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Priority Substances List Assessment Report for Acrylonitrile
2.0 Summary of Information Critical to Assessment of "Toxic" Under CEPA 1999 (continue)
2.4 Effects characterization
2.4.2 Abiotic atmospheric effects
Worst-case calculations were made to determine whether acrylonitrile has the potential to contribute to depletion of stratospheric ozone, ground-level ozone formation or climate change (Bunce, 1996).
The Ozone Depletion Potential (ODP) was calculated to be 0, since acrylonitrile does not contain chlorine or bromine atoms.
The Photochemical Ozone Creation Potential (POCP) was estimated to be 25 (relative to the value of an equal mass of the reference compound ethene, which has a POCP of 100), based on the following formula:
POCP = (kACN / kethene) × (Methene / MACN) × 100
where:
- kACN is the rate constant for the reaction of acrylonitrile with OH radicals (4 × 10-12 cm3/mol per second),
- kethene is the rate constant for the reaction of ethene with OH radicals (8.5 × 10-12 cm3/mol per second),
- Methene is the molecular weight of ethene (28.1 g/mol), and
- MACN is the molecular weight of acrylonitrile (53.1 g/mol).
The Global Warming Potential (GWP) was calculated to be 4.3 × 10-4 (relative to the reference compound CFC-11, which has a GWP of 1), based on the following formula:
GWP = (tACN / tCFC-11) × (MCFC-11 / MACN) × (SACN / SCFC-11)
where:
- tACN is the lifetime of acrylonitrile (0.0099 years),
- tCFC-11 is the lifetime of CFC-11 (60 years),
- MCFC-11 is the molecular weight of CFC-11 (137.5 g/mol),
- MACN is the molecular weight of acrylonitrile (53.1 g/mol),
- SACN is the infrared absorption strength of acrylonitrile (2389/cm2 per atmosphere, default), and
- SCFC-11 is the infrared absorption strength of CFC-11 (2389/cm2 per atmosphere).
Actual contribution to formation of photochemical ozone depends on both reactivity and concentration in an area or region. The POCP value indicates a moderate potential for photochemical ozone formation. However, acrylonitrile is released from only a few point sources in Canada, and, importantly, levels of acrylonitrile in ambient urban air have generally been below detection levels of 0.9 µg/m3 (Bell et al., 1991; OMOE, 1992a; Ng and Karellas, 1994), which indicates that acrylonitrile is likely to be only a very minor contributor to photochemical ozone formation. The absence of chlorine and bromine atoms in the molecule means that the potential contributions of acrylonitrile to stratospheric ozone depletion and climate change are both negligible.
2.4.3 Experimental animals and in vitro
2.4.3.1 Acute toxicity
The acute toxicity of acrylonitrile is relatively high, with four-hour LC50s ranging from 300 to 900 mg/m3 (Knobloch et al., 1971, 1972) and LD50s ranging from 25 to 186 mg/kg-bw (Maltoni et al., 1987). Signs of acute toxicity include respiratory tract irritation and central nervous system dysfunction, resembling cyanide poisoning. Superficial necrosis of the liver and hemorrhagic gastritis of the forestomach have also been observed following acute exposure.
Acrylonitrile-induced neurotoxicity following acute exposure has been described as a two-phase phenomenon. The first phase, which occurs shortly after exposure and is consistent with cholinergic overstimulation, has been likened to toxicity caused by acetylcholinesterase inhibition. Cholinomimetic signs in rats exposed to acrylonitrile have included vasodilation, salivation, lacrimation, diarrhea and gastric secretion. These effects are maximal within one hour of dosing. The second phase of toxicity is delayed by four or more hours and includes signs of central nervous system disturbance, such as trembling, ataxia, convulsions and respiratory failure (TERA, 1997). The acetylcholine-like toxicity is thought to be caused by acrylonitrile, while the central nervous system depression is caused by cyanide (the latter does not cause acetylcholine-like effects).
2.4.3.2 Short-term toxicity
Available short-term inhalation studies are restricted to a few investigations involving administration of single dose levels and, for one, examination of clinical signs only. Exposure response has not, therefore, been well characterized. There were effects on biochemical parameters, clinical signs and body weight, although no histopathological effects on principal organs, following exposure of rats to 280 mg/m3 (Gut et al., 1984, 1985).
In short-term studies by the oral route, effects on the liver, adrenal and gastric mucosa have been observed, with effects on the gastric mucosa occurring at lowest doses in all studies in which they were examined. Effects on the adrenal cortex observed in short-term repeated-dose toxicity studies from one laboratory have not been noted in longer-term investigations in animals exposed to higher concentrations. In investigations by Szabo et al. (1984), effects on the non-protein sulphydryl in gastric mucosa and hyperplasia in the adrenal cortex have been reported at levels as low as 2 mg/kg-bw per day administered by drinking water and gavage, respectively, for 60 days. Effects on hepatic glutathione were also observed by these authors at similar doses administered by gavage but not in drinking water (2.8 mg/kg-bw per day for 21 days), although Silver et al. (1982) noted only slight biochemical effects but no histopathological effects in the liver at doses up to 70 mg/kg-bw per day (drinking water, 21 days). Significant increases in proliferation in the forestomach but no changes in the liver or glandular stomach have been observed at 11.7 mg/kg-bw (Ghanayem et al., 1995, 1997).
Effects of pretreatment with inducers of the mixed-function oxidase system or antioxidants on toxicity in short-term studies have been consistent with metabolism to the epoxide 2-cyanoethylene oxide being the putatively toxic metabolic pathway.
2.4.3.3 Subchronic toxicity
Results of identified subchronic toxicity studies are limited to an early 13-week inhalation study in rats and dogs that has not been validated (IBT, 1976) and a preliminary brief report of the results of a 13-week gavage study in mice (NTP, 1996). Lack of validation and inadequate detail limit the utility of these studies for hazard evaluation or characterization of dose-response.
