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Chapter 6 ANILINE = Aniline (also called aminobenzene or benzenamine) is a colorless, oily liquid that freezes at -6.2°C and boils at 184°C. It is combustible and is moderately soluble in water. At 25°C, aniline has a vapor pressure of O. .67 mm Hg. Aniline is one of the most important organic bases and is the parent compound for more than 300 chemical products. It is typically produced by the catalytic hydrogenation of nitrobenzene. The gas-phase reaction of hydrogen and nitrobenzene over a catalyst at temperatures below 350°C yields more than 98% aniline. Aniline as a free base is a relatively unstable compound, which is rapidly oxidized in the presence of air and light to ~ complex mixture of quinoneimines, quinones, and highly colored polymers of unknown composition. It is a weak base that is readily converted to a water-soluble, stable salt in acid solution (a hydrochloride) (International Agency for Research on Cancer, 1974; Radomaki, 1979} 123 .
PRODUCTION Table 6-1 lists the current producers of aniline, their locations, and their annual capacities. Three of the companies are planning to increase production in the near future. Rubicon Chemicals, Inc. plans to expand its capacity at Geismer, Lie. by an additional 9,100 metric tons per year dur ing 1980 (Chemical Marketing Reporter, 1979 ~ . Anger lean Cyanamid Co. will increase the capacity of its Willow Island, W. Va . facility to a total of 50,000 metric tons per year during 1980 (Chemical Marketing Reporter, 1979) . file Polyurethane Division of Mobay Chemical Corp. in New Martinsville, W. Va., plans to begin recovery of aniline from its iron oxide plant in the first quarter of 1981. Capacity will be 12,000 metric tons. By 1985, Mobay's polyurethane capac ity is expected to reach 18, 000 metr ic tons (Chemical }economics Handbook, 1978) . USES 6-2 . U.S. consumption patterns of aniline in 1979 are shown in Table 124
Table 6-1 ANILINE PRODUCERS AND CAPACITIESa - Producer and Location Capac ity (103 metric tons) Rubicon Chemicals, Tnc Geismar, La. . E. I. du Pont de Nemours & Co., Inc. Beaumont, Tex. Gibbstown, N.J. First Chemical Corp., subsidiary of First MiSSiSSippi Corp. Pascagoula, Miss. Ame r ican Cyanamid Co Bound Brook, N.J. . Orga n ic Chemica 1 s D iv i s ion Willow Island, W. Va. Bombay Chemical Corp., Industrial Chemicals Division New Martinsville, W. Va. 1~27 118 73 114 27 23 45 Total annual U. S. aniline production for recent years: b Thousands of metr ic tons 1975 1976 1977 1978 247.2 265.5 275.4 309.7 a SRI (Standard Research Inst itute ), 1979 . b U.S. International Trade Commission, 1976, 1977, 1978, 1979. 125
Table 6-2 ANILINE CONSUMPTION PA]~ERNSa, b Percent of Use total 103 metric tons Intermediate for monomer ic 50 155 and polymeric isocyanates .. Intermediate for rubber 27 84 chemica Is Dyes and dye intermediates 6 19 Hydroquinone 5 15 Intermediate for pharmaceuticals 3 9 Miscellaneous 9 28 l a Chemical Marketing Reporter, 1979. b Total U. S. consumption is considered equal to U.S production; imports and exports are negligible. 126
An iline is used as an intermediate in the production of E~,E~'-methylenediphenyl diisocyanate (NDI) and polymeric MDI, which are used primarily in the manufacture of rigid polyurethane foam for building insulation (Chemical Bconomice Handbook, 19783. Me U.S. producers of MDI include Hobay Chemical Corp. in Cedar Bayou, Tex., and New Martinsville, W. vat, Rubicon Chemicals, Inc. in Geismar, La., and the Upjohn Co. in L;a Porte, Tex. (Stanford Re~earcb Institute, 1979 ~ . The chemicals derived from aniline are used in rubber manufacture as vulcanization accelerators, antioxidants, and antidegradants {Northcott, 1978~. file Host commercially significant are 2-mercaptobenzothiazole and N~cyclohexyl-2-benzothiazole (Chemical Economics Handbook , 1978 I, produced by Amer icon Cyanamid Co. in Bound Brook , N . J . the B. F. Good r ich Co. in Henry , 111 ., Monsanto Co. in Nitro, W. Va ., Pennwalt Corp. in Wyandotte, Mich., and Un iroyal, Inc. in Geismar, La . 2-Mercaptobenzott~iazole is also produced by Eastman Kodak Co. in Rochester , N. Y., and the Goodyear Tire and Rubber Co. in Niagara Falls, N.Y. (Stanford Research Institute, 1979 ~ . Dyes prepared from aniline and aniline derivatives are included in the following four dye classes: azo, tr iphenylmetbane, anthraquinone, and safranines {International Agency for Research on Cancer, 1974~. The Colour Index (1971) lists 174 dyes that can be prepared from aniline, and more than 700 dyes that can be prepared from aniline derivatives. Because of the increased use of synthetic 127
