesions in rats. Particular caution is needed in extrapolating olfactory effects from rats to humans for risk assessment.

No behavioural signs of vestibular dysfunction were observed and animal studies do not provide any convincing evidence of learning deficits. While chronic exposure to acrylonitrile can increase glial cell brain tumors in rats, epidemiology studies suggest that acrylonitrile is not a potent CNS tumorigen in humans.  Finally, the neurotoxicity observed with acrylonitrile varies considerably across different species.

While the molecular mechanisms of acrylonitrile neurotoxicity are poorly understood, it is known that treatment selectively induces oxidative stress. The role of the parent compound, cyanide production, GSH conjugation and depletion, CYP2E1, GST and SOD activities, and of other enzymes and metabolites associated with the metabolism of acrylonitrile, provide many potential ways in which neurotoxicity can be expressed.  Several mechanisms may therefore be involved in acrylonitrile neurotoxicity. The role the cholinergic system plays also unclear. The electrophilic nature of acrylonitrile enables binding to nucleophilic sites on macromolecules, and it has been suggested that adducts could serve as biomarker of exposure or could be seen as a putative molecular mechanism of action for electrophilic neurotoxicants. It is conlcuded that the mechanism of acrylonitrile-induced neurotoxicity is still unknown and most likely multifaceted.

Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 424 (Neurotoxicity Study in Rodents)
Deviations:
yes
Remarks:
Only 2 test substance concentrations, only 10 rats per group single sex, and no neurohistopathalogy.
GLP compliance:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Adult male Sprague-Dawley rats, weighing approximately 150g, obtained from the Laboratory Animal Center of Fudan University. Rats were housed two per cage, and acclimated for 2 weeks before exposure. Rats were divided randomly into three groups of 10 rats each. The animals were maintained at room temperature (22-26oC) on a 12:12 hour light:dark cycle. Chow from Double-lion Experiment Animal Fodder Science and Technique Service Co., Ltd (China), and tap water were provided ad libitum.
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Acrylonitrile was added to the drinking water of the rats, at concentrations of 50ppm and 200ppm. Treated water was provided ad libitum.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analytical verification of concentration is not reported, however an estimated dosage was calculated based on water intake.
Duration of treatment / exposure:
12 weeks.
Frequency of treatment:
Daily in ad libitum drinking water.
Dose / conc.:
0 ppm
Remarks:
Drinking water
Dose / conc.:
50 ppm
Remarks:
Drinking water
Dose / conc.:
200 ppm
Remarks:
Drinking water
No. of animals per sex per dose:
10 male rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rats were exposed to one of two acrylonitrile concentrations in drinking water (provided ad libitum) for 12 weeks. Observations were made during the exposure period.
Observations and clinical examinations performed and frequency:
Food intake was recorded daily, and water consumption was recorded every other day. Individual body weights were recorded prior to exposure, and once every 2 weeks thereafter. Gross observations were made of the rats during the exposure period, no further information is provided regarding the detail and frequency of these observations.
Specific biochemical examinations:
A minimum of 5 rats were randomly selected from each treatment group at Week 12. 24-hour urine was collected from these rats. The urine was frozen and stored at -70oC. The levels of thiocyanate in the urine were determined according to the method of Haque and Bradbury (1999).
Neurobehavioural examinations performed and frequency:
Neurobehavioural testing took place at 0, 4, 8 and 12 weeks of exposure. Technicians carrying out the tests were trained, and blinded to the treatment groups of the rats.
Testing was conducted at weeks 0, 4, 8 and 12 weeks of exposure. Tests were: the open field test, the rotarod test, and the spatial water maze (see below for descriptions).
Sacrifice and (histo)pathology:
Not examined/reported.
Other examinations:
No further examinations were made.
Positive control:
Not examined.
Statistics:
One-way and repeated measures ANOVA were used to determine the effects of treatment over time both within and between groups. Significance was accepted at the 5% level.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Details on results:
There were no reported mortalities throughout the 12 week exposure period. Clinical signs appeared during the 6th week of acrylonitrile administration; 3 rats in the 50ppm group and 5 rats in the 200ppm showed behavioural changes. The reported main behavioural changes were head twitching, trembling, circling, backwards pedalling, and decreased homecage activities. The coat appearance of all acrylonitrile exposed rats became soiled.