2.4.3.4 Chronic toxicity and carcinogenicity
In the descriptions of the following studies, tumour types are reported as described by the authors. However, it should be noted that the histopathology of the tumours may be unclear (see footnote 2).
2.4.3.4.1 Inhalation
Quast et al. (1980b) conducted a bioassay in which Sprague-Dawley (Spartan substrain) rats (100 per sex per group) were exposed by inhalation to average concentrations of 0, 20 or 80 ppm (0, 44 or 176 mg/m3) of acrylonitrile six hours per day, five days per week, for two years. Non-neoplastic histopathological changes related to the treatment were found in the nasal turbinates and the central nervous system of both males and females. In the brain, the changes were characterized by focal gliosis and perivascular cuffing at the highest concentration. The inflammatory changes in the nasal turbinates were considered to be due to acrylonitrile irritation. These effects were not observed at 20 ppm, and this dose is considered as a No-Observed-Effect Level (NOEL). An early onset of chronic renal disease in the 20 ppm group was observed upon histopathological examination. The renal effect was not apparent at the high dose because of early mortality. The chronic renal disease was considered a secondary effect caused by increased water intake and is commonly observed in older rats of this strain. A pair-fed control study was not performed, and further clinical analyses are required to understand the chronic renal effect.
In both sexes, there was an increase in the combined incidence of malignant and benign tumours of the brain and spinal cord (Table 5) and benign and malignant tumours of the Zymbal gland at the high dose. In males, the combined incidence of benign and malignant tumours of the small intestine and the tongue was increased at the high dose. The incidence of adenocarcinoma of the mammary gland was increased at the high dose in females (Quast et al., 1980b).
In an earlier study, Maltoni et al. (1977) exposed Sprague-Dawley rats to 0, 5, 10, 20 or 40 ppm (0, 11, 22, 44 and 88 mg/m3) acrylonitrile for four hours per day, five days per week, for 52 weeks. Increases in the incidence of tumours were observed in the mammary gland in males and females, in the forestomach in males and in the skin in females. The authors concluded that because of the lack of a dose-related response in tumour incidence, the results could be evaluated as "borderline carcinogenic effects." Low concentrations of acrylonitrile, short exposure time and small group size (n = 30) limit the sensitivity of the study.
In a follow-up study by Maltoni et al. (1987, 1988), 54 female Sprague-Dawley rat breeders and male and female offspring were administered 60 ppm (132 mg/m3) by inhalation for 4-7 hours per day, five days per week. The breeders and some of the offspring were exposed for 104 weeks; the remaining offspring were exposed for 15 weeks only. The non-neoplastic treatment-related changes included slight, but significant, increases in the incidence of encephalic glial cell hyperplasia and dysplasia in offspring exposed for 104 weeks. A significantly increased incidence of various tumours was observed in the exposed offspring, both males and females. Tumours with increased incidence included mammary gland tumours in females, Zymbal gland tumours in males, extrahepatic angiosarcoma in both males and females, hepatomas in males and encephalic gliomas in both males and females. The most pronounced acrylonitrile-related tumour was encephalic glioma (in control and exposure groups, respectively: 2/158 and 11/67 in males; 2/149 and 10/54 in females) in the offspring treated with acrylonitrile for 104 weeks.
2.4.3.4.2 Drinking water
Quast et al. (1980a) administered acrylonitrile in drinking water to groups of 48 Sprague-Dawley rats of each sex (n = 80 for controls) for two years at dose levels of 0, 35, 100 or 300 ppm (based upon data for water consumption and body weight, the authors reported intakes of 0, 3.4, 8.5 or 21.2 mg/kg-bw per day for males and 0, 4.4, 10.8 or 25.0 mg/kg-bw per day for females). There was treatment-related hyperplasia and hyperkeratosis of the squamous epithelium of the forestomach in females at all dose levels and in males at 100 and 300 ppm. In the brain of females, there was a significantly increased incidence of focal gliosis and perivascular cuffing in the 35 and 100 ppm groups. Other changes were not considered to be directly treatment related, but, rather, secondary to decreased food and water consumption, although supporting information from pair-fed controls was not available.
Sacrifice and necropsy were carried out on moribund animals. Tumours (including astrocytomas) were observed as early as 7-12 months in females in the high-dose group; in other dose groups, they appeared initially in the 13- to 18-month period. In both males and females, the combined incidence of benign and malignant tumours of the brain and spinal cord was significantly increased in a dose-related manner at all levels of exposure (Table 6). The incidence of carcinoma of the Zymbal gland was significantly increased at the highest dose in males and at the two highest doses in females (Quast et al., 1980a).
In a study conducted by Bio/Dynamics Inc. (1980a), groups of 100 male and 100 female Sprague-Dawley rats were administered acrylonitrile at dose levels of 0, 1 or 100 ppm in drinking water (0, 0.09 and 8.0 mg/kg-bw per day for males and 0, 0.15 and 10.7 mg/kg-bw per day for females, based upon body weight and water consumption 1) for 19 and 22 months. The mean absolute and relative weights of the kidneys in the high-dose females were increased (not always significantly) at all sacrifice intervals. There was an increase in testicular weight to body weight ratio in the high-dose males at the 12- and 18-month sacrifices and at the end of the experiment. No such changes were evident at 1 ppm. This concentration can be considered as a NOEL and 100 ppm as a Lowest-Observed-Adverse-Effect Level (LOAEL) for non-neoplastic effects.