f ibers and str toter controls imposed by the Food and Drug Administration (FDA), very few of these dyes are currently produced in commercially significant quantities (Northcott, 1978~. One of the more significant of the aniline~based dyes, from ~ commercial standpoint is C.~. (Color Index) Vat Blue 1, used widely to dye cotton f ibers such an those used in denim. C. I. Vat Blue 1 (DSC Blue No. 6) has also been used as a colorant for surgical sutures. (Bauer, 1979; 21 CFR 74~. This dye is produced by BASE Wyandotte Corp. in Parsippany, N. J., and Buffalo Color Corp. in Buffalo, N.Y. (Standford Research Institute, 1979~. Among the commercially more signif icant dye intermediates derived from aniline are E' - itroaniline, which is produced by Monsanto Co. in Sauget, Ill., Amer ican Color & Chemical Corp. in Lock Haven Pa ., and the Signal Compan ies Inc . in Shrevepor t, La .; N,N-diethylaniline1 and N,N-dimethylaniline, both produced by American Cyanamid Co. in Bound Brook, N.J., Buffalo Color Corp. in Buf falo, N.Y., and E . I . du Pont de Nemours ~ Co. in Deepwater , N.Y.; and o-, m-, and E'-chloroan il ine, which is produced 1 Also used to make 2-chloro-2 ' ,6 '-die'chyl-N- {methoxymethyl) acetanilide, an herbicide marketed under the trade name Lasso (Chemical Economics Handbook, 197 8 ~ . 128
by E. I . du Pant de Nemours ~ Co., Inc. in Deepwater, N. J. o- and Chloroaniline are also produced by Monsanto Co. in Luling, I.a. (Stanford Research Institute, 1979; Colour Index, 1971) . Aniline is also involved in the production of t~ydroc~uinone, which is used primarily an a developing agent for black-and-white photography tWoodlief , 1973), and as a polymerization shortstop in styrene-butadiene rubber production (Bauer, 1979 ~ . Hydroguinone is produced by Eastman Kodak Co. in Kingsport, Tenn. and the Goodyear Tire & Rubber Co. in Bayport, Tex. (Stanford Research Institute, 19 79 . In the pharmaceutical industry, aniline is used in the production of acetanilide, which was once widely included in analgesic and antipyretic formulations: it is currently used as an intermediate in the manufacture of most sulfanilamide drugs {Northcott, 19781. Pharmaceutical aniline is produced by Eastman Kodak Co. in Rochester, N.Y., Merck & Co., Inc. in Albany, Gal, Salisbury Laboratories in Charles City, Iowa, and Syntex Corp. in Newport, Tenn. {Stanford Research Institute, 1979 ~ . There are a number of miscellaneous applications of aniline. It is used in the production of intermediates for herbicides, fung ic ides, insecticides, an imal repellents, and defoliants (Northcott, 1978) and in the production of cyclobexylamine (formerly an intermediate in the manufacture of cyclamate synthetic sweeteners and presently an intermediate in the production of a 129
var iety of other chemicals and as a corrosion inhibitor) . Cyclobexylamine and its derivatives are produced by Abbott Laboratories in Wichita, Kane., Honeanto Co. in Sauget, Ill., and Virginia Chemicals Inc. in Bucks, Ala. and Portsmouth, Va. (Stanford Research Institute, 1979~. Aniline is also used. in the production of ~,p'-methylenedianiline, an intermediate for the commercial synthesis of a polyamide fiber marketed under the trade name Quiana. The sole producer of Quiana is E.I. du Pont de Nemours ~ Co., Inc. The monomer is produced at Belle, W. Va., and the polymer is spun into yarn at the plant in Chattanooga, Tenn. (Chemical Economics Handbook, 1977 ~ . EXPOSURE As demonstrated above, aniline is produced in large quantities and has numerous applications. Although the potential for human exposure is correspondingly large, there are no quantitative estimates of environmental exposures of the general public. Nonetheless, the National Institute on Occupational Safety and Health (NIOSH), based on results of a National Occupational Hazards Survey, has estimated that a potential 1.26 million workers could be exposed to an il ine . Exposure to aniline in the workplace is regulated by the Occupational Safety and Health Administration tOSHA). me health standards for occupational exposure to air contaminants require that an employee's exposure to aniline shall not exceed 130