The two acrylonitrile treated groups demonstrated reduced body weight gain as compared with controls. This difference became apparent in the 50ppm after 4 weeks exposure, and the difference reached statistical significance from 6 weeks until the end of the experiment. The rats in the 200ppm group showed a significant decrease in body weight gain at weeks 4 and 6, however mean body weights recovered at weeks 8, 10 and 12.

Average daily water consumption of rats in the 200ppm group significantly decreased compared to controls and the 50ppm group (6.73±1.87, 7.85±1.77, and 8.06±2.18 ml/day/100g body weight, respectively). There was no difference between the control and the 50ppm group. Estimated acrylonitrile consumption (based on water intake) in the 50ppm rats was 4.03mg/day/kg body weight, and 13.46mg/day/kg body weight in the 200ppm rats.
At week 12, the levels of thiocyanate in urine in the control, 50ppm and 200ppm groups were 2.79±2.48, 6.10±4.38, and 25.03±4.22mg/g creatinine, respectively.

In the open field test (Table 1), the acrylonitrile treated groups appeared to show prolonged startup latencies compared to controls. At week 0 the 50ppm group showed significantly shorter start up latencies than the control group, but with increasing exposure duration the start up latency gradually increased and showed a tendency to be longer than in controls. The 200ppm group showed higher activity (locomotion) than the controls at weeks 0, 8 and 12. There were no other treatment-related differences, however at week 0 (i.e. pretreatment) rats in the 50ppm group reared less and groomed less than controls.

In the rotarod test at week 8 (Table 2), there were significant decreases in the maximum and total falling latencies in the acrylonitrile treated rats compared with controls. The maximal and total latency to fall steadily increased with time in the control group. From week 4 in the 50ppm group, maximal and total latency to fall decreased. In the 200ppm group, the maximal and total latency to fall was always shorter than their pretreatment values.

In the spatial water maze (Table 3), the training times and training duration gradually increased over the exposure period, but the decrease in the acrylonitrile groups was less than that of the controls. In weeks 4 and 8 the training times and training duration in the 200ppm were significantly increased compared to the control and 50ppm groups.
Dose descriptor:
NOAEC
Basis for effect level:
other: both concentrations (50 and 200ppm) tested caused effects
Remarks on result:
not determinable
Remarks:
no NOAEC identified

Effects of acrylonitrile on performance in the open field test (mean±SD, n=10)

Parameters

Acrylonitrile

(ppm)

Treatment duration (weeks)

0

4

8

12

Startup latency (s)

0

3.93±2.85

5.55±6.43

2.40±3.12

1.10±0.39

50

1.30±1.49*

4.50±4.24

2.95±2.83

2.10±2.64

200

3.80±2.82

3.10±4.65

1.80±1.62

2.75±2.03

Locomotor (sections crossed)

0

55.70±23.73

72.00±35.52

44.00±28.79

70.00±53.45

50

80.80±21.63

71.00±35.86

51.40±27.80

95.80±70.74

200

97.10±17.80*

88.10±48.83

90.90±44.51*

97.70±56.10*

Rearing (No. of times)

0

10.40±3.72

5.90±3.60

2.40±3.10

3.90±3.90

50

5.60±3.41*

5.00±3.46

1.60±2.06

2.70±3.02

200

10.00±5.06

3.70±3.37

3.60±2.88

7.40±7.49

Grooming bouts (No. of times)

0

1.90±1.79

1.80±1.87

2.90±2.69

3.80±3.85

50

1.20±1.13*

2.30±2.11

4.60±2.50

0.10±0.32

200

1.40±0.84

1.50±1.43

2.60±2.80

2.30±1.49

* versus control p<0.05

Effects of acrylonitrile on performance in the rotarod test (mean±SD, n=10)

Parameters

Acrylonitrile

(ppm)

Treatment duration (weeks)

0

4

8

12

Maximum latency from falling (s)