| Animal data | Parameter estimates | Human equivalent values |
||
|---|---|---|---|---|
| Dose | Incidence | |||
| Males: Brain and/or spinal cord, benign and malignant; excluding animals dying or sacrificed before 6 months |
control | 0/98 | TC052 = 52 mg/m3 95% LCL3 = 29 mg/m3 Chi-square = 0.73 degrees of freedom = 1 p-value = 1.00 |
TC054 = 8.9 mg/m3 95% LCL = 5 mg/m3 |
| 44 mg/m3 (20 ppm) | 4/97 (4 astrocytoma) | |||
| 176 mg/m3 (80 ppm) | 22/98 (15 astrocytoma, 7 benign) | |||
| Males: Brain and/or spinal cord, benign and malignant; excluding animals dying or sacrificed before 10 months (TERA, 1997) |
control | 0/975 | TC052 = 51 mg/m3 95% LCL = 33 mg/m3 Chi-square = 0.00 degrees of freedom = 1 p-value = 1.00 |
TC054 = 8.7 mg/m3 95% LCL = 5.6 mg/m3 |
| 44 mg/m3 (20 ppm) | 4/935 | |||
| 176 mg/m3 (80 ppm) | 15/835 | |||
| Females: Brain and/or spinal cord, benign and malignant; excluding animals dying or sacrificed before 6 months |
control | 0/99 | TC052 = 35 mg/m3 95% LCL = 26 mg/m3 Chi-square = 0.65 degrees of freedom = 2 p-value = 0.72 |
TC054 = 6 mg/m3 95% LCL = 4.5 mg/m3 |
| 44 mg/m3 (20 ppm) | 8/100 (4 astrocytoma, 4 benign) | |||
| 176 mg/m3 (80 ppm) | 21/99 (17 astrocytoma, 4 benign) | |||
| Females: Brain and/or spinal cord, benign and malignant; excluding animals dying or sacrificed before 6 months (TERA, 1997) | control | 0/995 | TC052 = 35 mg/m3 95% LCL = 26 mg/m3 Chi-square = 0.69 degrees of freedom = 2 p-value = 0.71 |
TC054 = 5.9 mg/m3 95% LCL = 4.4 mg/m3 |
| 44 mg/m3 (20 ppm) | 8/995 | |||
| 176 mg/m3 (80 ppm) | 21/995 | |||
1 Quast et al. (1980b).
2 For this study, the resulting TC05s were multiplied by (6 hours per day/24 hours per day) × (5 days per week/7 days per week) to adjust for intermittent to continuous exposure.
3 95% LCL = lower 95% confidence limit.
4 To scale from rats to humans, the TC05s were multiplied by (0.11 m3 per day/0.35 kg-bw) × (70 kg-bw/23 m3 per day), where 0.11 m3 per day is the breathing rate of a rat, 0.35 kg-bw is the body weight of a rat, 23 m3 per day is the breathing rate of a human and 70 kg-bw is the body weight of a human.
5 These incidence data could not be verified in an examination of mortality data in Quast et al. (1980b).
In high-dose males, increased incidences of squamous cell carcinoma of the stomach and carcinoma of the Zymbal gland were observed at the 12-month sacrifice. In high-dose females, astrocytoma of the brain and carcinoma of the Zymbal gland were increased at 12 months. At the high dose, there was an increased cumulative incidence of astrocytoma of the brain, carcinoma of the Zymbal gland and papilloma/carcinoma of the stomach in both males and females. In females, the incidence of astrocytoma of the spinal cord was significantly increased at the high dose. The spinal cord tissue of the males was not examined, although overall histological examination was rather extensive (Bio/Dynamics Inc., 1980a).
A bioassay in Fischer 344 rats exposed to acrylonitrile in drinking water was also conducted by Bio/Dynamics Inc. (1980b). Rats (200 per sex, control group; 100 per sex per dose group) were administered acrylonitrile in drinking water for approximately two years. The dose levels were 0, 1, 3, 10, 30 and 100 ppm acrylonitrile (0, 0.1, 0.3, 0.8, 2.5 and 8.4 mg/kg-bw per day for males and 0, 0.1, 0.4, 1.3, 3.7 and 10.9 mg/kg-bw per day for females, as reported by U.S. EPA, 1985).
Serial sacrifices were conducted at 6, 12 and 18 months (20 per sex per control group and 10 per sex per treated group). To ensure at least 10 rats per sex per group for histopathological evaluation, all females were sacrificed at 23 months, owing to low survival. The males were continued on test until the 26th month.
The consistently elevated mortality in the highest dose groups was a consequence of tumours. Other changes observed primarily in the highest exposure group included consistently lower body weights in females and males and consistent reduction in hemoglobin, hematocrit and erythrocyte counts in females throughout the study. A decrease in water intake was also observed, while food consumption was comparable for all groups (Bio/Dynamics Inc., 1980b).
| Animal data | Parameter estimates | Human equivalent values |
||
|---|---|---|---|---|
| Dose | Incidence | |||
| Males: brain and/or spinal cord, benign and malignant; excluding animals dying or sacrificed before 6 months |
control | 1/79 (1 astrocytoma) | TD05 = 0.84 mg/kg-bw per day 95% LCL2 = 0.68 mg/kg-bw per day Chi-square = 3.68 degrees of freedom = 2 p-value = 0.16 |
TD05 = 0.84 mg/kg-bw per day 95% LCL = 0.68 mg/kg-bw per day |
| 3.4 mg/kg-bw per day (35 ppm) | 12/47 (8 astrocytoma, 4 benign) | |||
| 8.5 mg/kg-bw per day (100 pm) | 23/47 (19 astrocytoma, 4 benign) | |||
| 21.2 mg/kg-bw per day (300 ppm) | 31/48 (23 astrocytoma, 8 benign) | |||
| Females: Brain and/or spinal cord, benign and malignant; excluding animals dying or sacrificed before 6 months |
control | 1/80 (1 astrocytoma) | Parameter estimates excluding high-dose group: TD053 = 0.56 mg/kg-bw per day 95% LCL = 0.44 mg/kg-bw per day Chi-square = 4.77 degrees of freedom = 1 p-value = 0.08 |
TD052 = 0.56 mg/kg-bw per day 95% LCL = 0.44 mg/kg-bw per day |
| 4.4 mg/kg-bw per day (35 ppm) | 22/48 (17 astrocytoma, 5 benign) | |||
| 10.8 mg/kg-bw per day (100 ppm) | 26/48 (22 astrocytoma, 4 benign) | |||
| [25.0 mg/kg-bw per day (300 ppm)] |
[31/47 (24 astrocytoma, 7 benign)] |
|||
1Quast et al. (1980a).
