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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 200
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 202
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 203
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 204
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 205
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 206
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 207
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 208
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 209
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 210
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 211
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 212
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Page 215
Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Suggested Citation:"2,4-Diaminotoluene." National Research Council. 1981. Aromatic Amines: An Assessment of the Biological and Environmental Effects. Washington, DC: The National Academies Press. doi: 10.17226/664.
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Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Chapter 8 2,4-DIAMINOTOLUENE NH2 \ H3C of/ \ - NH2 ~ . 2,4-Diaminotoluene (toluene-2,4-diamine; 2,4-DT) is a colorless crystal that melts at 99°C. It is soluble in hot water. 2,4-DT synthesis takes place in three steps, beginning with the nitration of toluene in a mixture of nitric and sulfuric acids at 30°C to 70°C. The resultant mononitrotoluene mixture is then nitrated again in a somewhat stronger acid medium to a mixture of dinitrotoluene isomers, of which the largest fraction is 2,4-dinitrotoluene {75.89~. A number of processes can then be used to produce 2, 4-DT from 2,4-dinitrotoluene. All of these methods involve catalytic hydrogenation, followed by pur if ication, to remove unwanted isomers and byproducts, PRODUCTION Table 8-1 lists the current producers of 2,4-DT, their locations, and (where available) their estimated annual capacities (Chemical Economics Handbook, 1977-1978; Stanford Research Institute International 1979; United States International Trade Commission, 1979~. 198

TABLE 8-1 Producers of 2,4-DT Company and Plant location Air Products and Chemicals, Inc. Industr ial Gases Div. Middlesex, N . J. Pasadena, Tex. A1 1 fed Chemica 1 Corpora tion Specialty Chemicals Division Moundsville, W. Va. Amer lean Cyanamid Co. Organic Chemicals Division Bound Brook, N. J. BASE Wyandotte Corporation Polymers Group Urethane Division Geismar, La. E. I . du Pont de Nemours & Company, Inc. Elastomer Chemicals Department Deepwater, N. J. Hobay Chemical Corporation Estimated Annual Capacity as of January 1, 1979a mousends of metric tons (owned by Bayer AG, Federal Rep. of Germany} Polyurethane Division Cedar Bayou, Tex. New Martinsville, W. Va. Olin Corporation O1 ins Chemicals Group Ast~tabula, Ohio Brandenburg, Ky. Lake Charles, La. Rochester, N. Y. Rubicon Chemicals Inc. (jointly owned by Uniroyal, Tnc., and Imperial Chemical Industr ies Limited, United Kingdom) Geismar, La. NIP 57a 30 Not 36 25 47 36 11 NOR 36 NIP 15 Estimates of production capacities are based on an assumed 2,4- diaminotoluene capacity of O .8 mete ic tons for each aetr ic ton of eoluene diisocyanate capacity, except for Air Products and Cbemicals, Inc., which does not produce toluene diisocyanate. b Not repor ted . 199

Total U.S. production of 2,4-DT has declined in recent years (U. S. International Trade Cc~mmisaion, 1976-1978) as shown in Table 8-2 . USES 2,4-DT is used almost exclusively for the production of toluene d i isocyanate . Other V. S. . consumption is estimated to amount to less than 230 metr ic tons annually (Chemical Economics Mandbook, 197 7-197 8 ) . Mixtures of isomers of 2, 4-DT are normally used to produce toluene diisocyanate. The most important mixture contains 80% of the 2 ,4-isomer and 209 of the 2,6-isomer. Eight U.S. companies produced 284, 072 . 4 metr ic tons of the 80/20 toluene di isocyanate mixture in 1978 (International Trade Commission, 1978~. A mixture containing 65% 2 ,4-isomer and 351 2,6-isomer is also used in significant quantities. Only a small quantity of 2,4-DT is isolated for oonver s ion to pure 2, 4 -toluene di i~ocyanate . The Chemical Economics Handbook (1977-1978 ~ estimated the U. S . consumption pattern for toluene diisocyanate in 1978 as Down in Table 8-3. 2, 4-DT can be used to produce (approximately} 60 dyes, which are used to color silk, wool, paper, boat f ibers, cellulosic f ibers, and cotton. The following nine dyes are believed to have been produced from 2,4-DT in the United States during 1978: Basic Brown 4, Basic 200

