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Toxicology of methacrylonitrile
Toxico[ogy, 65 (1991) 239-250 Elsevier Scientific Publishers Ireland
Ltd.
Review Paper
Toxicology Mohammed
of methacrylonitrile
Y.H. Farooqui*
and Mohammad
M. Mumtaza
Division qf Environmental To.yicology, Deportment qf Biological Sciences. The Unirersi/y of Texas Pun American, Edinburg. TX 78539 and ‘Office <$ R erearch : and Droelopmmt. Environmental Critrrio and Assessment Office. United States Environmental Protection Agenqv, Cincinnati. OH 4526X (U.S. A.) (Received
June 26th.
1990: accepted
October
17th. 1990)
Summary The chemistry, industrial reviewed. Methacrylonitrile,
usage. general toxicity and experimental a reactive, unsaturated and methylated
use of methacrylonitrile are briefly aliphatic nitrile. has industrial ap-
plications in a variety of organic processes related to the polymer industry. Its general toxic effects are primarily related to the release of cyanide and formation of reactive metabolites containing a double bond between carbon 2 and 3. Methacrylonitrile has been given by a variety of routes to mammalian species to study its toxic effects. More recent in vivo and in vitro experimental work concerning its toxicity and metabolism are summarized and possible mechanisms of this chemical’s toxic action are discussed. Keja words: Methacrylonitrile; adverse effect level
Toxicity;
Metabolism;
No observed
adverse
effect level: Lowest observed
Introduction
Aliphatic nitriles have a wide variety of uses in organic synthesis and in the chemical and pharmaceutical industries. They are also natural constituents of a number of foodstuffs. Hence, there is a large human population at risk, either through direct occupational exposure or ambient environmental exposure, or from contact with products containing nitriles. Toxic effects of aliphatic nitriles have been attributed to the in vivo liberation of cyanide since the early work of Lang [l].
*Address ull correspondence and reprint requests to: Dr. Mohammed Farooqui. Associate Professor, Division of Environmental Toxicology, Department of Biological Sciences, The University of Texas Pan American, Edinburg, TX 78539, U.S.A. The views of this paper are those of the authors and do not necessarily reflect the views or policies of the Unites States Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
0300-483x/91/$03.50 0 1991 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
239
Methacrylonitrile (MeAN) is an industrial nitrile that has a history of widespread use. Products containing MeAN and other related nitriles are shown to produce various toxic effects in laboratory animals, and some cases of potential human exposure to MeAN have been reported. Reviews on comparative toxicity and metabolism of nitriles other than MeAN exist in the literature [2,3] but the information on MeAN, has not been compiled thus far into a review. The intent of this paper is to summarize the chemistry, industrial usage and general toxicity of MeAN, review the previous and more recent experimental use of MeAN and discuss possible toxic mechanisms as defined by recent experimental work. Chemistry MeAN is a colorless liquid having the physical and chemical properties listed in Table I. It is a highly reactive unsaturated alkyl nitrile that readily polymerizes in the absence of a stabilizer. The commercial product contains 50 ppm hydroquinone monoethyl ether as a stabilizer. Because of its double bond, additional reactions are possible with biological molecules. Production and industrial use MeAN is produced TABLE
commercially
by the vapor-phase
reaction
of isobutylene
with
I
CHEMICAL
AND
PHYSICAL
PROPERTIES
OF METHACRYLONITRILE”
CAS number Synonyms
126-98-7 2-methyl-2-propenenitriie. methyl acrylonitrile. -methacrylonitrile, isopropene cyanide. isopropenyl nitrile. 2-cyano-I-propene. 2-cyanopropene, MAN. MeAN
Molecular
C,H,” H?C=C (CH,)-CN 67.09 Colorless liquidb -35.8”C
formula
Structural formula Molecular weight Physical state: Melting point Boiling point Flash point Vapor pressure
90.3”C 55°F (12.7”2(3)’ 40 torr @ 13°C. 65 torr @ 25°C and 100 torr @ 3 3”Cb
Specific gravity Refractive index Solubility in water Solubility in organic
0.800 at 20”Ch “20 = 1.4007 D 2.57 g/l00 g water @ 20°C Miscible with acetone octane 0.68d
Log octanoliwater coefficient “Adapted bAdapted ‘Adapted dAdapted
240
solvents partition
from [8] unless indicated from [ 511. from 191. from [ 27 1.
otherwise.
and toluene
at 2tk25”C
ammonia and oxygen in the presence of a catalyst. Acetonitrile, hydrogen cyanide and acrolein are known by-products of this reaction; they are removed by scrubbing, distillation and fractionation H’ith a yield of approximately 70% specification-grade MeAN [4,5]. The domestic production of MeAN has been reported to be between 1.Ol and 10.1 million pounds in 1977 [6]. The U.S. International Trade Commission (USITC) [7] did not report domestic production of MeAN for the years 1981 through 1987, because the Commission reports production of only those chemicals that are manufactured by at least three producers and MeAN is produced by only two producers. Therefore, the actual production data for MeAN is not accurately available. The producers with U.S. patents are Shell, Allied and Goodrich [8]. The other known United States producers are Kodak, Araphose Chemicals and Fike Chemicals. MeAN is used in the preparation of homo- and copolymers, elastomers, coatings and plastics [8,9] and as a chemical intermediate in the preparation of acids, amides, amines, esters and other nitriles [8]. It is also used as a replacement for acrylonitrile in an acrylonitrile-butadiene-styrene-like polymer, since MeAN improves barrier properties to gases like carbon dioxide in carbonated containers [4]. Occupational
and public exposure
According to a NIOSH report, 28 workers were potentially exposed to MeAN in the workplace in 1980 [lo]. The actual frequency, level or duration of MeAN exposure to workers is not available. A time-weighted average (TWA) threshold limit value of 1 ppm (3 mg/m3) for MeAN exposure was adopted by the American Conference of Governmental Industrial Hygienists [ 1l-121 and is also reported in other sources [ 13-161. This TWA recommendation includes a “skin” notation, indicating a potential for absorption of this compound dermally (including mucous membranes and eyes, either by airborne or direct contact). An atmospheric odor threshold that workers can detect is reported to be 7 ppm [ 171. MeAN has been identified as a component of the mainstream smoke of unfiltered cigarettes made from air-cured, flue-cured or a blend of these tobaccos; the concentration of MeAN in the smoke was 3 &cigarette [18]. In the United States, under the Resource Conservation and Recovery Act, MeAN has been designated as a hazardous waste (Waste No. U152) in Appendix VIII, a listing of chemicals that have been shown to have toxic, carcinogenic, mutagenic or teratogenic effects on humans or other life forms [ 19). U.S. Food and Drug Administration has established a limit of no more than 41% of MeAN-derived polymer in the MeAN-grafted butadiene copolymers for use in the preparation of resinous and polymeric coating materials [20]. Similarly, the limits of 0.5 mg MeAN per square inch of food-contact surface and 50 ppm for water containers have been established in the chloroform-soluble coating components [20]. Human toxicity Only one report is available in the literature about the toxicity of MeAN to humans in which groups of eight to nine subjects were exposed by inhalation twice to a series
241
(24, 14, 0, 7, 14, 24, 7, 2, 0 and 2 ppm in that order) of MeAN concentrations for periods of I min each, with 45-min or longer intervals between exposures [21]. Among subjects exposed to 24 ppm, only 6-22% experienced nose. throat, or eye irritation; no irritation was noted at the other concentrations. A majority of subjects (88-89%) could detect odor at 14 or 24 ppm, but only 47% could detect odor at 7 ppm. In other studies, exposure of 9 subjects to 2 ppm vapor for 10 min or 7 subjects to 14 ppm for 10 min caused nose, throat and eye irritation [21]. No data are available on the epidemiological evidence, case reports. chemical disposition and biochemical. carcinogenic, mutagenic or teratogenic effects of MeAN in humans. Toxicity
and biochemical
effects in experimental
animals
Signs of toxicit) MeAN is shown to be highly toxic in mice, rats and rabbits by dermal. respiratory and oral routes [21-241. Acute toxicity data on MeAN in laboratory animals is summarized in Table II. Pozzani et al. [21] have reported acute inhalation studies in A/J mice, Harlan-Wistar rats, albino guinea pigs and rabbits. The animals were exposed to unspecified concentrations of MeAN vapor for 4-h periods. In all of the species tested, death was preceded by loss of consciousness and tonic-clonic convulsions. At necropsy, no gross treatment-related changes were observed in animals that died or in those surviving a 14-day observation period. In albino rabbits intravenous injection of 6.25 and 12.5 mgikg MeAN produced significant but not dose-related amounts of cyanide into blood which began to fall within 4 h of injection [21]. Inhalation exposure (7 h/day. 5 days/week over a YO-day period) of male beagle dogs to 0, 3.2, 8.8 or 13.5 ppm of MeAN produced dose-related cyanide only at the latter two doses which disappeared with a maximum half-life of 3 days [21]. In subchronic studies, female Harlan-Wistar rats exposed to (& 109.3 ppm of MeAN
TABLE
II
ACUTE
TOXICITY
OF MeAN
IN LAHORAT-ORY
ANIMALS
Species
Route
Toxic do\c
Rat Rat
Oral Inhalation
LII,,,. 700 mg/kg” h LC,,,: 32x 700 ppr1P
Mouse
Oral Inhalation Inhalation Skm
LD<,,: Lc‘,,,: LC,,,: LC,,,:
Oral
LD,,,: 4 mg/kg”
Mouse Guinea Rabbit
pig
Gerbil
from [ 2 I ] from [ 521. ‘Range of doses depending on body weight. ‘Adapted from [ 27). ‘Determmed in our laboratory according to Weil 1531.