295% LCL = lower 95% confidence limit.
3 Excludes high-dose group. A dose-related increase in mortality was observed for females, resulting in a plateau in the dose-response function and lack of fit of the model to brain/spinal tumours. However, when the model was refit excluding the highest dose group, this lack of fit was no longer apparent.
An increase in the relative organ weights of the liver and kidney was noted at the highest dose levels; however, the mean absolute weights for these organs were either comparable to those in the controls or only slightly increased. At terminal sacrifice, the absolute liver and heart weights were elevated in females exposed to 30 ppm, but body weight was comparable to that in controls. A LOAEL of 100 ppm and a Lowest-Observed-Effect Level (LOEL) of 30 ppm for non-neoplastic effects can be designated. In both males and females, the incidence of astrocytoma of the brain (Table 7) and the incidence of carcinoma of the Zymbal gland were significantly increased at the two highest dose levels (Bio/Dynamics Inc., 1980b).
In a multigeneration reproductive study, 0, 100 or 500 ppm acrylonitrile (0, 14 or 70 mg/kg-bw per day; Health Canada, 1994) was administered in drinking water to breeders (F0) and the offspring of Charles River Sprague-Dawley rats (Litton Bionetics Inc., 1980). Rats of the F1b generation in the high-exposure group had a significantly increased incidence of astrocytomas and Zymbal gland tumours. For control, low-exposure and high-exposure groups, the incidence of astrocytomas was 0/20, 1/19 and 4/17 (p < 0.05), respectively, and the incidence of Zymbal gland tumours was 0/20, 2/19 and 4/17 (p < 0.05), respectively. The tumour incidence was low, but the exposure and observation period (approximately 45 weeks) was also relatively short. Not all tissues were examined histopathologically.
| Animal data | Parameter estimates | Human equivalent values |
||
|---|---|---|---|---|
| Dose | Incidence | |||
| Males: Nervous system, combined incidence, astrocytoma and focal gliosis, excluding animals dying or sacrificed before 6 months | control | 5/182 (3 strocytoma, 2 benign) | TD052 = 1.8 mg/kg-bw per day 95% LCL3 = 1.2 mg/kg-bw per day Chi-square = 3.0 degrees of freedom = 3 p-value = 0.39 |
TD05 = 2.3 mg/kg-bw per day 95% LCL = 1.6 mg/kg-bw per day |
| 0.08 mg/kg-bw per day (1 ppm) | 2/90 (2 astrocytoma) | |||
| 0.25 mg/kg-bw per day (3 pm) | 1/89 (1 astrocytoma) | |||
| 0.84 mg/kg-bw per day (10 ppm) | 2/90 (2 astrocytoma) | |||
| 2.49 g/kg-bw per day (30 ppm) | 10/89 (10 astrocytoma) | |||
| 8.37 g/kg-bw per day (100 ppm) | 22/90 (21 astrocytoma, 1 benign) |
|||
| Females: Brain and/or spinal cord, benign and malignant; excluding animals dying or sacrificed before 6 months | control | 1/178 (1astrocytoma) | TD05 = 2.3 mg/kg-bw per day 95% LCL = 1.4 mg/kg-bw per day Chi-square = 1.8 degrees of freedom = 3 p-value = 0.62 |
TD05 = 2.3 mg/kg-bw per day 95% LCL = 1.4 mg/kg-bw per day |
| 0.10 mg/kg-bw per day (1 ppm) | 1/90 (1 astrocytoma) | |||
| 0.40 mg/kg-bw per day (3 ppm) | 2/90 (2 astrocytoma) | |||
| 1.30 mg/kg-bw per day (10 ppm) | 5/88 (4 astrocytoma, 1 benign) | |||
| 3.70 mg/kg-bw per day (30 ppm) | 6/90 (6 astrocytoma) | |||
| 10.90 mg/kg-bw per day (100 ppm) | 26/90 (24 astrocytoma, 2 benign) | |||
1Bio/Dynamics Inc. (1980b).
2The experimental length for this study was 24 months for females and 26 months for males, so the resulting TD05s for males were multiplied by (26 months/24 months) × (26 months/24 months)2, where the first term amortizes the dose to be constant over the standard lifetime of a rat (24 months) and the second factor, suggested by Peto et al.(1984), corrects for an experimental length that is unequal to the standard lifetime.
3 95% LCL = lower 95% confidence limit.
More recently, Bigner et al. (1986) observed neuro-oncogenic effects in Fischer 344 rats administered 0, 100 or 500 ppm acrylonitrile in drinking water (0, 14 and 70 mg/kg-bw per day; Health Canada, 1994). Each exposure group consisted of 50 male and 50 female rats. A fourth group of 300 rats (147 males, 153 females) was exposed to 500 ppm acrylonitrile. Although the protocol of the study indicated that rats were exposed for their lifetime, results were presented for an 18-month observation period. There was a dose-related significant reduction in body weight in both males and females at 500 ppm. In rats exposed for 12-18 months, neurological signs such as decreased activity, paralysis, head tilt, circling and seizures were observed in the 100 and 500 ppm groups. In control, low-exposure and two high-exposure groups, the incidence of neurological signs was 0/100, 4/100, 16/100 and 29/300, respectively. Histopathological examination of 215 animals in the 500 ppm group revealed 49 primary brain tumours, which were difficult to classify.2 Other tumours frequently observed included Zymbal gland tumours, forestomach papillomas and subcutaneous papillomas. No further details, however, were presented. The authors reported that the increase in incidence of the primary brain tumour in the highest exposure group was significant (p-values were not reported, data poorly presented). Other endpoints were not examined. The results are inadequate, therefore, for establishing effect levels for non-neoplastic effects or for characterizing exposure-response for tumours.