0

26.70±10.81

29.80±13.84

54.10±19.35

51.40±17.72

50

51.40±28.69*

24.50±9.63

37.50±18.03*

46.30±15.57

200

49.00±36.48

27.10±15.95

23.80±15.36*

25.20±21.41*

Total latency of three trials (s)

0

55.40±19.82

69.40±39.08

126.20±41.46

118.00±37.36

50

106.30±41.66*

48.60±14.50

80.70±35.44*

110.00±37.60

200

105.00±72.25

63.40±42.03

52.40±31.83

61.90±55.72*

* versus control p<0.05

Effects of acrylonitrile on performance in the spatial water maze (mean±SD, n=10)

Parameters

Acrylonitrile

(ppm)

Treatment duration (weeks)

0

4

8

12

Training times (No.)

0

10.20±6.36

5.40±3.48

4.50±5.76

2.90±2.60

50

8.00±2.50

7.90±4.98

4.70±5.25

2.40±0.84

200

11.40±3.81

11.10±4.91*

5.80±2.82

2.70±1.57

Training duration (s)

0

17.64±6.55

20.03±18.52

9.18±6.81

11.58±5.12

50

30.12±15.33*

11.65±7.11

7.15±2.33

14.45±11.31

200

21.81±6.20

22.44±9.31

13.71±6.03*

11.58±7.50

* versus control p<0.05

Conclusions:
Rats exposed to acrylonitrile in their drinking water for 12 weeks exhibited behavioural changes and there were effects on performance in 3 standard neurobehavioural tests, compared to control rats.
Executive summary:

Male Sprague-Dawley rats were exposed to two concentrations (50 and 200 ppm) of acrylonitrile in the drinking water for 12 weeks. Three neurobehavioural tests (open field, rotarod and spatial water maze) were conducted at weeks 0, 4, 8 and 12 of exposure, and compared to results for concurrent controls. Clinical behavioural signs were reported and included head twitching and circling. There were no consistent changes in the open field test, although rats in the 200 ppm group showed significantly higher levels of locomotion compared to controls. The 50 ppm group showed a significantly decreased startup latency, decreased rearing frequency and fewer grooming bouts than controls at week 0 (i.e pretreatment). The authors do not discuss the fact that behavioural differences prior to the start of acrylonitrile exposure may have confounded the results following exposure, nor do they comment on possible explanations for the behavioural differences in this group. Performance in the rotarod test was affected by time of exposure, particularly in the 200 ppm group, however there were also significant differences at week 0 between groups that were not discussed. Acrylonitrile also negatively affected performance in the spatial water maze, however the treated rats appeared to recover towards the end of the experiment. This study provides evidence that acrylonitrile may affect performance in tests which can be used to asses neurological effects. However it is noted that the dose levels used in this study were likely to result in general subacute toxicity, therefore it is unclear whether the nervous system is specifically targeted.

Endpoint:
neurotoxicity
Remarks:
other: review
Type of information:
other: expert review / secondary source
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline available
Principles of method if other than guideline:
Review and assessment of the published literature.
GLP compliance:
no
Remarks:
: not applicable
Limit test:
no
Species:
other: various
Strain:
other: various
Sex:
male/female
Details on test animals and environmental conditions:
Limited methodological details for individual studies are reported in the EU RAR
Route of administration:
other: various
Details on exposure:
A number of different studies are reviewed
Duration of treatment / exposure:
A number of different studies are reviewed
Frequency of treatment:
A number of different studies are reviewed
No. of animals per sex per dose:
A number of different studies are reviewed

The EU RAR notes that the central nervous system has been identified as a target organ in studies in various animal species by a number of authors. It notes that symptoms consistent with an effect on cholinergic transmission are seen in the rat at dose levels of 20-80 mg/kg in rat, but that no clinical symptoms of neurotoxicity were identified in a mouse study at similar dose levels.