“Adapted
bAdapted
242
17 mg/kg” 36 ppm’l XX ppm,’ 168 mg/kg.‘.h
vapors, 7 h/day, 5 days/week for 91 days, showed loss of consciousness and increase in relative liver weight at higher doses, although no microscopic lesions were observed [21]. In a similar study over the same time period, male beagle dogs exposed to t&-13.5 ppm MeAN vapors developed central nervous system effects at the highest dose, manifested by tonic convulsions and loss of control over the hind limbs that were accompanied histopathologically with microscopic brain lesions [21]. In white mice, white rats and rabbits, toxic actions of MeAN were attributed to inactivation of cytochrome oxidase leading to disturbances of tissue respiration [23,24]. The principal clinical manifestation of the toxic effects of lethal and threshold MeAN concentration was an injury to the central nervous system [23,24]. In Wistar rats, exposure to MeAN (3180 - 5700 ppm) for 30 min. the signs of toxicity were related to the in vivo liberation of cyanide [25]. In contrast, the signs of toxicity observed in the same species after acrylonitrile exposure, where metabolic cyanide plays a minor role, were not cyanide related. The acute toxicity of MeAN was antagonized with cyanide antidotes, 4-dimethyaminophenol plus sodium thiosulfate and N-acetyl-cysteine which reacted directly with unsaturated nitriles. Observations on signs of toxicity of MeAN in male Sprague-Dawley rats have recently been reported [26] (Table III). Within 1 h following the oral administration of MeAN, one or more of the male Sprague-Dawley rats in each treatment group developed ataxia, trembling, convulsions. mild diarrhea and irregular breathing. Unlike acrylonitrile (AN), cholinomimetic signs were negligible or absent in MeAN-treated rats. Another important sign of MeAN toxicity, which has not been reported earlier, was the retention of urine in the bladder. Up to 58% of the MeAN (I LDo,, 100 mg/kg) treated animals had their bladder partially or fully distended at the time of sacrifice [26]. Based upon these studies the NOAEL (no observed adverse effect level) and the LOAEL (lowest observed adverse effect level) for MeAN are 50 mg/kg (0.25 LD,,) and 100 mg/kg (0.5 LD,,). respectively.
TABLE SIGNS
111 OF TOXICITY
FOLLOWING
Signs of toxicity”.b.’
I-D,,,
Ataxia Trembling Convulsions Irregular breathing Diarrhea Urine retentior8 “Adapted from bSigns observed
[26]. for 6 hours
ORAL
OF MeAN
(mg/k&)
0.25
0.5
(50)
(100)
I.0 (200)
2.0 (300)
I I I
2 3 3
3 4
2 0 II
3
0 0 0 0 0 0
following
ADMINISTRATION
1 58
4 4
I NT
oral administration.
‘Severity of Signs graded on a scale of I < 2 < 3 < 4 [ 541. ‘Shown as percent of total number of rats with urine retention. NT = not tested.
243
Cyanide liberation Increased blood and urine concentrations of thiocyanate have been reported after MeAN administration [27,28]. Tanii and Hashimoto [27] have reported that pretreatment of male ddY mice with carbon tetrachloride, a known modulator of hepatic mixed function oxygenase system, resulted in much lower concentrations of cyanide than controls and greatly reduced the toxicity of MeAN. Cavazos et al. [28], using radioactive MeAN, found that, after 5 days of dosing rats orally with 100 mgkg, the total urinary excretion of thiocyanate was about 12X, whereas the total urinary excretion of 14C was about 43X, indicating presence of MeAN metabolites other than thiocyanate. In the livers of MeAN ingested rats, thiocyanate levels remained up to 35 nmol/g for initial 6 h and gradually returned to normal. A similar trend was observed in kidney, heart, lung and brain. In plasma, the thiocyanate concentration increased significantly from 26.3 pmol/l at 1 h to 8 1.2 pmolil at 6 h of dosing [29]. Glutathione depletion Soluble thiol contents, including glutathione (GSH), are depleted after MeAN administration. However, MeAN is a less potent depleter of GSH than AN. Day et al. [30] have shown that following the administration of 100 mg/kg MeAN, the maximum depletion was noticed in the liver (39% of control) and in kidney, lung, heart, brain and spleen, ranging between 26 and 34% of control. In vitro studies by Day et al. [30] have shown that addition of MeAN (O--40 mM) to a solution of 0.3 mM GSH at pH 7.4, resulted in a time as well as MeAN concentration dependent depletion of GSH. Thin layer chromatography analysis of incubation mixtures of MeAN with GSH and cysteine showed the appearance of distinct spots representing the adducts S-(2-cyanopropyl) GSH and S-(2-cyanopropyl) cysteinc [30]. Pharmacokinetics
and metabolism
Chemical disposition and covalent binding of MeAN Wolff [3 1,321 has reported pharmacokinetics of AN and two of its analogs MeAN and crotononitrile (CRN) in rats given 5-60 mg/kg intraperitoneally. In this study, MeAN and CRN concentrations followed a first order kinetic elimination and the AN demonstrated a biphasic kinetic elimination. Cavazos et al. [29] have reported that in male Sprague-Dawley rats, following an oral administration of 100 mg/kg MeAN (8 PCilkg [2,3- 14C]MeAN), the primary route of excretion was the urine (43% of dose). Substantial amounts of radioactivity were also found in the feces (15%) and the expired air (2.5”/0). The total 14C-activity excreted in 5 days was 59% of the dose administered. This indicates that more than 40% of MeAN was retained in the body either bound to macromolecules or in the form of unexcretable conjugates. The red blood cells retained significant amounts of radioactivity for more than 5 days after administration, whereas the i4C-activity in plasma declined sharply. More than 50”% of the radioactivity in erythrocytes was detected as covalently bound to cytoplasmic (hemoglobin) and membrane proteins. A small amount of radioactivity was also found associated with the heme fraction. This study indicated that MeAN may damage red blood cells by mechanisms other than release of cyanide.
244
Tissue distribution studies using radioactive MeAN have shown that it is absorbed extensively through the gastrointestinal tract and distributed in all the tissues of the rats [26]. The gastrointestinal tract, however, contained the highest levels up to 48 h, suggesting hepato-intestinal recirculation and resecretion of MeAN, cyanide or other metabolite(s) by the gastrointestinal mucosa. Despite extensive incorporation of MeAN-derived radioactivity in tissue macromolecules, the blood concentration of 14C-activity was significantly greater than tissue concentrations even after 96 h of dosing. Substantial accumulation of radioactivity in the bone was another important observation in this study [26]. Retention of i4C-activity in the bone might lead to an interaction of MeAN with bone marrow thus affecting the rate of hemopoiesis. In vitro metabolism of MeAN to cyanide Tanii and Hashimoto [27] have reported the kinetic constants for the in vitro liberation of cyanide from MeAN in ddY mouse hepatic microsomal enzyme system. The K,,, value for MeAN was 0.133 mM, which ranked 5th from the lowest value reported for 13 aliphatic nitriles, and the V,,, for MeAN was 417 ng cyanide formed/15 min per mg protein [27]. In liver fractions from male Sprague-Dawley rats, the metabolism of MeAN to cyanide was localized in the microsomal fraction and required NADPH and oxygen for maximal activity [33]. Metabolism of MeAN was increased in microsomes obtained from phenobarbital-treated rats and decreased after cobaltous chloride and SKF 525 A treatments. Addition of the epoxide hydratase inhibitor I,l,l-trichloropropane 2,3-epoxide, decreased the formation of cyanide from MeAN. Addition of GSH, cysteine, D-penicihamine or 2-mercaptoethanol enhanced the release of cyanide from MeAN. However, when the cytosolic fraction was added to the incubation medium along with microsomes the formation of cyanide was significantly reduced [34], indicating a role of GSH S-transferase provided by the cytosol. These findings indicated that MeAN is metabolized to cyanide by a cytochrome P-450-dependent mixed-function oxidase system. Nitriles differ remarkably in their capacity to release cyanide both in vivo and in vitro [34,35]. Among AN and two of its analogs CRN and MeAN, when incubated with rat liver microsomes, the highest amount of cyanide was released from MeAN (57.9 nmol cyanideimg protein per min) followed distantly by CRN and AN (8 and < 1 nmolimg protein per min, respectively) [33,34,36] and was influenced by the presence of GSH in the incubation medium [34]. Such differences in their metabolism appear to be involved in the toxicity of nitriles. For example, the signs of toxicity in the case of AN are far less cyanide-related than both of its analogs MeAN and CRN where treated animals show distinct cyanide-related nervous effects including depression, convulsions and respiratory failure. In contrast, the early signs of AN toxicity are distinctively cholinomimetic including salivation, lacrimation, vasodilation and diarrhea with central nervous system signs developing very late. Silver et al. [35] have suggested that the length of carbon chain, presence of substituents at the carbon, position of double bonds and, for some compounds, route of administration, are important factors influencing the release of cyanide from nitriles. Recently, the vehicles used to administer nitriles, have been shown to influence the absorption and toxicity of nitriles [37].