5 ppmor l9 mg/m3 air in any 8-hour workday of ~ 40-bour workweek (Occupational Safety and Health Administration, 1980~. In 1979, the A'ner ican Conference of Governmental Industr ial Hygienists (ACGIH) adopted a threshold limit value time weighted average for dermal exposure to aniline and its homologs of 2 ppm or 10 mg/m3 a ir for any 8-hour workday or 40-hour workweek: and a threshold limit value, short-term exposure limit of 5 ppm or 20 mg/m3 air for a period of up to 15 minutes, not to occur more than 4 times per day {Amer ican Conference of Governmental Industr ial Hyg fen ists, 1979 ~ . Because of aniline ' s widespread use, it is generally considered to be a likely component of many industr ial wastewater discharges. However, the committee found only one reference (Jungclaus et al., 1978J in which aniline concentrations had actually been measured in such a discharge; the aniline concentration in the was tewater discharge. These investigators reported that of a specialty chemicals plant was O .02 ppm. file compound was not detected downstream of the plant nor in the stream sediment. Aniline is biodegradable. It is susceptible to treatment in wastewater with activated sludge (Joel and Grady, 1977) . In air, it is subject to attack by the hydroxyl radical (Spicer et al., 1974), but its overall half-1 if e in a ir is riot known . No information could be found on the presence of an i 1 ine in consumer products, and no 131
evidence was found that aniline is covered by Food and Drug Administration regulations. The Interagency Testing Committee, established under section 4 (e) of the Toxic Substance Control Act {TSCA}, has added aniline to its Pr for ity List of Chemicals despite the previous National Cancer Institute (NCI, 1978) test. Chemicals on this list are considered for testing by the U. S. Environmenta1 Protection Agency (EPA) in accordance with section 4 (a) of TSCA. Within 12 months of such a recommendation, the EPA must initiate rulemaking to require testing of chemical or publish its reasons for not doing so. The committee recommended studies to determine the carcinogenicity, mutagenicity, teratogenicity, chronic effects, environmental effects, and epidemiology of aniline. 132
ANALYTIC t4E:THODS In addition to the general analytic procedures for primary aromatic amides, discussed in Chapter 1, the following additional information from recent literature should help in Methods selection. I"ination and Bromination The sensitivity of aniline in electron capture-gas . chromatography {EC-GC) assays is greatly enhanced by iodination or Bromination of the molecule . gofman et al . {1979), descr ibed the following process: For iodination, the compound in 1 N hydrochlor to acid is treated with Sodium nitrite at 0°C, iodinated with potassium iodide at room temperature, and boiled at reflex. Nate iodine derivative is extracted with hexane; iodination efficiency is 871. Bromination of aniline is carried out in 1 M sulfuric acid with mesidine, potassium bromide, and potassium bromate. ate reaction product {2,4,6-tribromoaniline) is extracted with toluene after alkalinization with 10 N sodium hydroxide. Efficiency of Bromination is 99.61. Cigarette Smoke file amines from cigarette smoke were trapped in dilute hydrochloric acid and enriched together with the basic portions, der ivatized to pentafluoropropionasudes, and determined by EC-GC with a nickel-63 electron-capture detector. Me detection limit 133
was approximately 50 pg of an iline per cigarette (Patr ianakos and Hof Flynn, 19 79 ~ . Ani 1 ine and i ts Metabol ites Sternson and Dewitte (1977) reported a high-pressure liquid cbra~natography (HP=) method for determining nanomole quantities of aniline and its metabolites, o- and ~-aminopbenol. phenylhydro~lamine, nitrosobenzene, nitrobenzene, azobenzene and azoxybenzene, which form nonenzymatically by condensation of reactive metabolites. The compounds were separated by reverse-phase HPLC on a Bondapak carbon-18 column and detected spectrophotometrically. The eluent for the first four components was methanol-water (15:85) Containing 0.26 M ammonium acetate and 0.015-M nickel acetate. me remaining components were elated with a solution of methanol and water (50:50~. Air and Personal Sampling Wood and Anderson {1975) described procedures for assaying airborne vapors of aniline and related compounds. The vapors were absorbed on silica gel, elated from the gel with 95% ethyl alcohol containing 0.1% heptanol, and separated and analyzed by gas chromatography with a column of OV-25. Boukun et al. (1974) devised a simple but sensitive and selective Method for determining aniline vapors in air. The air sample W86 drawn through an indicator tube 134