Gallagher et al. (1988) investigated the carcinogenicity of acrylonitrile administered via drinking water at 0, 20, 100 or 500 ppm (approximately 0, 2.8, 14 and 70 mg/kg-bw per day; Health Canada, 1994) to male Sprague-Dawley rats (20 per group) for two years. There was no survival in the 500 ppm exposure group at two years. Ingestion of acrylonitrile at concentrations up to and including 100 ppm did not increase mortality. The necropsy results revealed a significant increase in Zymbal gland tumours at 500 ppm (0/18, 0/20, 1/19 and 9/18 [p < 0.005] in control, low-, mid- and high-dose groups, respectively). No increase in tumours of other organs including brain was observed, although four rats developed papillomatous proliferation of the epithelium of the forestomach in the high-exposure group.
It is of interest to note that whereas Gallagher et al. (1988) reported increased incidence of tumours of the Zymbal gland only at a dose level of 70 mg/kg-bw per day in Sprague-Dawley rats, Bio/Dynamics Inc. (1980a) reported increased incidence of astrocytoma of the brain, carcinoma of the Zymbal gland and papilloma/ carcinoma of the stomach in the same strain of rats at 8 mg/kg-bw per day.
2.4.3.4.3 Gavage
Groups of 100 male and 100 female Sprague-Dawley (Spartan substrain) rats were exposed in another Bio/Dynamics Inc. (1980c) study to acrylonitrile in deionized water by intubation at 0, 0.1 or 10 mg/kg-bw per day for five days per week for 20 months. The non-neoplastic effects in the high-dose group included consistently higher mortality in both males and females and decreased body weights in males. Relative liver weight was increased in males at the high dose. The dose of 10 mg/kg-bw per day is proposed as a LOAEL, based upon decreased body weight and increased liver to body weight ratio in male rats. In both males and females at the high dose, there was an increased incidence of astrocytoma of the brain, squamous cell carcinoma of the Zymbal gland and papilloma/carcinoma of the stomach. In both sexes, squamous cell papilloma of the stomach was reported at the high dose as early as 12 months. At the 18-month sacrifice, squamous cell carcinoma of the stomach was reported in males at the high dose. Astrocytoma of the brain and carcinoma of the Zymbal gland were reported in high-dose females at the 18-month sacrifice.
Maltoni et al. (1977) exposed 40 Sprague-Dawley rats of each sex by gavage to acrylonitrile in olive oil at 0 or 5 mg/kg-bw per day, three days per week, for 52 weeks. In females, there was some evidence of increases in mammary gland carcinomas (7/75 and 4/40 in control and exposed groups, respectively) and forestomach epithelial tumours (0/75 and 4/40 in control and exposed groups, respectively) in females. However, a high spontaneous incidence of mammary gland tumours in this strain of rats, the single dose level and the short duration of exposure limit the adequacy of the study.
2.4.3.5 Genotoxicity
2.4.3.5.1 in vitro studies
In the Salmonella mammalian microsome assay, acrylonitrile has induced reverse mutations in strains TA1535 (Lijinsky and Andrews, 1980), TA1535 and TA100 (Zeiger and Haworth, 1985), but only when hamster or rat S9 was present. Weak positive results were also reported in several Escherichia coli strains in the absence of metabolic activation (Venitt et al., 1977).
In mammalian cells, acrylonitrile induced hprt mutations in human lymphoblasts without metabolic activation (Crespi et al., 1985), but not at the same locus in Chinese hamster V79 cells (Lee and Webner, 1985). In several studies, acrylonitrile was positive at the TK locus in mouse lymphoma L5178 TK+/- cells, either with or without rat S9 (Amacher and Turner, 1985; Lee and Webber, 1985; Myhr et al., 1985; Oberly et al., 1985), and in mouse lymphoma P388F cells with metabolic activation (Anderson and Cross, 1985). It was also mutagenic at the TK locus in human lymphoblasts with metabolic activation (Crespi et al., 1985; Recio and Skopek, 1988).
Acrylonitrile induced structural chromosomal aberrations either with or without metabolic activation in Chinese hamster ovary cells (Danford, 1985; Gulati et al., 1985; Natarajan et al., 1985) and without metabolic activation in Chinese hamster lung cells (Ishidate and Sofuni, 1985).
Acrylonitrile induced sister chromatid exchanges in Chinese hamster ovary cells with or without metabolic activation (Gulati et al., 1985) or only with metabolic activation (Brat and Williams, 1982; Natarajan et al., 1985). In human lymphocytes, results for sister chromatid exchanges were mixed, with one positive study with phenobarbital sodium-induced or 5,6-benzoflavone-induced rat liver (Perocco et al., 1982) and one negative study with Aroclor-induced rat liver (Obe et al., 1985). Sister chromatid exchanges were induced in human bronchial epithelial cells in the absence of S9 (Chang et al., 1990).
Results of in vitro assays for DNA single strand breaks and DNA repair (unscheduled DNA synthesis) were mixed but more commonly negative in a range of cell types from rats and humans, with and without activation. Cell transformation in mouse and hamster embryo cells has also been investigated, with mixed results.
Binding of 2-cyanoethylene oxide to nucleic acids has also been reported in in vitro studies at high concentrations (Hogy and Guengerich, 1986; Solomon and Segal, 1989; Solomon et al., 1993; Yates et al., 1993, 19943). The formation of DNA adducts is increased substantially in the presence of metabolic activation. Under non-activating conditions involving incubation of calf thymus DNA with either acrylonitrile or 2-cyanoethylene oxide in vitro , 2-cyanoethylene oxide alkylates DNA much more readily than acrylonitrile (Guengerich et al., 1981; Solomon et al., 1984, 1993). Incubation of DNA with 2-cyanoethylene oxide yields 7-(2-oxoethyl)-guanine (Guengerich et al., 1981; Hogy and Guengerich, 1986; Solomon and Segal, 1989; Solomon et al., 1993; Yates et al., 1993, 1994) as well as other adducts. Compared with studies with rat liver microsomes, little or no DNA alkylation was observed with rat brain microsomes (Guengerich et al., 1981). DNA alkylation in human liver microsomes was much less than that observed with rat microsomes (Guengerich et al., 1981).