It is postulated that the neurotoxic effects of acrylonitrile may be mediated by cyanide liberated as a result of oxidative metabolism. Other workers, however, have suggested however that the toxicity of acrylonitrile is due not to liberated cyanide but to acrylonitrile itself. This conclusion was based on the observation that a reduction of the acrylonitrile concentration in the blood by L-cysteine (which may have been caused by cyanoethylation of acrylonitrile with L-cysteine) was an effective protectant. Pre-treatment of rats and mice with thiosulphate did not afford protection against lethal doses and cyanide blood level remained well below levels associated with specific cyanide symptoms. These results indicate that, in addition to the release of cyanide, another mode of action plays a role in the acute toxicity of acrylonitrile and there is evidence that cyanide antidotes are effective in preventing acute toxicity in some animal models but are totally ineffective in others.

The acute toxicity of nitriles in general depends on the complex interplay of a number of factors including as the rate of cyanide liberation and detoxification, the dose of cyanogen, route of administration, species and the presence of other bioreactive sites within the nitrile molecule. While acrylonitrile is cyanogenic, it is also metabolised to the reactive epoxide 2 -cyanoethylene oxide (CEO) and the parent molecule is also capable of non-enzymatically cyanoethylating essential functional groups. All of these factors may therefore contribute to the overall toxicity of acrylonitrile.

Effects on cholinergic transmission in the rat may result from an inactivation of acetylcholinesterase by the cyanoethylation of the hydroxyl group of one serine residue, or may be due to damage to acetylcholine muscarinic receptors by acrylonitrile or its metabolites. Cholinergic effects are particularly marked in glutathione-depleted animals. Acrylonitrile produces cholinomimetic actions (salivation, diarrhoea and increased gastric secretion) which are prevented by pre-treatment with atropine It has also been demonstrated that acrylonitrile causes acute gastric haemorrhage and mucosal erosions. A possible mechanism may involve the interaction of acrylonitrile with critical sulphydryl groups on acetylcholine muscarinic receptors, leading to gastric haemorrhagic. Pre-treatment of rats with atropine was shown to afford significant protection. Treatment of rats with atropine after the first appearance of neurotoxic signs prevented further progress of toxicity.

Two distinct phases of acute neurotoxicity have been reported in rats administered acrylonitrile. Early signs of salivation, lacrimation, miosis, diarrhoea, polyuria and peripheral vasodilation peak within 60 minutes of dosing. Other signs of toxicity include flushing of the face, ears and extremities. The early phase is followed by a delayed phase (>4 hours) which includes central nervous system effects such as respiratory depression, convulsions and death. Signs of neurotoxic signs are dose-dependent regardless of the route of administration.

Conclusions:
It is concluded that acrylonitrile causes symptoms consistent with neurotoxicity in animal studies, however the mechanism underlying these findings is complex and unclear and is likely to involve a number of factors.
Executive summary:

The EU RAR reviews the literature on the neurotoxicity of acrylonitrile. It is concluded that the symptoms of acute acrylonitrile poisoning (such as general convulsions and paralysis of the lower limbs) support the conclusion that acrylonitrile acts both on the central and peripheral nervous systems. The EU RAR notes two distinct phases of acute neurotoxicity reported in rats administered acrylonitrile. Early signs of salivation, lacrimation, miosis, diarrhoea, polyuria and peripheral vasodilation peak within 60 minutes of dosing. Other signs of toxicity include flushing of the face, ears and extremities. The early phase is followed by a delayed phase (>4 hours) which includes central nervous system effects such as respiratory depression, convulsions and death. Signs of neurotoxic signs are dose-dependent regardless of the route of administration. It is noted that the acute toxicity of nitriles in general depends on the complex interplay of a number of factors including as the rate of cyanide liberation and detoxification, the dose of cyanogen, route of administration, species and the presence of other bioreactive sites within the nitrile molecule. While acrylonitrile is cyanogenic, it is also metabolised to the reactive epoxide 2 -cyanoethylene oxide (CEO) and the parent molecule is also capable of non-enzymatically cyanoethylating essential functional groups. The EU RAR concludes that all of these factors may contribute to the neurotoxicity of acrylonitrile.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A non-standard published study is avaialable.