Table 8-2 Annual Production of 2, 4-DTa . . Year 1976 1977 1978 Thousands of metr ic tons 105 e 9 lOle 1 63 e3 a Data from U. S. International Trade Commission, 1976-1978. 201

Table 8-3 U.S. Consumption of Toluene Di-isocyanate in 1978a Use ~ _ Flexible polyurethane foams Polyurethane surface coatings Polyurethane elastomers Other (including rigid polyurethane foam) Total Percent of Total 83% 6 3 8 100% a Data from Chemical Economics Handbook, 1977-1978. 202

Orange 1, Direct Brown 2, Direct Brown 154, Direct Black 4, Leuco Sulphur Orange 1, Leuco Sulphur Brown 10, Solvent Brown 12, and Sulphur Black (Colour Index, 1975; U.S. International Trade Commission, 1978) . They are used in spirit varnishes and wood stains as indicators, in the manufacture of pigments, and as biolog to s ta ins . 2,4-DT is used as a developer for direct dyes, particularly to obtain black, dark blue, and brown shades, and to obtain navy blue and black colors on leather. It is also used to dye furs and was an ingredient in hair dye formulations until banned in 1971 (International Agency for Research on Cancer, 19781. EXPOSURE More than 99% of the 2,4-DT produced in the United States is used to produce toluene di isocyanate, generally at the site of production. The s ingle exception is the Air Products and Chemicals Plant at Pasadena, Tex., which produces no toluene diisocyanate and conversely, a 45,000 metric ton/year capacity toluene diisocyanate plant at Freeport, Tex. operated by Dow Chemical, which does not produce its own 2,4-DT. Therefore, the main sources from which humans are exposed will be the plants listed in Table 8-1. However there is no information on which to base an analysis of either occupational or general exposures from plant emissions. Because the substance is solid at ambient temperatures, air emissions from 2,4-DT production do not appear to be significant. 203

However, contamination of wastewater discharges from the plants is a likely route of exposure. The hydrogenation of dinitrotoluene yields 4 mol of water for every mat of 2 ,4-DT produced (600 1 of water per metric ton of 2,4-DT) . This water is separated in a dehydration column at 100°C-150°C at atmospheric pressure (Milligan and Gilbert, 1978~. Because the vapor pressure of 2,4-DT is 11 mm Hg at 150°C and it is soluble in hot water, the separated water is undoubtedly highly contaminated. This wastewater is probably treated before it is discharged. Some 2 ,4-DT could find its way into consumer products as an impurity in dyes. However, such exposure is nearly impossible to quantify. Under the U. S . Food and Drug Administration (FDA) regulation concerning 2,4-DT, (21 CFR 177) is listed under the category of ant ioxidants and ant iozonants . The total of these components is not to exceed S ~ by we ight of rubber product. The U.S. Occupational Safety and Health Administration does not have an occupational standard covering the exposure of workers to 2,4-Dr. 2()4

ANALYTIC METHODS Jones et al. (1978) reported separations of nondye components in the commercial food color preparation Brown FK. The dye, which is manufactured by the reaction of diazotized sulfanilic acid with a mixture of m-phenylenediamine and 2,4-diaminotoluene, has been shown to consist of six major colored components. High-pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) procedures were evaluated for their ability to separate a mixture of the six dye components as well as the starting compounds (e.g., 2,4-DT) used in the synthesis. Two different columns and solvent systems were used in the HPLC investigations with a W absorption detector set at 254 nm. One of the systems, which consisted of a column of Partisil 5 loaded with 7% aminopropyl phase, was subjected to a 30-minute linear gradient from acetonitrile-water (2:3) to acetonitrile-water (2:3 containing sodium biphosphate (2 g/1~. Only partial resolution of the components was obtained. The other HPLC system, which consisted of a column of Chromosorb Si 100 loaded with a 21% octadecyl phase subjected to a 17-minute linear gradient from 5% to 40% acetonitrile in water (containing 1.2 and 2.4 g/1 of sodium biphosphate and sodium hypophosphate, respectively} was more successful: all nine components of the mixture were separated with essentially baseline resolution. Unger and Friedman (1979) developed an HPLC procedure to assay 2,6- and 2,4-DT. Their technique was adaptable to biomonitoring and metabolic studies with samples of urine and plasma from rats. A normal-phase silica column was used with a mobile phase consisting 205