2.4.3.5.2 In vivo studies
Limitations of the few in vivo studies conducted in which the genotoxicity of acrylonitrile has been investigated preclude definitive conclusions.
Exposure to acrylonitrile in drinking water resulted in increased frequency of mutants at the hprt locus in splenic T-cells (Walker and Walker, 1997).4 Female F344 rats were exposed to 0, 33, 100 or 500 ppm (0, 8, 21 or 76 mg/kg-bw per day; Health Canada, 1994) in drinking water for up to four weeks. Serial sacrifices were carried out throughout exposure and up to eight weeks post-exposure. At four weeks post-exposure, the average observed mutant frequency in splenic T-cells was increased in a dose-related manner (significant at the two highest doses).
Results of a range of assays for structural chromosomal aberrations, micronuclei in bone marrow and micronuclei in peripheral blood cells have been negative or inconclusive, although there was no indication in the published accounts of three of the four studies that the compound reached the target site. These include studies in Swiss (Rabello-Gay and Ahmed, 1980), NMRI (Leonard et al., 1981) and C57B1/6 (Sharief et al., 1986) mice and a collaborative study using multiple routes of exposure in mice and rats (Morita et al., 1997).
Results of dominant lethal assays were inconclusive in mice (Leonard et al., 1981) and negative in rats (Working et al., 1987).
In assays for unscheduled DNA synthesis in rats, results were positive only for the liver (Hogy and Guengerich, 1986), equivocal in lung, testes and gastric tissues (Ahmed et al., 1992a,b; Abdel-Rahman et al., 1994) and, notably, negative in the brain (Hogy and Guengerich, 1986). In these studies, however, unscheduled DNA synthesis was measured by liquid scintillation counting to determine 3H-thymidine uptake in the cell population, which does not discriminate between cells undergoing repair and those that are replicating. Results for unscheduled DNA synthesis in rat liver and spermatocytes were negative when 3H-thymidine uptake in individual cells was determined by autoradiography, which eliminates replicating cells from the analysis (Butterworth et al., 1992).
Urine from acrylonitrile-exposed rats and mice was also mutagenic in Salmonella typhimurium following intraperitoneal administration of acrylonitrile to rats and mice (Lambotte-Vandepaer et al., 1980, 1981). In both species, mutagenic activity occurred without activation. Mutagenic activity was also observed in urine of rats administered acrylonitrile by stomach intubation (Lambotte-Vandepaer et al., 1985). Thiocyanate, hydroxyethylmercapturic acid and cyanoethylmercapturic acid were not believed to be responsible for urinary mutagenicity.
In in vivo studies in F344 rats administered 50 mg acrylonitrile/kg-bw intraperitoneally, 7-(2-oxoethyl)-guanine adducts were detected in liver (Hogy and Guengerich, 1986). Incorporation of acrylonitrile into hepatic RNA was observed following intraperitoneal administration to rats (Peter et al., 1983). However, no DNA adducts were detected in the brain, which is the primary target for acrylonitrile-induced tumorigenesis, in this or a subsequent study in which F344 rats received 50 or 100 mg acrylonitrile/kg-bw by subcutaneous injection (Prokopczyk et al., 1988). In contrast, in three studies from one laboratory, exposure of SD rats to 46.5 mg [14C]acrylonitrile/kg-bw (50 µCi/kg-bw) resulted in apparent binding of radioactivity to DNA from liver, stomach, brain (Farooqui and Ahmed, 1983), lung (Ahmed et al., 1992a) and testicles (Ahmed et al. 1992b). In each tissue, there was a rapid decrease in radioactivity of DNA samples collected up to 72 hours following treatment.
It is not clear why acrylonitrile-DNA binding was detected in the brain in these studies and not by Hogy and Guengerich (1986) or Prokopczyk et al. (1988). The DNA isolation protocols and method for correcting for contaminating protein in the DNA sample used by Hogy and Guengerich (1986) may have allowed a more stringent determination of DNA-bound material. Alternatively, the methods used to achieve greater DNA purity might have caused the loss of adducts or inhibited the recovery of adducted DNA; more likely, 7-(2-oxoethyl)-guanine and cyanoethyl adducts are of little consequence in the induction of acrylonitrile-induced brain tumours. Indeed, investigation of the role of cyanohydroxy-ethylguanine in the induction of these tumours seems warranted.
2.4.3.6 Reproductive and developmental toxicity
Consistent effects on the reproductive organs of male or female animals have not been observed in repeated-dose toxicity and carcinogenicity studies conducted to date. In a specialized investigation in CD-1 mice, however, degenerative changes in the seminiferous tubules and associated decreases in sperm counts were observed at 10 mg/kg-bw per day (NOEL, 1 mg/kg-bw per day) (Tandon et al., 1988). Although epididymal sperm motility was reduced in a 13-week study with B6C3F1 mice, there was no dose-response and no effect upon sperm density at doses up to 12 mg/kg-bw per day by gavage, although histopathological results were not reported (Southern Research Institute, 1996). In a three-generation study in rats exposed via drinking water (14 or 70 mg/kg-bw per day), adverse effects on pup survival and viability and lactation indices were attributed to maternal toxicity (Litton Bionetics Inc., 1980).
In two studies by inhalation, developmental effects (fetotoxic and teratogenic) were not observed at concentrations that were not toxic to the mothers (Murray et al., 1978; Saillenfait et al., 1993a). In the investigation in which concentration-response was best characterized (four exposure concentrations and controls with two-fold spacing), the LOEL for maternal toxicity and for fetotoxicity was 55 mg/m3; the NOEL was 26.4 mg/m3 (Saillenfait et al., 1993a).