Effect on neurotoxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: chronic oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Neurological examinations in rats following oral and inhalational exposure to acrylonitrile
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
No further information
Route of administration:
oral: unspecified
Vehicle:
not specified
Details on exposure:
Rats were exposed orally to acrylonitrile once a day, 5 days per week for 12 weeks. The results of this study prompted a second study where rats were exposed to acrylonitrile vapour for 6 hours a day, 5 days per week for 24 weeks.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No information available/
Duration of treatment / exposure:
Oral: 12 weeks.
Inhalation: 24 weeks.
Frequency of treatment:
Oral: once a day, 5 days per week for 12 weeks.
Inhalation: 6 hours per day, 5 days per week for 24 weeks.
Dose / conc.:
12.5 mg/kg bw/day
Remarks:
Oral gavage
Dose / conc.:
25 mg/kg bw/day
Remarks:
Oral gavage
Dose / conc.:
50 mg/kg bw/day
Remarks:
Oral gavage
Dose / conc.:
25 ppm
Remarks:
Inhalation
Dose / conc.:
50 ppm
Remarks:
Inhalation
Dose / conc.:
100 ppm
Remarks:
Inhalation
No. of animals per sex per dose:
No information
Control animals:
not specified
Details on study design:
No information available
Observations and clinical examinations performed and frequency:
Oral and inhalation exposure : Motor and sensory conduction velocities (MCV & SCV), and amplitudes of the sensory and motor action potentials (ASAP & AMAP) of the tail nerve.
Specific biochemical examinations:
No information available
Neurobehavioural examinations performed and frequency:
Oral and inhalation exposure: Neurophysiological examination
Sacrifice and (histo)pathology:
No information available
Other examinations:
No information available
Positive control:
No information available
Statistics:
No information available
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
not specified
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Details on results:
Mean bodyweights were below controls in the mid and high dose animals given acrylonitrile by gavage and in the high dose group exposed via inhalation. Rats given acrylonitrile orally developed behavioural sensitisation to subsequent administration.

One week after onset of treatment the rats developed salivation, locomotor hyperactivity, and moderately intense sterotypies associated with fur wetting. These effects started shortly after gavage dosing, lasted two to three hours and become more pronounces as treatment continued. High dose animals developed hindlimb weakness associated with decreases in sensory conduction velocities and amplitudes of sensory action potentials and could not rear from the 9th week on, but the weakness abated during the recovery period.

Rats exposed to acrlonitrile vapour exhibited time- and concentration-dependent decreases in motor and sensory conduction velocities and amplitudes of sensory action potentials. These changes were only partially reversible after an eight week recovery period. Related nitriles (i.e., methAN, trans-3-pentenenitrile, 3-methyl-2-butenenitrile, and 4-penetenitrile) did not cause abnormal behavioral or electrophysiological changes in spite of an obvious general toxicity.
Dose descriptor:
NOAEL
Remarks:
[gavage]
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Hindlimb weakness, changes in peripheral nerve conduction velocity and action potential
Remarks on result:
other:
Dose descriptor:
LOAEL
Remarks:
[gavage]
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Hindlimb weakness, changes in peripheral nerve conduction velocity and action potential
Remarks on result:
other:
Dose descriptor:
LOAEC
Remarks:
[inhalation]
Effect level:
25 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Changes in nerve conduction velocity and action potential.
Remarks on result:
other:
Dose descriptor:
NOAEC
Effect level:
25 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Changes in nerve conduction velocity and action potential.
Remarks on result:
not determinable
Remarks:
no NOAEC identified

Rats given acrylonitrile orally developed behavioural sensitisation characterised by salivation, locomotor hyperactivity and moderately intense stereotypies. Moreover, rats in the high dose group developed weaknesses in hindlimbs associated with decreases in SCV and ASAP. Rats exposed to acrylonitrile vapour exhibited time- and concentration-dependent decreases in MCV, SCV, and ASAP, which were partially reversible after 8 weeks of recovery. For oral exposures, this study identifies NOAEL and LOAEL values of 25 and 50 mg/kg bw/d for hindlimb weakness and changes in peripheral nerve conduction velocity and action potential. For inhalation exposures, this study identifies a LOAEL value of 25 ppm, based on changes in nerve conduction velocity and action potential.