245
Species dl]Jerence in toxicity and metabolism Table II shows that there is a clear species difference in the toxicity of MeAN in terms of LD,, values, gerbils being the most susceptible species followed by mice and rats. Studies in our laboratory have shown that there is a distinct species difference in the metabolism of MeAN to cyanide among gerbils, mice and rats, both in terms of amount of cyanide formed and the time of peak levels in blood. The data in Fig. 1 shows that following a single oral dose of 0.5 LD,,, MeANikg, the blood cyanide concentrations in rats were maximal at 3 h after treatment. However, in gerbils and mice the blood cyanide concentrations were maximal at 1 h. In all species studied, cyanide levels gradually declined to negligible levels by 24 h. In addition, the gerbils metabolized greater amounts of MeAN to cyanide than either the mice or rats. This explains the extremely low LD,, of MeAN for mice and gerbils. Species difference has also been reported for other nitriles including AN [38] and dimethylaminopropionitrile [39]. Variations in biotransformation between species may be either qualitative or quantitative differences. The qualitative differences may involve metabolic routes and are due either to a species defect or to a reaction peculiar to a species [40]. The quantitative variations may result from differences in enzymic levels or natural inhibitors in the balance of reverse enzyme reactions. Most. but not all, nitriles are metabolized via a mixed-function oxidase system. Some variations may be due to extrahepatic metabolism and the biological half-life of metabolites formed. The gerbils are among unique animals that use amino acids [40], such as taurine, for conjugation reactions rather than the usual hepatochelating agent GSH. Gastrointestinal flora may also play a role in species differences since not all animals are germ free. There may be numerous other factors involved in species difference in the toxicity of nitriles and it needs to be explored as to what specific factors are involved in the case of MeAN.
Fig. 1.Time course of blood cyanide concentrations in rats (circles). gerbils (diamonds) and mxe (triangles) rats. after single oral dose of MeAN. MeAN was administered orally at 0.5 LD,,, (male Sprague-Dawley 100 mg/kg; Albino-Swiss mice, I7 mgikg; Mongolian gerbils. 4 mg/kg). Each value is the mean f S.11. of 3 animals. Cyanide levels were determined by microdiffusion technique 1331.
246
Metabolic
pathways for MeA N
Because of its similarity to acrylonitrile, it is likely that MeAN is metabolized by two pathways as proposed in Fig. 2. One major pathway of MeAN metabolism is the direct conjugation with GSH, which is catalyzed by GSH S-transferases. The product S-(2-cyanopropyl) GSH [30] may be further metabolized to N-acetyl-S(2-cyanopropyl) cysteine. Another major pathway involves an epoxide intermediate and is catalyzed by the hepatic microsomal P-450 system [33]. Further metabolism of epoxide by GSH epoxide transferase may result in another GSH conjugate depending on the site of nucleophilic attack on the epoxide molecule. Alternatively, the epoxide intermediate could produce cyanide and other metabolites by rearrangement or by catalysis with epoxide hydratase. Further metabolism of GSH conjugates may lead to the excretion of various mercapturic acids. Mutagenicity
and reproductive toxicity
In two independent studies, MeAN was non-mutagenic in the Salmonellaimicrosomal assay in strains TA97, TA98, TAlOO, TA1535 and TA1537 with or without either hamster or rat liver activation [41]. In Drosophila assay, MeAN fed to larvae at 5950 or 6077 ppm did not induce sex-linked recessive lethal mutations [41]. In pregnant Sprague-Dawley rats, oral administration of 50 mgikg per day MeAN (0.25 LD,,) during first or second week of gestation and 100 mgikg per day (0.5 LD,,) only during second week of gestation, resulted in a dose-dependent significant reduction in maternal body weight gain [42]. In this study, control rats delivered nor-
QH QH CHzCCN _ epoxide hydratax micrcaomal oxidation
/
oxidation pdUCfS
CH3
/ -0. ~~~~“g.EllI~tlt~
SCN-
OXIDATTVE PATHWAY macmmokcules GSCH2-F(OH)CN
CV, = C-CN
___,
GSCH2-q=O-
CH3 mcrcapturic acid
cyanoisop&pyl
mcrcapturic acid
,
l ,,,,g,
~~:~~~?c
-
Fig. 2. A
proposed
metabolic
pathway
1. A. Nucleic acids (NH OH) 9. Pmteins (NH2, OH, SH) C. Lipids (OH, NH, ha) = 2. Bzological Neumtransmwxs: A. adrenahn and its analogs 9. semtonm C. amincbutyric acid D. histammc 3. Other nucleophilic components of tissues
for MeAN.
247
TABLE ORGAN
IV TOXICITY
OF ALIPHATIC
NITRILES
Nitrile
Organ
toxicity
Acrylonitrile Propionitrile Malononitrile Methacrylonitrile Crotononitrile Acetonitrile
Adrenal apoplexy [ 391 Duodenal ulcers [ 40) Nuclear changes in neurons and satellite ganglia Skin poison [7], urine retention [26] Highly corrosive action [IS] Thyroid hyperemia and hyperplasia (421
Dimethyl amino-propionitrile Allylnitrile
Neurological bladder Long-term dyskinesia
dysfunction [46]
141 I
[43--15]
ma1 size litter and MeAN-treated rats failed to maintain pregnancy and aborted; these abortions may be cyanide-related [43]. At the end of gestation, the rats fed with MeAN developed dose-dependent mild to severe edema in the fallopian tubes [42]. These findings suggest that MeAN has a potential for reproductive toxicity. Based upon these studies, the LOAEL for the reproductive effects of MeAN appears to be 50 mg/kg (0.25 LD,,). Conclusions In conclusion, we have attempted to summarize the basic chemical and toxicologic information available on MeAN. MeAN belongs to the family of aliphatic nitriles, each of which, despite structural similarities, exhibit entirely different biochemical effects. Although most of the aliphatic nitriles are potent neurotoxins, their toxic actions on other organ systems differ remarkably (Table IV) [ 17,39,44-511. Aliphatic nitrile biotransformation (Fig. 2) requires activation of the molecule before release of the cyanide group. The instability of the cyanohydrin formed suggests that a nascent form of aldehyde may be formed; it may escape the detoxication mechanisms of the cells and, perhaps, have a role in the mechanism of toxicity of these compounds. Despite the amount of information available on MeAN, further extensive work on various aspects of MeAN metabolism, toxicology and pharmacology is indispensable to understand the mechanism underlying its toxicity. Acknowledgement This work was supported by Grant No. SORR08038 from the National of Health Minority Biomedical Research Support Program.
Institutes
References 1 2
248
S. Lang, Uber die Umwandlung des Acetonitrils und seine, Homologen im Tierkorper. Arch. Exp. Pathol. Pharmakol., 34 (1894) 247. A.E. Ahmed and N.M. Trieff, Aliphatic nitriles; metabolism and toxicity. in J.W.Bridge and L.F. Chasseud (Eds.). Progress in Drug Metabolism, 7 (1983) 229.
A.E. Ahmed, M.Y.H. Foreign Compounds.
Farooqui Academic
and N.M. Trieff, Nitriles. in M.W. Anders Press. New York, 1985 pp. 485-518.
(Ed.). Bioactivation
of
D.M. Cosidine (Ed.), Chemical and Process Technology Encyclopedia. McGraw-Hill Book Company. New York, 1974, pp. 30-34. J.W. Nemec and L.S. Kirch, Methacrylic acid and derivatives. in Kirk-Othmer Encyclopedia of Chemical Technology. 3rd edn.. Wiley. New York. 15. 1981. pp. 346. 363, 376. USEPA, U.S. Environmental Protection Agency. Computer printout (CICIS): Production Statistics for Chemicals in the Nonconfidential Initial TSCA Chemical Substance Inventory, Washington. DC. Office of Toxic Substances and Pesticides. 1987. USITC, U.S. International Trade Commission. Synthetic Organic Chemicals. United States Produc-
8 9 10
II I2
I3
tion and Sales. Publication Nos. 1292. 1422. 1401, 1588, 1548, 1745, 1892, and 2009. Washington. DC, Government Printing Office, 1982-87. M. Windholz (Ed.), The Merck Index. 10th edn., Merck and Company, Rahway. NJ, 1980. p. 850. N.I. Sax and R.J. Lewis Jr.. Hawley’s Condensed Chemical Dictionary, I Ith edn.. Van Nostrand Reinhold Company, New York, 1987, p. 751. NIOSH, National Occupational Exposure Survey (198&83) [database]. Cincinnati, OH: Department of health and Human Services. National Institute for Occupational Safety and Health, 1984. ACGIH, Threshold Limit Values and Biological Exposure Indices for 198687. Cincinnati, OH: American Conference of Governmental Industrial Hygienists. 1986. p, 23. ACGIH, Documentation of the Threshold Limit Values and Biological Exposure Indices, 5th edn. Cincinnati, OH: American Conference of Governmental Industrial Hygienists, 1986. p. 370. Registry of Toxic Effects of Chemical Substances (RTECS), (RTECS UD14CJOOOO).Vol. 4, 1985-86, p. 3856.