Similarly, in two studies by the oral route, developmental effects have not been observed at doses that were not also toxic to the mothers (lowest reported effect level in the mothers, 14 mg/kg-bw per day) (Murray et al., 1978; Litton Bionetics Inc., 1980). Reversible biochemical effects on the brain but not functional neurological effects were observed in offspring of rats exposed to 5 mg/kg-bw per day (a dose that did not impact on body weight of the dams); dose-response was not investigated in this study (Mehrotra et al., 1988).
Results of in vitro studies in rat embryos indicate that developmental effects may be due to monooxygenase-mediated liberation of cyanide (Saillenfait et al., 1992, 1993b).
2.4.3.7 Neurological effects and effects on the immune system
In recently published studies in rats exposed by inhalation to 25 ppm (55 mg/m3) acrylonitrile and above for 24 weeks, there were partially reversible time- and concentration-dependent reductions in motor and sensory conduction (Gagnaire et al., 1998).
In the few identified investigations of the immunological effects of acrylonitrile, effects on the lung following inhalation (Bhooma et al., 1992) and on the gastrointestinal tract following ingestion (Hamada et al., 1998) have been observed at concentrations and doses at which histopathological effects have also been observed.
2.4.3.8 Toxicokinetics and mode of action
2.4.3.8.1 Toxicokinetics
Based on studies conducted primarily in laboratory animals, acrylonitrile is rapidly absorbed and distributed throughout examined tissues. However, there appears to be little potential for significant accumulation in any organ, with most of the compound being excreted primarily as metabolites in urine in the first 24-48 hours following administration.
Acrylonitrile is metabolized primarily by two pathways: conjugation with glutathione to form N-acetyl-S-(2-cyanoethyl)cysteine and oxidation by cytochrome P-450 to form remaining urinary metabolites. Oxidative metabolism of acrylonitrile leads to the formation of 2-cyanoethylene oxide, which is either conjugated with glutathione or directly hydrolysed by epoxide hydrolase.
Available data5 are consistent with conjugation with glutathione being the major detoxification pathway of acrylonitrile, while the oxidation of acrylonitrile to 2-cyanoethylene oxide can be viewed as an activation pathway, producing a greater proportion of the total metabolites in mice than in rats. Available data also indicate that there are route-specific variations in metabolism. Based on studies in which 2-cyanoethylene oxide has been administered, there is no indication of preferential uptake or retention in specific organs, including the brain.
Liver microsomes from rats, mice and humans produced 2-cyanoethylene oxide at a greater rate than lung or brain microsomes, suggesting that the liver is the major site of 2-cyanoethylene oxide formation in vivo (Roberts et al., 1989; Kedderis and Batra, 1991). Studies in subcellular hepatic fractions indicate that there is an active epoxide hydrolase pathway for 2-cyanoethylene oxide in humans, which is inactive, although inducible, in rodents (Kedderis and Batra, 1993). Studies with inhibitory antibodies in human hepatic microsomes indicate that the 2E1 isoform of cytochrome P-450 is primarily involved in acrylonitrile epoxidation (Guengerich et al., 1991; Kedderis et al., 1993).
A physiologically based pharmacokinetic model has been developed and verified for the rat (Gargas et al., 1995; Kedderis et al., 1996), and work is under way to scale it to humans. In a recent, although incompletely reported, study, Kedderis (1997) estimated in vivo activity of epoxide hydrolase in humans based on the ratio of epoxide hydrolase to P-450 activity in subcellular hepatic fractions multiplied by the P-450 activity in vivo. Human blood to air coefficients for acrylonitrile and 2-cyanoethylene oxide have also been recently determined, although incompletely reported at present (Kedderis and Held, 1998). Research is in progress to determine partition coefficients for other human tissues.
2.4.3.8.2 Mode of action
Data on the genotoxicity of acrylonitrile are addressed in Section 2.4.3.5.
There are some suggestions from in vitro studies reported as abstracts that free radicals (·OH, H2O2, O2·) may be directly implicated in the oxidation of acrylonitrile and DNA damage. Formation of free radicals may be partially related to the release of cyanide or other mechanisms responsible for cellular and DNA damage (Ahmed and Nouraldeen, 1996; Ahmed et al., 1996; El-zahaby et al., 1996; Mohamadin et al., 1996).
In more recent investigations, the results of which have been presented incompletely at this time, Prow et al. (1997) reported that acrylonitrile inhibited gap junctional intercellular communication in a rat astrocyte cell line in a dose-dependent manner, possibly through an oxidative stress mechanism. Similarly, Zhang et al. (1998) assayed acrylonitrile with Syrian hamster embryo cells, with and without an antioxidant, and concluded that oxidative stress contributed to morphological transformation in the cells. Jiang et al. (1998) assayed acrylonitrile with a rat astrocyte cell line and reported oxidative damage (indicated by the presence of 8-hydroxy-2'-deoxyguanosine) at all concentrations tested.
Jiang et al. (1997) exposed male Sprague-Dawley rats to 0 or 100 ppm acrylonitrile in drinking water for two weeks. Endpoints examined were levels of glutathione and reactive oxygen species in brain and liver, presence of 8-hydroxy-2'-deoxyguanosine (indicative of oxidative DNA damage) in several tissues and determination of activation of NF-KB (a transcription factor strongly associated with oxidative stress). Glutathione in brain was decreased. (Whysner et al. [1998a] reported no effects upon concentrations of glutathione in the brain of male Sprague-Dawley rats exposed to 3, 30 or 300 ppm acrylonitrile in drinking water for three weeks.) In addition, reactive oxygen species were increased four-fold, levels of 8-hydroxy-2'-deoxyguanosine were increased three-fold and activation of NF-KB was observed in the brain.