filled with porcelain powder treated with a mixture of alcohol ammonium bexanitrocerate (IV) solution and aqueous potassium persulfate solution. The concentration of aniline in the air was determined by the length of the indicator mass, which had changed colors. Gromiec and Adamlak-Ziemba (1974) determined vapors of N-ethylaniline and aniline in admixture in air. Both compounds were adsorbed in 21 ethyl alcohol, and the sum of the two amides was determined calorimetrically by using an indophenol procedure. S imu 1 taneou s ly, an i 1 ine was de termined by d iazot i za tion and coupl ing with N-l-naphthylethylenediamine. m e concentration of N-ethylaniline was calculated by difference. The determinable limit - for N-ethylaniline and aniline was 1.0 and 0.37 mg/ml, respectively. A rather unique method for personal monitoring was reported by Schaffernicht and Schreinicke (19741. A personal sampler connected to a telemetric system was placed directly on a workman, thereby permitting continuous measurement of the toxic substances in his breathing zone. The toxic substances were colorimetrically determined by absorption in a tube of the personal sampler. Electrolytic current was proportional to the concentration of the toxic substance and was used as the basis for a frequency-modulated telemetric signal. After demodulation on the receiving side, the data were recorded by a strip recorder. The system is suitable for measuring sulfur dioxide, hydrogen sulfide, hydrogen cyanide, phenol, and aniline in the range'; likely to occur in industrial situations. The person being monitored is free to move within a radius of 150 meters around the receiver. 135
Volumetric Analysis Madra icon et al. (1973 ~ reported an iodo~etr ic method for determining aniline or Anesthesia {ethylene aminobenzoate) using neutral iodine-bromide solution. me procedure is reported to be sensitive to 5 p9 of aniline in 0.00051 solutions and 40 p9 of Anesthesin in 0.00251 solutions. me preparations were treated with 5 to 6 ml of 1-M hydrochloric acid and 50 ml of 0.1-M iodine-bromine solution and heated at 40°C to 50°C for 3 minutes. After the addition of both 10 ml of ethanol and 10% potassium iodide solution, the released iodine was titrated with a sodium thiosulfate solution, with starch as an indicator. Other Analytic Methods for Aniline Ascik et al. (1975) determined toxic compounds in pulp and paper mills. Hey discussed methods of sampling and instrumental analysis and tabulted maximum permissible concentrations for several compounds, including aniline. Zaugol tnikov et al. (1975 ~ determined several environmental contaminants, including aniline, and used nomograms and equations to determine maximum permissible concentrations of the compounds in industr ial environments, city air, and municipal water reservoirs. Hartstein and Eorshey (1974) repot ted on exper iments per formed by the Bureau of Hines to investigate products formed on thermal oxidative degradation of selected compounds under both dynamic and static conditions. Four broad classes of mater ials were studied: polyvinyl chlor ide 136
compounds, neoprene compounds, rigid urethane foam, and variously treated woods. n~ermogravi'setric and differential thermal analyses were performed to explore the feasibility of using these analyses to identify "serials. Sixteen toxic products including aniline were detected and measured. Dutkiewics and l;zy~nska (19733 employed thin-layer chromatography (TLC) to analyze the urine from rats given an oral dose of hydrazobenzene 4200-400 ag/kg). Aniline was one of the products detected in the or ine . 137
HEALTH EFFECTS The primary exposure of humans to aniline is occupational, however, exposure to aniline in the environment may also occur. Aniline is a volatile liquid at room temperature and is rapidly absorbed when inhaled {Dusk iewicz and Piotrowsk i, 1961; Vas ilenko et al., 19721. It is also rapidly and efficiently absorbed through the skin and from the gastrointestinal tract following oral ingestion. These properties led to the establishment of a threshold limit value (TLV) of 5 ppm (19 mg/m3~. Metabolism The metabolism of aniline is complex and multifaceted. As with many other compounds, the metabol ic process seems to take place in two stages. The first stage, which is mediated by microsomal enzymes in the liver, consists of oxidation (hydroxylation) of the 2 and 4 positions on the aromatic ring and the nitrogen atom (N-hydroxylation). Usually 4-hydroxylation predominates with the formation of p-aminophenol, the principal metabolite (Parke, 1960; Smith and Williams, 1949; Williams, 1959~. N-hydroxylation results in the formation of a possible biologically significant metabolite, phenylhydroxylamine. However, phenylhydroxylamine does not appear to be carcinogenic or mutagenic under test conditions where a series of other arylhydroxylamines were positive for both effects (Berman _ al., 1968) 138