I4 15 I6
Patty’s Industrial Hygiene and Toxicology, 3rd rev. edn.. Vol. 2C. 1982, pp. 48634888. The Sigma-Aldrich Library of Chemical Safety Data. edn. I. 1985, p. 1171. N.1. Sax and R.J. Lewis. Dangerous Properties of Industrial Materials, 7th edn., Vol. III. 1989. p. 2251.
17
J.E. Amoore and E. Hautala. Odor as an aid to chemical safety: odor thresholds compared with threshold limit values and volatilities for 214 industrial chemicals in air and water dilution. J. Appl. Toxicol.. 6 (1983) 272. R.R. Baker. H.F. Dymond and P.K. Shillabeer. Determination of a&unsaturated compounds formed
I8 I9 20
21 22 23 24 25 26 27 28 29
by a burning cigarette. Anal. Proc., 21 (1984) 135. USEPA, U.S. Environmental Protection Agency. 40 CFR 261. Identification and listing of hazardous waste. Section 261.33 and Appendix VIII, 1986. pp. 359, 384, 398. USFDA, Food and Drug Administration, 21 CFR Subpart C-Substances for Use as Components of Coatings, 175.300. Resinous and Polymeric coatings. Washington. DC., Office of the Federal Register. 1986. pp. 141. 143. 152, 153. lJ.C. Pozzani. E.R. Kinkead and J.M. King, The mammalian toxicity of methacrylonitrile. Am. Ind. Hyg. Assoc. J.. 29 (1968) 202. W.A. McOmie. Comparative 31 (1949) 113. S.A. Kurzaliev. [Characteristics 5 (1985) 37.
toxicity
of methacrylonitrile
and acrylonitrile.
of the toxic action of methacrylonitrile].
J. Ind. Hyg. Toxicol.,
Gig. Tr. Prof. Zabol. (USSR),
S.A. Kurzaliev and A.N. Zuev, [Toxic effect of methacrylonitrile on the body of experimental animals after chronic exposure]. Gig. Tr. Prof. Zabol. (USSR), 4 (1988) 51. H. Peter and H.M. Bolt. Effect of antidotes of the acute toxicity of methacrylonitrile. Int. Arch. Occup. Environ. Health, 55 (1985) 175. M.Y.H. Farooqui. R. Cavazos. M.I. Villarreal and E. Massa, Toxicity and tissue distribution of methacrylonitrile in rats. Ecotoxicol. Environ. Safety, (1990). in press. H. Tanii and K. Hashimoto. Studies on the mechanism of acute toxicity of nitriles in mice. Arch. Toxicol., 55 (1984) 47. R. Cavazos. M.Y.H. Farooqui, W.W. Day. M.I. Villarreal and E. Massa, Disposition of methacrylonitrile in rats and distribution in blood components. J. Appl. Toxicol.. 9 (1989) 53. R. Cavazos, M.Y.H. Farooqui and W.W. Day. Metabolism of methacrylonitrile to cyanide in rats. Toxicologist, 8 (1988) 210.
249
30 31 32 33 34 35 36 37
38 39
40
41 42 43 44
45 46 41 48
49 50 51 52 53 54
250
W.W. Day. R. Cavazos and M.Y.H. Farooqui. Interaction of methacrylonitrile with glutathionc. Res. Commun. Chem. Pathol. Pharmacol.. 62 (1988) 267. M.S. Wolff. Acrylonitrile Pharmacodynamics and Mutagenesis. Govt. Reports Announccmcnty and Index (GRA&I). NIOSH, Issue 23. 19X8. M.S. Wolff, Acrylonitrile pharmacodynamics and mutagenesis. NIOSH. Environmental Science Laboratory, Mount Sinai School of Medicine, NY. (1977). M.Y.H. Farooqui. R.G. Diaz and R. Cavazos. Metabolism of methacrylonitrile to cyanide ill ~rlro studies. J. Biochem. Toxicol., (1990). in press. M.Y.H. Farooqui and E. Massa, Effect of glutathione on in ~+/ro metabolism of unsaturated aliphatic nitriles. Bull. Environ. Contam. Toxicol.. 5 (1990) 109. E.H. Silver. S.H. Kuttab. T. Hasan and M. Hassan. Structural considerations in the metabolism 01 nitriles to cyanide in vivo. Drug Metab. Dispos., 10 (19X2) 495. M.E. Abreu and A.E. Ahmed. Metabolism of acrylonitrile to cyanide in vitro studies. Drug Mctab. Dispos., 8 (1980) 376. M.A. Wallig. D.H. Gould, J.V. Stcenhouse. M.J. Fettman and C.C. Willhitc. The relationship 01 vehicle to target organ toxicology induced by the naturally occurring nitrllc I-cyano-2-hydroxy-3.butcnc. Fundam. Appl. Toxicol.. I2 (19X9) 377. A.E. Ahmed and K. Pate]. Acrylonitrile: in vivo metabohsm in rats and mice. Drug Metab. Dispos.. 9 (1981) 219. M.M. Mumtaz, M.Y.H. Farooqui. B.I. Ghanayem and A.E. Ahmed. The urotoxlc mechanisms ol N.N-dimethylaminopropionitrile in rats and mice I. Biochemical and morphological characterlz+tion of the injury and its relationship to metabolism. J. Toxicol. Environ. Health. ( 1990). in press. I.G. Sipes and A.J. Gandolfi. Biotransformation of toxicants. in C.D. Klaahscn, M.O. Amdur and J. Doull (Eds.) Casaett and Doull’s Toxicology. The Basic Science of Poison\. 3rd cdn.. Macmillan. New York. 1986. pp. 64 98. E. Zeiger. B. Anderson, S. Haworth. T. Lawlor, K. Mortelmans and W. Speck. S&no&/tr mutagcmcity tests: 111. Results from testing of 255 chemicals. Environ. Mutagen.. 9 (19X7) I. M.I. Villarreal. R. Cavazos and M.Y.H. Farooqui, Reproductlvc toxicity of methacrylonitrilc in rats. Proc. NIH-MBRS Symp.. 16 (198X) XX. C.C. Willhite, V.H. Ferm and R.P. Smith, Teratogenic effect\ of aliphatic nitrilcs (1981) 317. S. Szabo. I. Hunter. K. Kovacs. E. Horvath. D. Szaho and H.C. Horncr. Pathogcncsis tal adrenal hemorrhagic necrosis: ultrastructural, hiochcmical, neuropharmacologic and tion studies with acrylonitrile in the rat. Lab. Invest.. 42 (IYXO) 533. S. Szaho and H. Seyle. Duodenal ulcers produced by propionitrilc. Arch. Pathol.. V.L. Van Breeman and J. Hirakoa. Ultrastructure of nerve and satellite of spinal ganglia
Teratology.
23
ofexperimenblood coagulaY3 (lY72) 390. of rats trcatcd
with malononitrile. Am. Zoo].. I (I961 ) 473. A.W. Spence and D. Marine. Production of thyroid hyperpla\ia in rats and mice by admlnistration of methyl cyanide. Proc. Sot. Exp. Biol. Med.. 29 (1932) 967. K. Kriess. D.H. Wegman, C.A. Niles. M.B. Siroky, R.J. Krame and R.G. Feldman. Neurological dysfunction of the bladders in workers exposed to dimethyl aminopropionitrilc. J. Am. Med. A\soc.. 243 (1980) 741. A.E. Ahmed and M Y.H. Farooqui, Metabolism and toxicity of N.N’-dimcthylaminopropionitrile in rats. Toxicologist. 26 (1984) 19X. H. Tanli. Y. Kurosaka. M. Hayashi, and K. Hashimoto, Ally]-nitrlle. a compound which Induces long-term dyskinesia in mice followmg a single administration. Exp. Neural.. 103 (1989) 64. K. Verschueren, Handbook of Environmental Data on Organic Chemicals. 2nd edn.. Van Nostrand Remhold Company, New York. 19X3. p. X15. H.F. Smyth Jr., C.P. Carpenter. C.S. Weil. U.C. Po/zani and J.A. Strlegel. Range-linding toxicity data: list VI. Am. lnd. Hyg. Asroc. J.. 23 (1962) Y5. C.S. Weil. Tables for convement calculation of median effective dose (LD,,) or ED,,,) and instructions in their use. Biometrics, X (1952) 249. A.E. Ahmed and M.Y.H. Farooqui, Comparative toxlclties of allphatic nitrilca. Toxicol. Lett. I? (1982) 157.