of acetonitrile-water saturated chloroform (8:2) and a 250 nm W absorption detector. The two compounds were resolved as 'sharp peaks in 3 minutes, and from 1 ng to 2 ng of each substance was quantitatede Dichloromethane extraction of urine and plasma "piked with 2,4-DT (10 to 200 ppm), yielded recoveries of about 90% or more. TEC tests were performed by using Inactivated silica gel G plates (layer thickness 0.25 mm) with a solvent system of phenol~ater (4 :1) . The starting materials of the manufacturing process {e . g ., 2 , 4-DT) were detected by spraying with 19 Ehrlich 's reagent in 50% acetic acid. Although the TLC system resolved for the six dye components, it was not suitable for the other compounds since sulfanilic acid was not resolved from one of the dye components. The two aromatic amines (2,6- and 2,4-DT) were not well resolved and ran close to the solvent front. Spectrophotofluor imetry {SPF} was the basis for a method described by Guthrie and Mckinney (1977) to analyze 2,4- and 2,6-DT in flexible polyurethane foams at levels as low as 1 ppm. me amines were extracted wi th methanol, separated by TLC, and assayed by SPF after reaction with Fluram reagent. The SPF assay was accomplished by uniformly spraying the developed and dried TLC plate with an 0.015% solution of Fluram in acetone and measuring the fluorescent spots (NEX = 390, XEm = 500 nm) with a "bin-film chromatographic scanner. The instability of the Fluram derivative required that quantitative measurements be completed within 1 hour. The nonuniform cbaracteristic of foams was determined by assaying sample extracts in duplicate at the 10 to 15 ppm level. Precision, 206

usually within +10% for a given extract, was +30% for six different samples of tbe same foam. Lepri et al. (1976} investigated the behavior of several coloring agents (including 2,4-DT} used in oxidation-type hair dyes on thin layers of various ion exchangers. Some of the systems provided un ique separations. Examples of such systems are AG 1-X4 (CH3co2-) developed with 0.1 mol acetate buffer in a 4:1 water-methanol mixture; BD-cellulose developed with 0.5 mol acetate buffer solution; Dowex 50-X4 (H+) with O .1 mo1 acetate buffer in a 1:1 mixture of water-methanol; Rexyn 102 (H+) developed with various mixtures of dimethy~formamide-water; and AG 3-X4A developed with 95% ethyl alcohol. Rf values were tabulated for some IS compounds . Two direct gas chromatography {GC) procedures for separation and analysis of isomeric diaminotoluenes were reported in 1968. Willeboordse _ al. (1968} separated mixtures of 2,3-, 3,4-, 2,4-, 2,5-, and 2,6-DT using a mixed partitioning agent of Carbowax 20M and Saponate DS-10 on base-loaded Chromosorb G. followed by Saponate DS-10 on the same solid support. Boufford (1968) separated a mixture of 3,4-, 2,3-, 2,4-, 2,5-, and 2,6-DT on a column of 5% Bentone 34 plus 155 Hyprose SP-80 [octakis(2-hydroxypropyl)-sucrose] on potassium-hydroxide-treated Chromosorb W at 170°C. Helium carrier gas and a hydrogen flame ionization detector (FID) were used. The analysis of 2,4-DT was specifically mentioned in several of the procedures described for primary aromatic amines. Additional 207

discussions in the literature (details unavailable) involve in situ TLC determinations of toluenediamine and methylenedianiline isomers in the products of hydrolytic degradation of polyurethanes (Lesiak and Orl ikowska, 1978 ~ and an ultrasonic method to assay amine ! solutions, including toluenediamine (Bogdanova et al., 1976) 4 208 .