The second stage consists of con jugation of these r ing hydroxyl groups with glucuronic and/or sulfuric acid. In addition, the unoxidized amine group may be conjugated with glucuronic acid with the formation of a N-glucuronide or with sulfuric acid with the for nation of an N-sulfate (sulfamate) (Boyland et al., 1957~. Phenylhydroxylamine may react with cysteine, leading eventually to the formation of mercapturic acid conjugates (Boyland et al., 1963) In all species except dogs, aniline is ~acetylated with the subsequent formation of a second series of metabolites containing the acetyl group (Williams, 1959~. Since deacetylation also occurs, these acetyl metabol ites are usually present in small quantities in ur ine . N-oxidation of the acetamide could result in the formation of an hydroxamic ac id, N-hydroxyacetan il ice, but evidence for its actual occurrence in tissues and urine is lacking. ~Hydroxylation forms N-acetyl-E~aminophenol (Wi 11 lams, 1959 ~ . Not all of these metabolites occur in all species, and the relative amounts formed vary considerably among species {Conney and Levin, 19741. However, it appears that ~aminophenol is excreted in the urine of all species as a glucuronic acid or sulfate conjugate {Gut and Becker, 1975; Williams, 1959~. In rats, 42.3% of the administered dose was recovered as p~an~inopheno1 in urine after acid hydrolysis (Bus et al., 1978} Concentrations of ~-aminophenol in the ur ine of workers has been measured as a rough means of estimating occupational exposure . An average of 39.44 mg/1 was associated with the occurrence of significant methemaglobinemia 139
(Pacseri, 1961). Phenylbydroxylamine is apparently also an important metabolite in mat species including humans, but its presence seems to be limited to the blood where it reacts with hemoglobin to form methemogiobin, after which it is oxidized to nitrosobenzene. It has never been detected in the urine of animals given aniline {Riese, 1966 ) . Both N-hydroxylation and ring hydroxylation are carried out by the mixed-function oxidase system of the liver microsomes and are stimulated by pretreatment of rat wit h either phenobarbital or aniline itself and inhibited by SKF 5 25A (Boobis and Powis, 1975; Conney and Levin, 1974; Patterson and Roberts, 1971; Wisniewska-Knypl and Jablonska, 197S Wisniewska-Knypl et al., 1975;~ . Aniline is very rapidly metabolized in rabbits and mice; its metabolic half-life in these species is approximately 40 minutes. It is metabolized less rapidly in rats and still less rapidly in dogs (Conney and Levin, 1974) . Acu te Tox ic i ty Mechanism of Methemoglobin Induction. Because of the widespread use of aniline in industry and its high vapor pressure, the occurrence of methemoglobinemia in chemical workers is a rather common exper fence. The mechanism of the induction of methemoglobinemia by aniline has been widely stud led. The bulk of the ava ilable evidence ind icates that aniline itself is not directly responsible for the induction of methemoglobinemia , but its metabolite, phenylhydroxylemine, is 140
responsible for this effect (Kiese, 1966; Lin and Wu, 1973; McLean et al., 1969 ; ~ . file phenylhydroxylamine, in the presence of oxygen, reacts with hemoglobin forming methe~globin and nitrosobenzene. Nitrosobenzene is in turn reduced by the diaphorase n loot inamide-aden ine d inucleotide pbospha te INADP ~ -aethemoglobin reductase ~ in the presence of NADP} back to phenylhydroxylamine, wh ich can in turn oxidize another molecule of hemoglobin . Th is cyclic reaction goes on until as many as 50 net of methemoglobin are produced from a single millimole of hydroxylamine {Kiese, 1966~. It appears that a small amount of nitrosobenzene is reduced all the way to the amine, and the reaction is then terminated. In addition, nitrosobenzene appears to be inactivated by glutathione, which is present in red blood cells (Aikawa et al., 1978; Eyer,1979; ~ . NADP-methemoglobin reductase is the enzyme ma inly responsible for the physiologic reduction of methemoglobin to hemoglobin. However, NADP-methemogiobin reductase teas a greater affinity for nitrosobenzene than for me/hemoglobin. This affinity inhibits the reconversion of methemaglobin back to hemoglobin a. long as the nitroso compound is present. Sensitivity to the induction of metbemoglobin by aromatic amines varies among species. On a mg/kg basis cats are the most sensitive, and humans are approximately 60% as sensitive; dogs are about 30% as sensitive, rats S%, and rabbits and monkeys seem to be quite resistant to aromatic-amine-induced methemaglobin (Hamblin, 1963~. Other evidence indicates that humans are roughly 70 tinges more 141