Ltd.
Review Paper
Toxicology Mohammed
of methacrylonitrile
Y.H. Farooqui*
and Mohammad
M. Mumtaza
Division qf Environmental To.yicology, Deportment qf Biological Sciences. The Unirersi/y of Texas Pun American, Edinburg. TX 78539 and ‘Office <$ R erearch : and Droelopmmt. Environmental Critrrio and Assessment Office. United States Environmental Protection Agenqv, Cincinnati. OH 4526X (U.S. A.) (Received
June 26th.
1990: accepted
October
17th. 1990)
Summary The chemistry, industrial reviewed. Methacrylonitrile,
usage. general toxicity and experimental a reactive, unsaturated and methylated
use of methacrylonitrile are briefly aliphatic nitrile. has industrial ap-
plications in a variety of organic processes related to the polymer industry. Its general toxic effects are primarily related to the release of cyanide and formation of reactive metabolites containing a double bond between carbon 2 and 3. Methacrylonitrile has been given by a variety of routes to mammalian species to study its toxic effects. More recent in vivo and in vitro experimental work concerning its toxicity and metabolism are summarized and possible mechanisms of this chemical’s toxic action are discussed. Keja words: Methacrylonitrile; adverse effect level
Toxicity;
Metabolism;
No observed
adverse
effect level: Lowest observed
Introduction
Aliphatic nitriles have a wide variety of uses in organic synthesis and in the chemical and pharmaceutical industries. They are also natural constituents of a number of foodstuffs. Hence, there is a large human population at risk, either through direct occupational exposure or ambient environmental exposure, or from contact with products containing nitriles. Toxic effects of aliphatic nitriles have been attributed to the in vivo liberation of cyanide since the early work of Lang [l].
*Address ull correspondence and reprint requests to: Dr. Mohammed Farooqui. Associate Professor, Division of Environmental Toxicology, Department of Biological Sciences, The University of Texas Pan American, Edinburg, TX 78539, U.S.A. The views of this paper are those of the authors and do not necessarily reflect the views or policies of the Unites States Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
0300-483x/91/$03.50 0 1991 Elsevier Scientific Publishers Printed and Published in Ireland
Ireland
Ltd.
239
Methacrylonitrile (MeAN) is an industrial nitrile that has a history of widespread use. Products containing MeAN and other related nitriles are shown to produce various toxic effects in laboratory animals, and some cases of potential human exposure to MeAN have been reported. Reviews on comparative toxicity and metabolism of nitriles other than MeAN exist in the literature [2,3] but the information on MeAN, has not been compiled thus far into a review. The intent of this paper is to summarize the chemistry, industrial usage and general toxicity of MeAN, review the previous and more recent experimental use of MeAN and discuss possible toxic mechanisms as defined by recent experimental work. Chemistry MeAN is a colorless liquid having the physical and chemical properties listed in Table I. It is a highly reactive unsaturated alkyl nitrile that readily polymerizes in the absence of a stabilizer. The commercial product contains 50 ppm hydroquinone monoethyl ether as a stabilizer. Because of its double bond, additional reactions are possible with biological molecules. Production and industrial use MeAN is produced TABLE
commercially
by the vapor-phase
reaction
of isobutylene
with
I
CHEMICAL
AND
PHYSICAL
PROPERTIES
OF METHACRYLONITRILE”
CAS number Synonyms
126-98-7 2-methyl-2-propenenitriie. methyl acrylonitrile. -methacrylonitrile, isopropene cyanide. isopropenyl nitrile. 2-cyano-I-propene. 2-cyanopropene, MAN. MeAN
Molecular
C,H,” H?C=C (CH,)-CN 67.09 Colorless liquidb -35.8”C
formula
Structural formula Molecular weight Physical state: Melting point Boiling point Flash point Vapor pressure
90.3”C 55°F (12.7”2(3)’ 40 torr @ 13°C. 65 torr @ 25°C and 100 torr @ 3 3”Cb
Specific gravity Refractive index Solubility in water Solubility in organic
0.800 at 20”Ch “20 = 1.4007 D 2.57 g/l00 g water @ 20°C Miscible with acetone octane 0.68d
Log octanoliwater coefficient “Adapted bAdapted ‘Adapted dAdapted
240
solvents partition
from [8] unless indicated from [ 511. from 191. from [ 27 1.
otherwise.
and toluene
at 2tk25”C
ammonia and oxygen in the presence of a catalyst. Acetonitrile, hydrogen cyanide and acrolein are known by-products of this reaction; they are removed by scrubbing, distillation and fractionation H’ith a yield of approximately 70% specification-grade MeAN [4,5]. The domestic production of MeAN has been reported to be between 1.Ol and 10.1 million pounds in 1977 [6]. The U.S. International Trade Commission (USITC) [7] did not report domestic production of MeAN for the years 1981 through 1987, because the Commission reports production of only those chemicals that are manufactured by at least three producers and MeAN is produced by only two producers. Therefore, the actual production data for MeAN is not accurately available. The producers with U.S. patents are Shell, Allied and Goodrich [8]. The other known United States producers are Kodak, Araphose Chemicals and Fike Chemicals. MeAN is used in the preparation of homo- and copolymers, elastomers, coatings and plastics [8,9] and as a chemical intermediate in the preparation of acids, amides, amines, esters and other nitriles [8]. It is also used as a replacement for acrylonitrile in an acrylonitrile-butadiene-styrene-like polymer, since MeAN improves barrier properties to gases like carbon dioxide in carbonated containers [4]. Occupational
and public exposure
According to a NIOSH report, 28 workers were potentially exposed to MeAN in the workplace in 1980 [lo]. The actual frequency, level or duration of MeAN exposure to workers is not available. A time-weighted average (TWA) threshold limit value of 1 ppm (3 mg/m3) for MeAN exposure was adopted by the American Conference of Governmental Industrial Hygienists [ 1l-121 and is also reported in other sources [ 13-161. This TWA recommendation includes a “skin” notation, indicating a potential for absorption of this compound dermally (including mucous membranes and eyes, either by airborne or direct contact). An atmospheric odor threshold that workers can detect is reported to be 7 ppm [ 171. MeAN has been identified as a component of the mainstream smoke of unfiltered cigarettes made from air-cured, flue-cured or a blend of these tobaccos; the concentration of MeAN in the smoke was 3 &cigarette [18]. In the United States, under the Resource Conservation and Recovery Act, MeAN has been designated as a hazardous waste (Waste No. U152) in Appendix VIII, a listing of chemicals that have been shown to have toxic, carcinogenic, mutagenic or teratogenic effects on humans or other life forms [ 19). U.S. Food and Drug Administration has established a limit of no more than 41% of MeAN-derived polymer in the MeAN-grafted butadiene copolymers for use in the preparation of resinous and polymeric coating materials [20]. Similarly, the limits of 0.5 mg MeAN per square inch of food-contact surface and 50 ppm for water containers have been established in the chloroform-soluble coating components [20]. Human toxicity Only one report is available in the literature about the toxicity of MeAN to humans in which groups of eight to nine subjects were exposed by inhalation twice to a series
241
(24, 14, 0, 7, 14, 24, 7, 2, 0 and 2 ppm in that order) of MeAN concentrations for periods of I min each, with 45-min or longer intervals between exposures [21]. Among subjects exposed to 24 ppm, only 6-22% experienced nose. throat, or eye irritation; no irritation was noted at the other concentrations. A majority of subjects (88-89%) could detect odor at 14 or 24 ppm, but only 47% could detect odor at 7 ppm. In other studies, exposure of 9 subjects to 2 ppm vapor for 10 min or 7 subjects to 14 ppm for 10 min caused nose, throat and eye irritation [21]. No data are available on the epidemiological evidence, case reports. chemical disposition and biochemical. carcinogenic, mutagenic or teratogenic effects of MeAN in humans. Toxicity
and biochemical
effects in experimental
animals
Signs of toxicit) MeAN is shown to be highly toxic in mice, rats and rabbits by dermal. respiratory and oral routes [21-241. Acute toxicity data on MeAN in laboratory animals is summarized in Table II. Pozzani et al. [21] have reported acute inhalation studies in A/J mice, Harlan-Wistar rats, albino guinea pigs and rabbits. The animals were exposed to unspecified concentrations of MeAN vapor for 4-h periods. In all of the species tested, death was preceded by loss of consciousness and tonic-clonic convulsions. At necropsy, no gross treatment-related changes were observed in animals that died or in those surviving a 14-day observation period. In albino rabbits intravenous injection of 6.25 and 12.5 mgikg MeAN produced significant but not dose-related amounts of cyanide into blood which began to fall within 4 h of injection [21]. Inhalation exposure (7 h/day. 5 days/week over a YO-day period) of male beagle dogs to 0, 3.2, 8.8 or 13.5 ppm of MeAN produced dose-related cyanide only at the latter two doses which disappeared with a maximum half-life of 3 days [21]. In subchronic studies, female Harlan-Wistar rats exposed to (& 109.3 ppm of MeAN
TABLE
II
ACUTE
TOXICITY
OF MeAN
IN LAHORAT-ORY
ANIMALS
Species
Route
Toxic do\c
Rat Rat
Oral Inhalation
LII,,,. 700 mg/kg” h LC,,,: 32x 700 ppr1P
Mouse
Oral Inhalation Inhalation Skm
LD<,,: Lc‘,,,: LC,,,: LC,,,:
Oral
LD,,,: 4 mg/kg”
Mouse Guinea Rabbit
pig
Gerbil
from [ 2 I ] from [ 521. ‘Range of doses depending on body weight. ‘Adapted from [ 27). ‘Determmed in our laboratory according to Weil 1531.