HEALTH EFFECTS Metabol ism 2,4-DT is rapidly absorbed after intraperitoneal in Section into rats and mice, and peak levels in blood or serum are attained within 1 hour after in jection (Grantham et al., 1979) . Less than 1.5% of the dose is excreted unchanged. Metabol ites are rapidly excreted, predominantly in ur ine (Grantham et al., 1979; War ing and Pheasant 1976 ~ . Unger et al . (1980 ~ conf irmed these observations in male B6C3F mice administered a single intraperitoneal dose of 2, 4- ~ 14C] DO . They noted rapid absorption from the peritoneal cavity with the dominant route of excretion via the kidneys; one hour af ter dosing almost 50% of the radioactivity was recovered in the urine. They also noted that the three tissues which have shown a carcinogenic response to 2,4-DT (hepatocarcinoma, rhabdomyosarcoma, and f ibrosarcoma), either acquired an initial high concentration of 2 ,4-DT (liver) or demonstrated an extended elimination half-life (muscle and skin). Major metabolic reactions include acetylation of one or both amino groups oxidation of the methyl group to the benzylic alcohol and benzoic acid functions and ring hydroxylation, primarily at the 3 and 5 pos itions. Glinsukon et al. (1975 ~ examined the abil ity of liver cytosol from var ious species to N-acetylate 2 ,4-DT. The liver cytosol f rom hamsters had the most enzymat ic activity followed by guinea-pig, rabbit, mouse and rat. Human liver cytosol formed only trace amounts of acetyl derivatives while dog liver cytosol had no 209

activity. Although the liver cytosol showed the greatest acetylating activity, the cytosol from the kidney, intestinal mucosa, and lung also were able to produce significant amounts of various acetylaminotoluenes. In this study 4-acetylamino-2-aminotoluene was the major metabolite and 2,4-diacetylaminotoluene the minor metabolite. Glucuronide and sulfate conjugation of these primary metabolize also occurs, varying with the species examined. No information is available on the mechanism of metabolic activation of 2,4-DT. Acute Toxicity National Institute of Occupational Safety and Health (1979 } reported that the oral LDLo (lowest published lethal dose) of 2,4-DT in rats was 500 mg/kg, and that the LDLo's by subcutaneous in Section to rats, dogs, and rabbits were 50, 200, and 400 mg/kg, respectively. More recent data on the acute toxicity of 2,4-DT after intraperitoneal injection in to male Fischer rats and female NTH Swiss strain mice indicate LD50 values of 32S mg/kg and 480 mg/kg, respectively (Grantham et al., 1979~. Gosselin et al. (1976) reported that no poisonings of humans by 2,4-DT were known. Nonetheless, they rated the compound as very toxic, reporting a probable oral LD~ in humans of 50-500 mg/kg. On the other hand, National Institute for Occupational Safety and Health (1979) lists the oral LDLo of 2,4-DT in humans as 50 mg/kg. 210

Teratogenicity No data are available on the embryotoxicity or teratogenicity of 2,4-DT. Care inogen to ity Subcutaneous Administration. Twenty rats (mixed strains and sex) were given 0.5 ml of 0.49 solution of 2,4-DT in propylene glycol subcutaneously at weekly intervals for approximately 8 months (Vmeda, 1955) . The survival rate at 8 months was only 451. All ra ts surviving 8 months developed subcutaneous sarcomas. The total dosage of 2,4-D07 ranged from 60-90 me per rat. No conte Honorary controls were used in this exper iment. Oral Administration. Ito et al. {1969) first reported the .. . carcinogenicity of 2,4-DT after oral administration. Hey observed carcinoma of the liver in male Wistar rats fed diets containing 600 or 1,000 ppm 2,4-DT for 33 to 34 weeks e Tumor incidences at 36 weeks were 9/9 (1001) after a dosage of 1,000 ppm 2,4-DT and 7/11 (64%) after a dosage of 600 ppm. Histologically, all the liver tumors were hepatocellular carcinomas. Many of the rats also had multiple metastatic tumors in lymph nodes, omentum, lungs, and epididymis. The livers of six control rats fed the basal diet without 2,4-DT were essentially normal. Weisburger et al. (1918) confirmed the hepatocarcinogenicity of 2,4-DT (as the dihydrochloride) after oral administration in CD~1 {Sprague-Dawley) rats and in HaM/lCR mice. 211