sensitive than rats to aniline itself (Jenkins et al., 1972} . Although it seems clear that phenylhydroxylamine is the primary metabolite of aniline responsible for methemoglobin induction, it is not necessarily the only one. For years, ~aminophenol wee regarded as the precursor of metheac~globin fornication, until it was found that pbenylhydroxylamine is approximately 20 times more potent. However, it is possible that ~-aminophenol, o-aminophenol, and even other metabolites of aniline may be involved (Smith et al., 1967) . E~Aminophenol also requires oxygen to oxidize hemoglobin to me/hemoglobin, resulting in the formation of the ~-quinoneimine, which may go back to ~-aminophenol in a manner analogous to the phenylhydroxylamine-nitrosobenzene cycle. However , only a few equivalents of methemoglobin are produced by one equivalent of Swami nophenol ~ Kiese, 196 6 ~ . Although mDP-methen~oglobin reductase is normally respons ible for the reduction of methemoglobin to hemoglobin, some individuals have a hereditary reduction or absence of this enzyme. Such individuals are hypersensitive to the induction of methemoglobinemia by nitrates. mis genetic conditon is due to an autosomal recessive allele and is manifested in homozygotes of both sexes (Goldstein et al. , 19681 . Such individuals are presumed to be more sensitive to aniline and related methen~globin inducers, but there is no conclusive evidence for this hypothesis at the present time. Although lack of the reductase may retard the conversion of methemoglobin back to hemoglobin, it may also prevent the reduction of the nitroso compounds back to the hydroxylamine (Radomski, 142
1979~. In contrast, the action of nitrite is a direct one, not involving HADP~ethe~globin reductase. Although generally regarded as a highly toxic compound for humans, the acute toxicity in laboratory animals is relatively low. me oral LD50 's in rats, mice, and cats are 440, 460, and 1,750 mg/kg, respectively. Dermal LD50 's are 1,400 eg/kg in rats and 1, 290 mg/kg in guinea pigs (National Institute for Occupational Safety and Health, 1977~. Fatal poisoning in bumans rarely occurs, even following severe exposure. me unfavorable reputation of . aniline as an intoxicant is undoubtedly due to the rapidity and eff iciency of its absorption through both the skin and the respiratory tract, resulting in the rapid induction of methemoglobinemia. This condition results in symptoms such as headache, nausea, and dizziness (Bamlin, 1963~. Indeed, four cases of methemoglobinemia, in which n~ethemoglobin values reached 17-26 9%, were reported from the wear ing of shoes dyed black with aniline (Ghir inghelli and Mol ins, 1951) . Methemoglobinenlia is a relatively benign and reversible condition, however, at least in normal individuals. Conversion of 75% of the hemoglobin in the body to met~her~lobin can occur without life-threatening results (Hamlin, 1963~. Experiments in dogs indicates that 4-aminobiphenyl is 10 to 20 times more potent than is aniline, even in the induction of aethe~globinemia . Experiments in cats have shown that ~nitro~obenzene is 50 to 80 times more potent than an il ine (Radomsk i, 1979 ~ . 143
Chron to Tox ic ity Carcinooenici~S: When Rehn f irst observed the induction of bladder cancer in chemical workers, he believed these tumors to be due to exposure to aniline and named them Aniline tumors.. mis unjustified misnomer persisted for many years until a careful epidemiologic investigation by Case et al. (1954) and Case and Pearson (1954 ~ convincingly attributed these tumors to 2-naphthylamine and to benzidine, rather than to aniline. Following these observations, aniline has been regarded as a noncarcinogenic substance. Unfortunately, aniline was never adequately tested in dogs, a test species often used for the evaluation of bladder carcinogens. In the only dog experiment reported in the literature, in which aniline was administered daily to three dogs for 4 years no tumors were observed (Gerhman et al. , 1948). Aniline, as the hydrochloride, was given to rats in drinking water in an amount calculated to provide a dose of 22 mg/day for 7SO days. One-half of the rats survived longer than 425 days. No tumors of the bladder, spleen, liver, or kidney were observed (Druckrey, 1950 ~ . Anil ine wan also tested both as the free base in lard (1 mg/mouse) or olive oil (8 x 5 mg/mouse) or as hydrochlor ide in water (13 x 4 mg/mouse). mese experiments indicated that 144