“Adapted
bAdapted
242
17 mg/kg” 36 ppm’l XX ppm,’ 168 mg/kg.‘.h
vapors, 7 h/day, 5 days/week for 91 days, showed loss of consciousness and increase in relative liver weight at higher doses, although no microscopic lesions were observed [21]. In a similar study over the same time period, male beagle dogs exposed to t&-13.5 ppm MeAN vapors developed central nervous system effects at the highest dose, manifested by tonic convulsions and loss of control over the hind limbs that were accompanied histopathologically with microscopic brain lesions [21]. In white mice, white rats and rabbits, toxic actions of MeAN were attributed to inactivation of cytochrome oxidase leading to disturbances of tissue respiration [23,24]. The principal clinical manifestation of the toxic effects of lethal and threshold MeAN concentration was an injury to the central nervous system [23,24]. In Wistar rats, exposure to MeAN (3180 - 5700 ppm) for 30 min. the signs of toxicity were related to the in vivo liberation of cyanide [25]. In contrast, the signs of toxicity observed in the same species after acrylonitrile exposure, where metabolic cyanide plays a minor role, were not cyanide related. The acute toxicity of MeAN was antagonized with cyanide antidotes, 4-dimethyaminophenol plus sodium thiosulfate and N-acetyl-cysteine which reacted directly with unsaturated nitriles. Observations on signs of toxicity of MeAN in male Sprague-Dawley rats have recently been reported [26] (Table III). Within 1 h following the oral administration of MeAN, one or more of the male Sprague-Dawley rats in each treatment group developed ataxia, trembling, convulsions. mild diarrhea and irregular breathing. Unlike acrylonitrile (AN), cholinomimetic signs were negligible or absent in MeAN-treated rats. Another important sign of MeAN toxicity, which has not been reported earlier, was the retention of urine in the bladder. Up to 58% of the MeAN (I LDo,, 100 mg/kg) treated animals had their bladder partially or fully distended at the time of sacrifice [26]. Based upon these studies the NOAEL (no observed adverse effect level) and the LOAEL (lowest observed adverse effect level) for MeAN are 50 mg/kg (0.25 LD,,) and 100 mg/kg (0.5 LD,,). respectively.
TABLE SIGNS
111 OF TOXICITY
FOLLOWING
Signs of toxicity”.b.’
I-D,,,
Ataxia Trembling Convulsions Irregular breathing Diarrhea Urine retentior8 “Adapted from bSigns observed
[26]. for 6 hours
ORAL
OF MeAN
(mg/k&)
0.25
0.5
(50)
(100)
I.0 (200)
2.0 (300)
I I I
2 3 3
3 4
2 0 II
3
0 0 0 0 0 0
following
ADMINISTRATION
1 58
4 4
I NT
oral administration.
‘Severity of Signs graded on a scale of I < 2 < 3 < 4 [ 541. ‘Shown as percent of total number of rats with urine retention. NT = not tested.
243
Cyanide liberation Increased blood and urine concentrations of thiocyanate have been reported after MeAN administration [27,28]. Tanii and Hashimoto [27] have reported that pretreatment of male ddY mice with carbon tetrachloride, a known modulator of hepatic mixed function oxygenase system, resulted in much lower concentrations of cyanide than controls and greatly reduced the toxicity of MeAN. Cavazos et al. [28], using radioactive MeAN, found that, after 5 days of dosing rats orally with 100 mgkg, the total urinary excretion of thiocyanate was about 12X, whereas the total urinary excretion of 14C was about 43X, indicating presence of MeAN metabolites other than thiocyanate. In the livers of MeAN ingested rats, thiocyanate levels remained up to 35 nmol/g for initial 6 h and gradually returned to normal. A similar trend was observed in kidney, heart, lung and brain. In plasma, the thiocyanate concentration increased significantly from 26.3 pmol/l at 1 h to 8 1.2 pmolil at 6 h of dosing [29]. Glutathione depletion Soluble thiol contents, including glutathione (GSH), are depleted after MeAN administration. However, MeAN is a less potent depleter of GSH than AN. Day et al. [30] have shown that following the administration of 100 mg/kg MeAN, the maximum depletion was noticed in the liver (39% of control) and in kidney, lung, heart, brain and spleen, ranging between 26 and 34% of control. In vitro studies by Day et al. [30] have shown that addition of MeAN (O--40 mM) to a solution of 0.3 mM GSH at pH 7.4, resulted in a time as well as MeAN concentration dependent depletion of GSH. Thin layer chromatography analysis of incubation mixtures of MeAN with GSH and cysteine showed the appearance of distinct spots representing the adducts S-(2-cyanopropyl) GSH and S-(2-cyanopropyl) cysteinc [30]. Pharmacokinetics
and metabolism
Chemical disposition and covalent binding of MeAN Wolff [3 1,321 has reported pharmacokinetics of AN and two of its analogs MeAN and crotononitrile (CRN) in rats given 5-60 mg/kg intraperitoneally. In this study, MeAN and CRN concentrations followed a first order kinetic elimination and the AN demonstrated a biphasic kinetic elimination. Cavazos et al. [29] have reported that in male Sprague-Dawley rats, following an oral administration of 100 mg/kg MeAN (8 PCilkg [2,3- 14C]MeAN), the primary route of excretion was the urine (43% of dose). Substantial amounts of radioactivity were also found in the feces (15%) and the expired air (2.5”/0). The total 14C-activity excreted in 5 days was 59% of the dose administered. This indicates that more than 40% of MeAN was retained in the body either bound to macromolecules or in the form of unexcretable conjugates. The red blood cells retained significant amounts of radioactivity for more than 5 days after administration, whereas the i4C-activity in plasma declined sharply. More than 50”% of the radioactivity in erythrocytes was detected as covalently bound to cytoplasmic (hemoglobin) and membrane proteins. A small amount of radioactivity was also found associated with the heme fraction. This study indicated that MeAN may damage red blood cells by mechanisms other than release of cyanide.
244
Tissue distribution studies using radioactive MeAN have shown that it is absorbed extensively through the gastrointestinal tract and distributed in all the tissues of the rats [26]. The gastrointestinal tract, however, contained the highest levels up to 48 h, suggesting hepato-intestinal recirculation and resecretion of MeAN, cyanide or other metabolite(s) by the gastrointestinal mucosa. Despite extensive incorporation of MeAN-derived radioactivity in tissue macromolecules, the blood concentration of 14C-activity was significantly greater than tissue concentrations even after 96 h of dosing. Substantial accumulation of radioactivity in the bone was another important observation in this study [26]. Retention of i4C-activity in the bone might lead to an interaction of MeAN with bone marrow thus affecting the rate of hemopoiesis. In vitro metabolism of MeAN to cyanide Tanii and Hashimoto [27] have reported the kinetic constants for the in vitro liberation of cyanide from MeAN in ddY mouse hepatic microsomal enzyme system. The K,,, value for MeAN was 0.133 mM, which ranked 5th from the lowest value reported for 13 aliphatic nitriles, and the V,,, for MeAN was 417 ng cyanide formed/15 min per mg protein [27]. In liver fractions from male Sprague-Dawley rats, the metabolism of MeAN to cyanide was localized in the microsomal fraction and required NADPH and oxygen for maximal activity [33]. Metabolism of MeAN was increased in microsomes obtained from phenobarbital-treated rats and decreased after cobaltous chloride and SKF 525 A treatments. Addition of the epoxide hydratase inhibitor I,l,l-trichloropropane 2,3-epoxide, decreased the formation of cyanide from MeAN. Addition of GSH, cysteine, D-penicihamine or 2-mercaptoethanol enhanced the release of cyanide from MeAN. However, when the cytosolic fraction was added to the incubation medium along with microsomes the formation of cyanide was significantly reduced [34], indicating a role of GSH S-transferase provided by the cytosol. These findings indicated that MeAN is metabolized to cyanide by a cytochrome P-450-dependent mixed-function oxidase system. Nitriles differ remarkably in their capacity to release cyanide both in vivo and in vitro [34,35]. Among AN and two of its analogs CRN and MeAN, when incubated with rat liver microsomes, the highest amount of cyanide was released from MeAN (57.9 nmol cyanideimg protein per min) followed distantly by CRN and AN (8 and < 1 nmolimg protein per min, respectively) [33,34,36] and was influenced by the presence of GSH in the incubation medium [34]. Such differences in their metabolism appear to be involved in the toxicity of nitriles. For example, the signs of toxicity in the case of AN are far less cyanide-related than both of its analogs MeAN and CRN where treated animals show distinct cyanide-related nervous effects including depression, convulsions and respiratory failure. In contrast, the early signs of AN toxicity are distinctively cholinomimetic including salivation, lacrimation, vasodilation and diarrhea with central nervous system signs developing very late. Silver et al. [35] have suggested that the length of carbon chain, presence of substituents at the carbon, position of double bonds and, for some compounds, route of administration, are important factors influencing the release of cyanide from nitriles. Recently, the vehicles used to administer nitriles, have been shown to influence the absorption and toxicity of nitriles [37].