Conclusions:
The nervous system of the rat appears to be a target following either oral or inhalation exposures of acrylonitrile.
Executive summary:

In this study, the authors investigated motor and sensory conduction velocities (MCV and SCV) and amplitudes of the sensory and motor action potentials (ASAP and AMAP) of the tail nerve in male Sprague-Dawley rats during chronic oral treatment with five unsaturated aliphatic nitriles. Rats were given doses of 12.5, 25 and 50 mg/kg bw acrylonitrile; 50, 70 and 90 mg/kg bw methacrylonitrile; 25, 50 and 100 mg/kg bw trans-3-pentenenitrile; 50, 100 and 200 mg/kg bw 3-methyl-2-butenenitrile or 4-pentenenitrile daily on 5 days per week for 12 weeks. Neurophysiological examinations were also carried out in rats exposed by inhalation to 25, 50 and 100 ppm of acrylonitrile for 6 hours a day, 5 days per week for 24 weeks. Rats given acrylonitrile orally developed behavioural effects characterized by salivation, locomotor hyperactivity and moderately intense stereotypies. Moreover, rats in the high dose group developed weakness in hindlimbs associated with decreases in SCV and ASAP. Rats exposed to acrylonitrile by inhalation exhibited time- and concentration-dependent decreases in MCV, SCV and ASAP which were partially reversible after 8 weeks of recovery. None of the other four nitriles caused any abnormal behaviour or any changes in the electrophysiological parameters in spite of an obvious general toxicity. Based upon these results, the authors conclude that the nervous system of the rat appears to be a target of acrylonitrile following either oral or inhalation exposure.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
58.4 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
A non-standard published study is avaialable.

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The neurotoxicity of acrylonitrile has been investigated in a number of non-standard studies.

Gagnaire et al. (1998) report reductions in nerve conduction velocity in rats exposed to acrylonitrile by inhalation for 6 hours a day, 5 days a week for 24 weeks. The exposure concentrations used in this study were 25, 50 and 100 ppm and some effects were reported at the lowest exposure concentration, albeit not consistently over the study period. The study has limitations in terms of its methodology and reporting; however the results are taken as an indication of neurotoxicity and are taken into account in derivation of the inhalation DNEL values.

Additional information

The neurotoxicity of acrylonitrile has been specifically investigated in a number of studies. 

Gagnaire et al. (1998) investigated motor and sensory conduction velocities (MCV and SCV) and amplitudes of the sensory and motor action potentials (ASAP and AMAP) of the tail nerve in male Sprague-Dawley rats during sub-chronic oral treatment with five unsaturated aliphatic nitriles. Rats were given doses of 12.5, 25 and 50 mg/kg bw acrylonitrile; 50, 70 and 90 mg/kg bw methacrylonitrile; 25, 50 and 100 mg/kg bw trans-3-pentenenitrile; 50, 100 and 200 mg/kg bw 3-methyl-2 -butenenitrile or 4-pentenenitrile daily on 5 days per week for 12 weeks. Neurophysiological examinations were also carried out in rats exposed by inhalation to 25, 50 and 100 ppm of acrylonitrile for 6 hours a day, 5 days per week for 24 weeks. Rats given acrylonitrile orally developed behavioural effects characterized by salivation, locomotor hyperactivity and moderately intense stereotypies. Moreover, rats in the high dose group developed weakness in hindlimbs associated with decreases in SCV and ASAP. Rats exposed to acrylonitrile by inhalation exhibited time- and concentration-dependent decreases in MCV, SCV and ASAP which were partially reversible after 8 weeks of recovery. None of the other four nitriles caused any abnormal behaviour or any changes in the electrophysiological parameters in spite of an obvious general toxicity. Based upon these results, the authors conclude that the nervous system of the rat appears to be a target of acrylonitrile following either oral or inhalation exposure.