aniline was not tumorigenic, when injected subcutaneously into mice in tests ranging from 12 to 24 months (International Agency for Research on Cancer, 19741. A subacute pilot study in rats was conducted primarily to determine the maximally tolerated dose (MTD) forte chronic feeding experiment. In this experiment, concentrations of 30, 100, 300, and 1,000 mg/kg body weight per day were administered in the diet to male and female Fisher-344 rats. Only the 1,000 mg/kg dose was clearly toxic, causing death in many female rats. In addition, the investigator observed depression of body weight gain, pathology of the liver, kidney, and spleen, and elevated methenoglobin concent ra t ions (Gra 1 la, 19 7 7 ~ . Until recently, these inadequate experiments plus the publication of a series of epidemiologic studies that exonerated aniline as a bladder carcinogen have led to the belief that aniline is not a carcinogen. In 1978, the results of a carcinogenesis bioassay in rats and mice given aniline hydrochloride were released. The compound was fed in the diet of male and female rats and mice for 103 weeks at two concentrations: 0. 6% and 0. at for rats and 1. 2% and 0. 6% for mice. Male rats had a significant number of hemangiosarcomas of the spleen. A significant increase in the combined incidence of f ibrosarcomas and sarcomas of the spleen Sand other organs was also observed in rats of both sexes. There was no evidence of 145
aniline-induced carcinogenicity in mice of either sex (National Cancer Institute, 1978~. This carcinogenesis bioassay was conducted according to the usual National Cancer Institute (NCI) protocol, which utilizes an MTD and one-half MOD as the doses for the study. Mutagenicity The data from mutagenicity tests of aniline are summarized in Table 6-3 . An il ine and an il ine-der ivatives (hydroxylamine and the nitroso der ivati~res) did not induce mutations in the Salmonella test system in four out of f ice studies, and weakly mutagenic in one (Mitchell, 1978 ~ . However, an iline was, in the presence of the comutagens" norharman and barman (,B-carboline derivatives), 146
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strongly mutagenic in the Sel~nella strain TA 98 (Nagao et al., 1977~. Aniline caused no DNA strand breakage in mammalian cells in culture (gwenberg et al., 1976) . We results are discussed below. Bacterial Tests. In an extensive study of the mutagenicity of chemical carcinogens in the Salnonella/microsome mutagenicity test, McCann _ al. (1975) found aniline to be nonmutagenic in all four tester strains (TA 1535, TA 1537, TA 98, TA 100 ~ . Similarly, Heabt _ al. (1979), tenting aniline a- well as the hydroxylamine and nitroso~erivatives, and Garner and Nutman (1977) also obtained negative results for aniline in the Salmonella test. Bowever, Mitchell (1978 ~ obtained weakly positive data on aniline mutagenicity in the Salmonella test by using a liquid-medium method, which involves a liquid incubation of the tester strain, metabolic activation system (S-9 or microsomes) and the test compound before plating the mixture on the agar . Under these conditions, the highest rate of mutagenicity observed was twice the number of spontaneous Stations. me signif icance of there results is highly questionable : no data were provided on the purity of the compound; no dose-respon~e relationship was observed for the mutagenicity of aniline; the mutagenic effect was independent of metabolic activation and the possibility that the aniline was oxidized under the test conditions to form a mutagenic oxidation - coupling product has not been ruled out. The non~utagen ic 5-carboline der ivative norharman has been shown to enhance the mutagenic activity of benzo (alpyrene, 148
dimethylaminoazobenzene, and tryptophan pyrolysates on Salmonella typhimurium strain TA 98 (Sugimura et al., 1977~. me mutagenicity of aniline and o-toluidine we. f irat demonstrated in the Salmonella system in the presence of norharman and S-9 fraction from rat liver {Nageo et al., 1977). me mutagenicity of aniline followed a clear dose response pattern in strain TA 98, when coincuba ted with norharman {200 pa/plate). The metabolic changes (activation) of aniline and/or norharman were necessary to demonstrate the mutagenicity of aniline in the presence of norharman, because no mutagenicity was observed without the S-9 fraction (Nagao et al., 1977) . Mammal fan Cell . Swenberq et al . (1976} evaluated the capacity of - an il ine to induce DNA strand breaks using an _-'ritro/alkaline elusion assay using Chinese hamster V79 cells with and without a liver microsomal activation system. No detectable chromo~omal damage, measured as an increase in the rate of DNA elusion was observed with aniline. Te ra tooen ic i TV No data were available to evaluate the potential teratogenicity or reproduct ive toxic ity of anil ine . CONCLUSIONS . . . Until the recent publication of the results of the NCI bioassay of a n i 1 ine, an i 1 ine had been cons ide red nones rc inogen ic . Conce r n 149