More recently, the National Cancer Institute (1979 ~ selected 2,4-DT for additional bioassay in the Carcinogenesis Testing Program. Groups of 50 F344 rats of each sex received 2,4-DT in feed at two dose levels. Time-weighted average doses were 79 ppm for 103 weeks (low dose) and 171 to 176 ppm for 79 to 84 Weeks (high dose). The incidence. of hepatocellular carcinomas or neoplastic nodules were dose related (males: controls 0/20, low-dose 5/49, bigh-dose 10/50; females: controls 0/20, low-dose 0/50, high-dose 6/49~. In addition, carcinomas or adenomas of the mammary gland occurred in female rats (controls 1/20, low-dose 38/50, high-dose 41/50~. The most common type of mammary tumor by far was the fibroadenoma. Other types of tumors appeared less frequently than did liver or mammary tumors, but were assumed to be related to exposure to 2,4-DT. These included lung tumors, squamous cell carcinoma of the skin and preputial gland, pancreatic acinar cell adenomas, subcutaneous f ibromas and f ibrosarcomas, and mesotheliomas. In parallel studies, groups of 50 B6C3F1 mice of each sex rece ived 2, 4-DT in feed at two dose levels, either 100 ppm or 200 ppm, for 101 weeks. Hepatocellular carcinomas occurred in female mice (control 0/19, low dose 13/47, high~ose 18/46). In addition, lympbomas occurred at a significant incidence in the low-dose female mice (controls 2/10, low~ose 29/47, high~ose 11/46) . No tumors occurred at significantly increased incidences in the dosed male mice. Skin Application. Two studies (Burnett et al., 1975; Giles et _., 1976) involving the skin painting of mice with hair dye 212

formulations that included 2, 4-DT have been performed. In one study (Giles et al., 1976), mice were given weekly topical applications of 0.05 ml of either a 69 solution of 2,4-DT alone or hair dye formulations containing 2,4-DT. me results of this study were judged inadequate (International Agency for Reserarch on Cancer, 1978) for an evaluation of the carcinogenicity of 2,4-DT because a large number of animals were unaccounted for in the final analysis of tumor incidence. The second study (Burnett et al., 1975) involved the painting of 0.05 ml of a hair dye formulation containing 0.2% of 2,4-DT on the skin of Swiss-Webster mice weekly or fortnightly for 18 months. Carcinogenicity of 2,4-DT could not be evaluated on the basis of this study because of the complexity of the applied mixture and because of the reported high incidence of tumors observed in control mice (International Agency for Research on Cancer, 1978~. Mutagenicity me data from mutagenicity and other genotoxic tests of 2,4-DT are summarized in Table 8-4. The substance was mutagenic in both the Salmonella and Escher ichia cold systems (Ames et al ., 1975 ; Aune et al., 1979; Pienta et al., 1917; Venitt, 1978) and in Drosophila melanogaster (Bli Eleven, 1977 ; Fahmy and Fahmy, 19771 . Negative results were obtained in Neurospora crassa tong, 1978) and in the micronucleus test in the rat tHossack and Richardson, 1977~. Potentially genotoxic effects (chromatic and chromosomal gaps and breaks in peripheral lymphocytes) from hair dyes which may have contained 2,4-DT, among other ingredients, were observed in professional hair colorists and others with a history of hair dye use (Kirkland _ al., 1978~. 213