Yuanqiong et al. (2011) report a non-monotonic dose-response relationship for the inhibition of acetylcholinesterase activity by acrylonitrile. This response is postulated to be due to the inhibition of acetylcholinesterase due to an interaction between acrylonitrile and cysteine residues on the enzyme. It is also suggested that the increase in acetylcholinesterase activity is due a stimulation of acetylcholine release. The authors further suggest that the biphasic response of brain acetylcholinesterase activity is hormetic; i.e. exposure to a low dose of acrylonitrile initiates a compensatory reaction that leads to a temporary increase in acetylcholinesterase activity. The influence of ethanol pre-treatment on response suggests an effect of acrylonitrile rather than a metabolite.

In a recent study of neuropathology aimed at elucidating the mechanism of carcinogenicity, Li et al.(2014) exposed groups of Wistar rats (6/sex) by gavaged with acrylonitrile at dose levels of 0, 5, 10 or 20 mg/kg bw on 5 days/week for 13 weeks. At termination, the brains of the rats were removed. The brain of each rat was examined by light microscopy, and four of the six brains in each sex of each group were examined by electron microscopy. Four brains from each group were examined for neuronal apoptosis by TUNEL. Six slides from each rat were examined to determine the amount and distribution of apoptotic neurons. DNA extracted from the rat brains was subjected to agarose gel electrophoresis. Bcl-2 protein, PCNA, and p53 protein were analysed immunohistochemically. Neuronal morphology with H&E staining was almost normal in rats administered 5 mg/kg bw/d acrylonitrile; occasional vacuolation was seen in this group.  At 10 mg/kg bw/d, neurons showed significant vacuolation; widening of spaces around the brain vessels was also observed.  More marked effects were seen in the brains of rats administered 20 mg/kg bw/d acrylonitrile.  Electron microscopy showed occasional malformed nuclei at 5 mg/kg bw/d. at 10 mg/kg bw/d, neurons showed disordered myelin sheaths, malformed nuclei, peripheral chromatin condensation and occasional apoptotic bodies.  At 20 mg/kg bw/d, neuronal nuclei were ‘obviously malformed’ with peripheral chromatin condensation.  TUNEL revealed a reduction in the number of apoptotic cells in all treated groups, but without a dose-response relationship. Agarose gel electrophoresis did not reveal the formation of DNA ladders in any group.  Immunohistochemistry is stated to have revealed an increase in the number of Bcl-2- positive cells in the treated groups; however figures are not reported.  There was no significant expression of p53 or PCNA in any group. The authors suggest that the suppression of spontaneous neuronal apoptosis by acrylonitrile may promote the growth of brain tumours in the rat. This conclusion is questionable given the lack of methodological detail and the absence of a dose-response relationship. The conclusion of the authors on a role for the inhibition of neuronal apoptosis in the aetiology of brain tumours in the rat is questionable as these tumours are demonstrated to be of microglial rather than neuronal origin.

Rongzhu et al. (2007) report a study in which male Sprague-Dawley rats were exposed to 50 or 200 ppm acrylonitrile in the drinking water for 12 weeks. Three neurobehavioural tests (open field, rotarod and spatial water maze) were conducted following exposure for 0, 4, 8 and 12 weeks and compared to results for concurrent controls. Clinical behavioural signs were reported and included head twitching and circling. There were no consistent changes in the open field test, although rats in the 200 ppm group showed significantly higher levels of locomotion compared to controls. The 50 ppm group showed a significantly decreased startup latency, decreased rearing frequency and fewer grooming bouts than controls at Week 0. The authors do not discuss the fact that behavioural differences prior to the start of acrylonitrile exposure may have confounded the results following exposure, nor do they comment on possible explanations for the behavioural differences in this group. Performance in the rotarod test was affected by time of exposure, particularly at 200 ppm; however there were also significant differences at Week 0 between groups that were not discussed. Acrylonitrile also negatively affected performance in the spatial water maze; however the treated rats appeared to recover towards the end of the experiment. This study provides evidence that acrylonitrile may affect performance in tests which are used to asses neurological effects. However it is noted that the dose levels used in this study were likely to result in subacute toxicity, therefore it is unclear whether the nervous system is specifically targeted.