about exposure to aromatic amines has always been focused on the induction of bladder cancer because this is the only form of cancer known to have been produced in humans by Come of these substances. There was no evidence of the induction of bladder tumors in either the rats or mice at the MTD in the NCT bioassay experiment. Whether the splen ic tumors observed in rats, but not in mice , at the large doses tested in this experiment indicate that aniline represents a carcinogenic threat to humans at a site other than the bladder cannot presently be ascertained. There is no evidence that primary splenic tumors result from the exposure of humans to aniline. On the other hand, there is evidence that a carcinogen may induce tumors in different tissues in different species, and this observation has been extended to the generalization that a substance inducing cancer in any tissue of one species may induce tumors in a dif ferent tissue in other species, including humans . Although this may be true as a generalization, there are undoubtedly exceptions since some types of tumors in test animals do not appear to be correlated with carcinogenic potential in humans. The present TLV for aniline in the United States is in line with that of most other countries, except the Soviet Union, which has lowered it to 0.1 mg/m3. Me basis for this action is unclear (Bardodej, 1975; Vasilenko, 1972; Winell, 1975) . The potential mutagenic activity of aniline has been evaluated extensively in the Salmonella test. The resulting data indicate 150
strongly the t anil ine alone is without mutagenic ef feet, although one study reports that aniline is weakly Mutagenic {Hitcbell, 1978~. However, in the presence of the ~co~autagen. norharman, aniline beckoned highly mutagenic in the Salmonella test. However. when an iline and norharman were given in the diet either alone or in combination to male Wistar rats there was no carcinogenic effect to the ur inary bladder or other organs which could be treatment related. Although this experiment we. terminated after only 80 weeks it does strongly suggest that norharman does not enhance aniline carcinogenicity as it does mutagenicity tHagiwara et al., 1980 ) . REC - MENDATI ONS Although considerable research has already been performed on aniline, gaps in our knowledge of its possible health effects continue to exist, and a compound of such industrial importance deserves to be more thoroughly studied. The carcinogenic effects (hemang iosarcomas of the spleen and sarcomas of the spleen and other organs) observed at the MTD in the nCI bioassay need to be stud fed further with another lifetime feeding study at 3 or 4 dose levels in a different strain of rat. This will greatly assist in interpreting the signif icance of the previously observed effects. A carcinogenicity study using Syrian golden hamsters may also be useful since these animals have been shown to develop bladder tumors after exposure to other aromatic amines. In addition, a long-term 151
(preferably 8 - 10 years), feeding study at the MTD should be conducted on a large number of dogs. Such an experiment will allay any suspicions concerning the possible role of aniline in the causation of burn bladder cancer. This test is desirable because the only test with dogs was conducted many years ago on a few anima Is for too short a tier - . Along with these studies, there should be further studies on the occurrence of netabolites of aniline in urine to explain the failure of this compound to induce bladder cancer in dogs ( if this fa ilure is confirmed) . Special attention should be pa id to N-hydroxylated urinary metabolites. In addition, studies on the potential for teratogenicity and reproductive toxicity need to be performed. Further epidemiologic investigations on workers exposed to aniline are also needed. Urine should be monitored and analyzed for aniline metabolites to confirm and quantitate exposures. 152
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