In recently published studies, levels of 8-oxodeoxyguanosine, cytochrome oxidase, glutathione and cyst(e)ine in the brain of rats exposed to acrylonitrile in drinking water in each of the three following protocols have been examined (Whysner et al., 1997, 1998a):
- In male Sprague-Dawley rats exposed for 21 days to 0, 3, 30 or 300 ppm (0, 0.42, 4.2 and 42 mg/kg-bw per day; Health Canada, 1994), there was a significant increase in 8-oxodeoxyguanosine in brain nuclear DNA at the two highest doses. Assays of brain for glutathione, cytochrome oxidase, catalase and glutathione peroxidase did not show differences between exposed and control groups. There was a higher concentration of cyst(e)ine at the highest dose. In the liver, nuclear DNA 8-oxodeoxyguanosine concentrations were significantly increased at the two highest doses. Although there was no significant change in hepatic glutathione or cyst(e)ine, there was a significant trend for increased hepatic cyst(e)ine. In the forestomach, glutathione and cyst(e)ine were significantly increased at the highest dose. In a bioassay with comparable dose levels, the incidence of brain and/or spinal cord tumours was significantly increased in male Sprague-Dawley rats exposed to 35 ppm (3.4 mg/kg-bw per day) acrylonitrile and higher for two years (Quast et al., 1980a).
- In male F344 rats exposed for 21 days to 0, 1, 3, 10, 30 or 100 ppm (0, 0.14, 0.42, 1.4, 4.2 or 14 mg/kg-bw per day; Health Canada, 1994), analyses were limited to the brain. There were no significant differences between groups for 8-oxodeoxyguanosine, cytochrome oxidase, glutathione or cyst(e)ine.
- In male Sprague-Dawley rats exposed for up to 94 days to 0 or 100 ppm (0 or 14 mg/kg-bw per day; Health Canada, 1994), concentrations of 8-oxodeoxyguanosine in the brain were significantly increased after three, 10 and 94 days of exposure. There were no effects upon glutathione or cytochrome oxidase. In liver, the concentration of 8-oxodeoxyguanosine was significantly increased at 10 days only. In the two-year drinking water bioassay with male Sprague-Dawley rats (Quast et al., 1980a), the incidence of brain and/or spinal cord tumours was significantly increased at 100 ppm (8.5 mg/kg-bw per day).
The endpoint for which changes were consistently observed in male Sprague-Dawley rats was the induction of oxidative DNA damage, including the accumulation of 8-oxodeoxy-guanosine in the brain. The authors drew correlations between these results and the incidence of brain/spinal cord tumours that had been reported in carcinogenicity bioassays in which male Sprague-Dawley rats were exposed to acrylonitrile via drinking water.
Increased levels of 8-oxodeoxyguanosine occur only in the anterior portion of the brain, which contains rapidly dividing glial cells (Whysner et al., 1998b).
2.4.4 Humans
In case reports of acute intoxication, effects on the central nervous system characteristic of cyanide poisoning and effects on the liver, manifested as increased enzyme levels in the blood, have been observed. There have also been reports that acrylonitrile is a skin irritant and sensitizer, the latter based on patch testing of workers.
In the few studies in which non-neoplastic effects of acrylonitrile have been investigated, only acute irritation has been reported consistently. In a cross-sectional investigation of workers exposed in acrylic fibre factories to approximately 1 ppm (2.2 mg/m3), there was no consistent evidence of adverse effects based on examination of a wide range of clinical parameters, including liver function tests (Muto et al., 1992). However, there was an increase in subjective symptoms of acute irritation, consistent with observations in another cohort of acrylic fibre manufacturing workers (Kaneko and Omae, 1992).
In a cross-sectional investigation of a smaller group of workers producing acrylic textile fibres for which quantitative data on exposure were not reported, there was no evidence of induction of hepatic cytochrome P-450 or genotoxicity of urine (Borba et al., 1996).
Although there was some evidence in primarily early limited studies of excesses of lung cancer (Thiess et al., 1980), "all tumours" (Zhou and Wang, 1991) and colorectal cancer (Mastrangelo et al., 1993), such excesses have not been confirmed in well-conducted and well-reported recent investigations in four relatively large cohorts of workers (Benn and Osborne, 1998; Blair et al., 1998; Swaen et al., 1998; Wood et al., 1998). Indeed, there is no consistent, convincing evidence of an association between exposure to acrylonitrile and cancer of a particular site that fulfils, even in part, traditional criteria for causality in epidemiological studies.
The largest of the recent cohort studies was that conducted by Blair et al. (1998), which included 25 460 workers from eight plants producing and using acrylonitrile. Although an excess of lung cancer was observed in the highest quintile of cumulative exposure, analysis of exposure-response did not provide strong or consistent evidence of a causal relationship. The exposure categories were:
0.01-0.13 ppm-years: 121 430 person-years
0.14-0.57 ppm-years: 69 122 person-years
0.58-1.50 ppm-years: 9 800 person-years
1.51-8.00 ppm-years: 63 483 person-years
>8.00 ppm-years: 44 807 person-years
It should be noted that the power to detect moderate excesses was small for some sites (stomach, brain, breast, prostate, lymphatic/hematopoietic) because of small numbers of deaths.
1Values presented here are the means of 23 (males) and 20 (females) intakes presented by Bio/Dynamics Inc. (1980a).
2"The brain tumours were remarkably similar from animal to animal, regardless of their size or anatomical location within the brain. They were also similar to, and probably indistinguishable from, a subset of spontaneously occurring rat-brain tumours that have been generally classified as astrocytomas or anaplastic astrocytomas by light-microscopic evaluation of H&E-stained slides. Despite this superficial similarity to astrocytomas, we have found no hard evidence on which to identify any of the neoplastic cells as astrocytic in lineage or relatedness" (Bigner et al., 1986).
3Yates et al. (1994) also reported single and double strand breaks in plasmid DNA incubated with 2-cyanoethylene oxide.
4Additional information was provided by the authors.
5Including results of short-term toxicity studies in which the oxidative pathway has been induced prior to administration with acrylonitrile or antioxidants have been administered concomitantly with acrylonitrile.