The EU RAR (2004) provides a comprehensive review of the literature available at that time on the neurotoxicity of acrylonitrile. It is concluded that the symptoms of acute acrylonitrile poisoning (such as general convulsions and paralysis of the lower limbs) support the conclusion that acrylonitrile acts both on the central and peripheral nervous systems. The EU RAR notes two distinct phases of acute neurotoxicity reported in rats administered acrylonitrile.  Early signs of salivation, lacrimation, miosis, diarrhoea, polyuria and peripheral vasodilation peak within 60 minutes of dosing.  Other signs of toxicity include flushing of the face, ears and extremities. The early phase is followed by a delayed phase (>4 hours) which includes central nervous system effects such as respiratory depression, convulsions and death. Signs of neurotoxicity are dose-dependent, regardless of the route of administration.  It is noted that the acute toxicity of nitriles in general depends on the complex interplay of a number of factors including as the rate of cyanide liberation and detoxification, the dose of cyanogen, route of administration, species and the presence of other bioreactive sites within the nitrile molecule. While acrylonitrile is cyanogenic, it is also metabolised to the reactive epoxide CEO and the parent molecule is also capable of non-enzymatically alkylating essential functional groups. The EU RAR concludes that all of these factors may contribute to the neurotoxicity of acrylonitrile.

Maurissen (2010) reviews the findings of a number of studies with specific focus on neurotoxicity and neuropathology. The author concludes that, while the CNS has been identified as a target organ for acrylonitrile in a number of animal studies, closer examination of a number of papers does not confirm the reported effects.  Signs of irritation have been consistently reported in animal studies and are also noted following human exposure.  Other signs and symptoms such as headache, fatigue and weakness have been documented in humans. Motor effects including decreased coordination and motor nerve conduction velocity have also been observed in studies in rats. In the sensory domain, nerve conduction velocity and action potential amplitude were probably affected at 50 mg/kg bw/d by gavage dosing and following inhalation of 100 ppm.  Acutely, acrylonitrile can reversibly decrease auditory sensitivity at 50 mg/kg bw and can potentiate the effects of noise in animals at a probably relevant noise level for humans, but at a dose level (50 mg/kg bw) and dose route (subcutaneous) without human relevance.  The documented synergy with noise, though, may be of relevance to humans and should not be overlooked.  Olfactory function can be reversibly affected in workers exposed to ABS products for several years.  Although not much can be concluded about acrylonitrile per se, it should be reminded that studies have identified nasal lesions in rats.  Particular caution is needed in extrapolating olfactory effects from rats to humans for risk assessment. No behavioural signs of vestibular dysfunction were observed and animal studies do not provide any convincing evidence of learning deficits. While chronic exposure to acrylonitrile can increase glial cell brain tumours in rats, epidemiology studies suggest that acrylonitrile is not a potent CNS carcinogen in humans.  Finally, it is observed that the neurotoxicity observed with acrylonitrile varies considerably across different species. While the molecular mechanisms of acrylonitrile neurotoxicity are poorly understood, it is known that treatment selectively induces oxidative stress. The role of the parent compound, cyanide production, GSH conjugation and depletion, CYP2E1, GST and SOD activities, and of other enzymes and metabolites associated with the metabolism of acrylonitrile, provide many potential ways in which neurotoxicity can be expressed.  Several mechanisms may therefore be involved in acrylonitrile neurotoxicity. The role the cholinergic system plays also unclear. The electrophilic nature of acrylonitrile enables binding to nucleophilic sites on macromolecules, and it has been suggested that adducts could serve as biomarker of exposure or could be seen as a putative molecular mechanism of action for electrophilic neurotoxicants. The author concludes, therefore, that the mechanism of neurotoxicity is still unknown and most likely multifaceted.

Justification for classification or non-classification

No classification is proposed for neurotoxicity. The animal data and acute human accidental exposures indicate potential for neurotoxicity, but this is consistent with and considered to be addressed by classification for acute toxicity. Evidence for the neurotoxicity of acrylonitrile following longer-term exposure is less convincing and not sufficiently robust for classification.