245
Species dl]Jerence in toxicity and metabolism Table II shows that there is a clear species difference in the toxicity of MeAN in terms of LD,, values, gerbils being the most susceptible species followed by mice and rats. Studies in our laboratory have shown that there is a distinct species difference in the metabolism of MeAN to cyanide among gerbils, mice and rats, both in terms of amount of cyanide formed and the time of peak levels in blood. The data in Fig. 1 shows that following a single oral dose of 0.5 LD,,, MeANikg, the blood cyanide concentrations in rats were maximal at 3 h after treatment. However, in gerbils and mice the blood cyanide concentrations were maximal at 1 h. In all species studied, cyanide levels gradually declined to negligible levels by 24 h. In addition, the gerbils metabolized greater amounts of MeAN to cyanide than either the mice or rats. This explains the extremely low LD,, of MeAN for mice and gerbils. Species difference has also been reported for other nitriles including AN [38] and dimethylaminopropionitrile [39]. Variations in biotransformation between species may be either qualitative or quantitative differences. The qualitative differences may involve metabolic routes and are due either to a species defect or to a reaction peculiar to a species [40]. The quantitative variations may result from differences in enzymic levels or natural inhibitors in the balance of reverse enzyme reactions. Most. but not all, nitriles are metabolized via a mixed-function oxidase system. Some variations may be due to extrahepatic metabolism and the biological half-life of metabolites formed. The gerbils are among unique animals that use amino acids [40], such as taurine, for conjugation reactions rather than the usual hepatochelating agent GSH. Gastrointestinal flora may also play a role in species differences since not all animals are germ free. There may be numerous other factors involved in species difference in the toxicity of nitriles and it needs to be explored as to what specific factors are involved in the case of MeAN.
Fig. 1.Time course of blood cyanide concentrations in rats (circles). gerbils (diamonds) and mxe (triangles) rats. after single oral dose of MeAN. MeAN was administered orally at 0.5 LD,,, (male Sprague-Dawley 100 mg/kg; Albino-Swiss mice, I7 mgikg; Mongolian gerbils. 4 mg/kg). Each value is the mean f S.11. of 3 animals. Cyanide levels were determined by microdiffusion technique 1331.
246
Metabolic
pathways for MeA N
Because of its similarity to acrylonitrile, it is likely that MeAN is metabolized by two pathways as proposed in Fig. 2. One major pathway of MeAN metabolism is the direct conjugation with GSH, which is catalyzed by GSH S-transferases. The product S-(2-cyanopropyl) GSH [30] may be further metabolized to N-acetyl-S(2-cyanopropyl) cysteine. Another major pathway involves an epoxide intermediate and is catalyzed by the hepatic microsomal P-450 system [33]. Further metabolism of epoxide by GSH epoxide transferase may result in another GSH conjugate depending on the site of nucleophilic attack on the epoxide molecule. Alternatively, the epoxide intermediate could produce cyanide and other metabolites by rearrangement or by catalysis with epoxide hydratase. Further metabolism of GSH conjugates may lead to the excretion of various mercapturic acids. Mutagenicity
and reproductive toxicity
In two independent studies, MeAN was non-mutagenic in the Salmonellaimicrosomal assay in strains TA97, TA98, TAlOO, TA1535 and TA1537 with or without either hamster or rat liver activation [41]. In Drosophila assay, MeAN fed to larvae at 5950 or 6077 ppm did not induce sex-linked recessive lethal mutations [41]. In pregnant Sprague-Dawley rats, oral administration of 50 mgikg per day MeAN (0.25 LD,,) during first or second week of gestation and 100 mgikg per day (0.5 LD,,) only during second week of gestation, resulted in a dose-dependent significant reduction in maternal body weight gain [42]. In this study, control rats delivered nor-
QH QH CHzCCN _ epoxide hydratax micrcaomal oxidation
/
oxidation pdUCfS
CH3
/ -0. ~~~~“g.EllI~tlt~
SCN-
OXIDATTVE PATHWAY macmmokcules GSCH2-F(OH)CN
CV, = C-CN
___,
GSCH2-q=O-
CH3 mcrcapturic acid
cyanoisop&pyl
mcrcapturic acid
,
l ,,,,g,
~~:~~~?c
-
Fig. 2. A
proposed
metabolic
pathway
1. A. Nucleic acids (NH OH) 9. Pmteins (NH2, OH, SH) C. Lipids (OH, NH, ha) = 2. Bzological Neumtransmwxs: A. adrenahn and its analogs 9. semtonm C. amincbutyric acid D. histammc 3. Other nucleophilic components of tissues
for MeAN.
247
TABLE ORGAN
IV TOXICITY
OF ALIPHATIC
NITRILES
Nitrile
Organ
toxicity
Acrylonitrile Propionitrile Malononitrile Methacrylonitrile Crotononitrile Acetonitrile
Adrenal apoplexy [ 391 Duodenal ulcers [ 40) Nuclear changes in neurons and satellite ganglia Skin poison [7], urine retention [26] Highly corrosive action [IS] Thyroid hyperemia and hyperplasia (421
Dimethyl amino-propionitrile Allylnitrile
Neurological bladder Long-term dyskinesia
dysfunction [46]
141 I
[43--15]
ma1 size litter and MeAN-treated rats failed to maintain pregnancy and aborted; these abortions may be cyanide-related [43]. At the end of gestation, the rats fed with MeAN developed dose-dependent mild to severe edema in the fallopian tubes [42]. These findings suggest that MeAN has a potential for reproductive toxicity. Based upon these studies, the LOAEL for the reproductive effects of MeAN appears to be 50 mg/kg (0.25 LD,,). Conclusions In conclusion, we have attempted to summarize the basic chemical and toxicologic information available on MeAN. MeAN belongs to the family of aliphatic nitriles, each of which, despite structural similarities, exhibit entirely different biochemical effects. Although most of the aliphatic nitriles are potent neurotoxins, their toxic actions on other organ systems differ remarkably (Table IV) [ 17,39,44-511. Aliphatic nitrile biotransformation (Fig. 2) requires activation of the molecule before release of the cyanide group. The instability of the cyanohydrin formed suggests that a nascent form of aldehyde may be formed; it may escape the detoxication mechanisms of the cells and, perhaps, have a role in the mechanism of toxicity of these compounds. Despite the amount of information available on MeAN, further extensive work on various aspects of MeAN metabolism, toxicology and pharmacology is indispensable to understand the mechanism underlying its toxicity. Acknowledgement This work was supported by Grant No. SORR08038 from the National of Health Minority Biomedical Research Support Program.
Institutes
References 1 2
248
S. Lang, Uber die Umwandlung des Acetonitrils und seine, Homologen im Tierkorper. Arch. Exp. Pathol. Pharmakol., 34 (1894) 247. A.E. Ahmed and N.M. Trieff, Aliphatic nitriles; metabolism and toxicity. in J.W.Bridge and L.F. Chasseud (Eds.). Progress in Drug Metabolism, 7 (1983) 229.
A.E. Ahmed, M.Y.H. Foreign Compounds.
Farooqui Academic
and N.M. Trieff, Nitriles. in M.W. Anders Press. New York, 1985 pp. 485-518.
(Ed.). Bioactivation
of
D.M. Cosidine (Ed.), Chemical and Process Technology Encyclopedia. McGraw-Hill Book Company. New York, 1974, pp. 30-34. J.W. Nemec and L.S. Kirch, Methacrylic acid and derivatives. in Kirk-Othmer Encyclopedia of Chemical Technology. 3rd edn.. Wiley. New York. 15. 1981. pp. 346. 363, 376. USEPA, U.S. Environmental Protection Agency. Computer printout (CICIS): Production Statistics for Chemicals in the Nonconfidential Initial TSCA Chemical Substance Inventory, Washington. DC. Office of Toxic Substances and Pesticides. 1987. USITC, U.S. International Trade Commission. Synthetic Organic Chemicals. United States Produc-
8 9 10
II I2
I3
tion and Sales. Publication Nos. 1292. 1422. 1401, 1588, 1548, 1745, 1892, and 2009. Washington. DC, Government Printing Office, 1982-87. M. Windholz (Ed.), The Merck Index. 10th edn., Merck and Company, Rahway. NJ, 1980. p. 850. N.I. Sax and R.J. Lewis Jr.. Hawley’s Condensed Chemical Dictionary, I Ith edn.. Van Nostrand Reinhold Company, New York, 1987, p. 751. NIOSH, National Occupational Exposure Survey (198&83) [database]. Cincinnati, OH: Department of health and Human Services. National Institute for Occupational Safety and Health, 1984. ACGIH, Threshold Limit Values and Biological Exposure Indices for 198687. Cincinnati, OH: American Conference of Governmental Industrial Hygienists. 1986. p, 23. ACGIH, Documentation of the Threshold Limit Values and Biological Exposure Indices, 5th edn. Cincinnati, OH: American Conference of Governmental Industrial Hygienists, 1986. p. 370. Registry of Toxic Effects of Chemical Substances (RTECS), (RTECS UD14CJOOOO).Vol. 4, 1985-86, p. 3856.
I4 15 I6
Patty’s Industrial Hygiene and Toxicology, 3rd rev. edn.. Vol. 2C. 1982, pp. 48634888. The Sigma-Aldrich Library of Chemical Safety Data. edn. I. 1985, p. 1171. N.1. Sax and R.J. Lewis. Dangerous Properties of Industrial Materials, 7th edn., Vol. III. 1989. p. 2251.
17
J.E. Amoore and E. Hautala. Odor as an aid to chemical safety: odor thresholds compared with threshold limit values and volatilities for 214 industrial chemicals in air and water dilution. J. Appl. Toxicol.. 6 (1983) 272. R.R. Baker. H.F. Dymond and P.K. Shillabeer. Determination of a&unsaturated compounds formed
I8 I9 20
21 22 23 24 25 26 27 28 29
by a burning cigarette. Anal. Proc., 21 (1984) 135. USEPA, U.S. Environmental Protection Agency. 40 CFR 261. Identification and listing of hazardous waste. Section 261.33 and Appendix VIII, 1986. pp. 359, 384, 398. USFDA, Food and Drug Administration, 21 CFR Subpart C-Substances for Use as Components of Coatings, 175.300. Resinous and Polymeric coatings. Washington. DC., Office of the Federal Register. 1986. pp. 141. 143. 152, 153. lJ.C. Pozzani. E.R. Kinkead and J.M. King, The mammalian toxicity of methacrylonitrile. Am. Ind. Hyg. Assoc. J.. 29 (1968) 202. W.A. McOmie. Comparative 31 (1949) 113. S.A. Kurzaliev. [Characteristics 5 (1985) 37.
toxicity
of methacrylonitrile
and acrylonitrile.
of the toxic action of methacrylonitrile].
J. Ind. Hyg. Toxicol.,
Gig. Tr. Prof. Zabol. (USSR),
S.A. Kurzaliev and A.N. Zuev, [Toxic effect of methacrylonitrile on the body of experimental animals after chronic exposure]. Gig. Tr. Prof. Zabol. (USSR), 4 (1988) 51. H. Peter and H.M. Bolt. Effect of antidotes of the acute toxicity of methacrylonitrile. Int. Arch. Occup. Environ. Health, 55 (1985) 175. M.Y.H. Farooqui. R. Cavazos. M.I. Villarreal and E. Massa, Toxicity and tissue distribution of methacrylonitrile in rats. Ecotoxicol. Environ. Safety, (1990). in press. H. Tanii and K. Hashimoto. Studies on the mechanism of acute toxicity of nitriles in mice. Arch. Toxicol., 55 (1984) 47. R. Cavazos. M.Y.H. Farooqui, W.W. Day. M.I. Villarreal and E. Massa, Disposition of methacrylonitrile in rats and distribution in blood components. J. Appl. Toxicol.. 9 (1989) 53. R. Cavazos, M.Y.H. Farooqui and W.W. Day. Metabolism of methacrylonitrile to cyanide in rats. Toxicologist, 8 (1988) 210.
249
30 31 32 33 34 35 36 37
38 39
40
41 42 43 44
45 46 41 48
49 50 51 52 53 54
250
W.W. Day. R. Cavazos and M.Y.H. Farooqui. Interaction of methacrylonitrile with glutathionc. Res. Commun. Chem. Pathol. Pharmacol.. 62 (1988) 267. M.S. Wolff. Acrylonitrile Pharmacodynamics and Mutagenesis. Govt. Reports Announccmcnty and Index (GRA&I). NIOSH, Issue 23. 19X8. M.S. Wolff, Acrylonitrile pharmacodynamics and mutagenesis. NIOSH. Environmental Science Laboratory, Mount Sinai School of Medicine, NY. (1977). M.Y.H. Farooqui. R.G. Diaz and R. Cavazos. Metabolism of methacrylonitrile to cyanide ill ~rlro studies. J. Biochem. Toxicol., (1990). in press. M.Y.H. Farooqui and E. Massa, Effect of glutathione on in ~+/ro metabolism of unsaturated aliphatic nitriles. Bull. Environ. Contam. Toxicol.. 5 (1990) 109. E.H. Silver. S.H. Kuttab. T. Hasan and M. Hassan. Structural considerations in the metabolism 01 nitriles to cyanide in vivo. Drug Metab. Dispos., 10 (19X2) 495. M.E. Abreu and A.E. Ahmed. Metabolism of acrylonitrile to cyanide in vitro studies. Drug Mctab. Dispos., 8 (1980) 376. M.A. Wallig. D.H. Gould, J.V. Stcenhouse. M.J. Fettman and C.C. Willhitc. The relationship 01 vehicle to target organ toxicology induced by the naturally occurring nitrllc I-cyano-2-hydroxy-3.butcnc. Fundam. Appl. Toxicol.. I2 (19X9) 377. A.E. Ahmed and K. Pate]. Acrylonitrile: in vivo metabohsm in rats and mice. Drug Metab. Dispos.. 9 (1981) 219. M.M. Mumtaz, M.Y.H. Farooqui. B.I. Ghanayem and A.E. Ahmed. The urotoxlc mechanisms ol N.N-dimethylaminopropionitrile in rats and mice I. Biochemical and morphological characterlz+tion of the injury and its relationship to metabolism. J. Toxicol. Environ. Health. ( 1990). in press. I.G. Sipes and A.J. Gandolfi. Biotransformation of toxicants. in C.D. Klaahscn, M.O. Amdur and J. Doull (Eds.) Casaett and Doull’s Toxicology. The Basic Science of Poison\. 3rd cdn.. Macmillan. New York. 1986. pp. 64 98. E. Zeiger. B. Anderson, S. Haworth. T. Lawlor, K. Mortelmans and W. Speck. S&no&/tr mutagcmcity tests: 111. Results from testing of 255 chemicals. Environ. Mutagen.. 9 (19X7) I. M.I. Villarreal. R. Cavazos and M.Y.H. Farooqui, Reproductlvc toxicity of methacrylonitrilc in rats. Proc. NIH-MBRS Symp.. 16 (198X) XX. C.C. Willhite, V.H. Ferm and R.P. Smith, Teratogenic effect\ of aliphatic nitrilcs (1981) 317. S. Szabo. I. Hunter. K. Kovacs. E. Horvath. D. Szaho and H.C. Horncr. Pathogcncsis tal adrenal hemorrhagic necrosis: ultrastructural, hiochcmical, neuropharmacologic and tion studies with acrylonitrile in the rat. Lab. Invest.. 42 (IYXO) 533. S. Szaho and H. Seyle. Duodenal ulcers produced by propionitrilc. Arch. Pathol.. V.L. Van Breeman and J. Hirakoa. Ultrastructure of nerve and satellite of spinal ganglia
Teratology.
23
ofexperimenblood coagulaY3 (lY72) 390. of rats trcatcd
with malononitrile. Am. Zoo].. I (I961 ) 473. A.W. Spence and D. Marine. Production of thyroid hyperpla\ia in rats and mice by admlnistration of methyl cyanide. Proc. Sot. Exp. Biol. Med.. 29 (1932) 967. K. Kriess. D.H. Wegman, C.A. Niles. M.B. Siroky, R.J. Krame and R.G. Feldman. Neurological dysfunction of the bladders in workers exposed to dimethyl aminopropionitrilc. J. Am. Med. A\soc.. 243 (1980) 741. A.E. Ahmed and M Y.H. Farooqui, Metabolism and toxicity of N.N’-dimcthylaminopropionitrile in rats. Toxicologist. 26 (1984) 19X. H. Tanli. Y. Kurosaka. M. Hayashi, and K. Hashimoto, Ally]-nitrlle. a compound which Induces long-term dyskinesia in mice followmg a single administration. Exp. Neural.. 103 (1989) 64. K. Verschueren, Handbook of Environmental Data on Organic Chemicals. 2nd edn.. Van Nostrand Remhold Company, New York. 19X3. p. X15. H.F. Smyth Jr., C.P. Carpenter. C.S. Weil. U.C. Po/zani and J.A. Strlegel. Range-linding toxicity data: list VI. Am. lnd. Hyg. Asroc. J.. 23 (1962) Y5. C.S. Weil. Tables for convement calculation of median effective dose (LD,,) or ED,,,) and instructions in their use. Biometrics, X (1952) 249. A.E. Ahmed and M.Y.H. Farooqui, Comparative toxlclties of allphatic nitrilca. Toxicol. Lett. I? (1982) 157.