TABLE 8-4 Mutagenicity Tests of 2,4-DT Observations Species/Strain Results Reference Gene Mutation Bacterial Salmonella, TA 1538, TA 98 pOsa bases et al. 1975 B. colt, 343/113 (erg+ reversion) polka Venitt, 1978 Yeast N. crassa, ad-3A ne ~ Ong, 1978 Insect D. aelanogaster, Oregon X, Berlin K pOnc Fahmy and Fahmy, 1977 Blijleven, 1977 Chromosome Damage Micronucleus test in rat Chromosome damage humans a Mutagenic in the presence of S-9 (metabolic activation) b Tested without metabolic activation. c X-Chromosome recessive (lethals and visibles). d Chromatid and chromosome gaps and breaks. 214 neg Hossack and Richardson, 1977 pomp neg Kirkland et al., 1978 .

Bacter ial Tests. The studies of Ames et al. (19751, Aune et al . _ _ _ (1979), and Pienta en al. (1977) clearly demonstrate that 2,4-DT is ~ potent mutagen in the Salmonella system. Like many other aromas ic amines, 2,4-DT requires metabolic activation {S-9 or microsomal fractions) for mutagenesis. Principally, it causes a frameshift type of change in strains TA 1538 and TA 98. Pienta et al. (1977) reported a correlation between the 2,4-DT-induced morphologic transformation in an In vitro carcinogenesis system (using secondary culture target cells prepared from cryopreserved, primary Syrian hamster embryo cells) and the mutagenicity of 2,4-DT in the Salmonella system. Venitt (1978) reported mutagenicity of 2,4-DT in the E. cold system. - Yeast. Ong (1978) used the adenine-3 (ad-3) forward-mutation system of N. crassia to test the mutagenicity of environmental agents and chemical carcinogens, including 2,4-DT. 2,4-DT was nonmutagenic in the N. crassa system; however, since no mammalian metabolic activation system was included in the test, these data must be regarded as inconclus ive . Insect. Fahmy and Fahmy (1977) examined the comparative mutagenic effect of 2,4-DT as well as that of phenylenediamine and benzidine in D. melanogaster. The compounds were injected at equ imolar dose range (5-20 - ) into ache abdomens of adult males, and mutagenicy was measured separately on the various stages of spermatogenes is. Germ. ~ c activity was assayed with respect to the ove ral 1 induction of the X-chromosome recessive ~ lethals 215

and visibles) mutations relative to the specific effects on ribosomal DNA (bobbed locus). All the compounds exerted mutagenicity, both on the X-chromosome and on the RNA genes, but activity on the different genie sites varied between compounds and as a function of cell stage. me mutagenic potency toward the bobbed locus was benzidine.2 ,4-DT~ 4-N-~phenylenediamine, which correlates with the carcinogenic potency of these compounds. 81ij~even (1977) observed similar mutagenicity by feeding 2,4-DT to male _. melanogaster and measuring sex-linked recessive lethal mutations. Metabolically active germ cells (spermatids and spermatocytes) bad the highest mutagenic activity. Chromosomal Damage. Micronucleus Test. Mossack and Richardson t1977) obtained negative results in micronucleus tests of 2,4-DT and 11 other hair dye constituents. Groups of five male and five female rats were gastricly intubated with the test compounds at 0.5. (w/~) in gum tragacanth containing 0.05% sodium sulpfite. The total dosages were close to the lethal doses and were administered in two equal parts, separated by an interval of 24 hours. The animals were killed 6 hours after the second dose and bone-marrow smears were prepared. me incidence of micronucleated cells per 2,000 polychromatic erythrocytes was compared with the values from the control group. me mean values and ranges of micronucleated cells were not 216

significantly different for the control and 2, 4--treated groups. Similar results were obtained for the other compounds. Chromosomal Damage in Humans . No data are available on the possible genotoxic effects of 2,4-DT Eer se in humans. However, in a recent epidemiologic study of the potentially genotoxic effects of hair dyes (Kirkland et al., 1978), chromosomal damage was investigated in peripheral-blood lymphocytes of professional hair colorists. The authors found no significant differences in chromosomal damage in cultured per ipheral-blood lymphocytes from 60 professional ha ir colorists as compared with those of 36 control subj ects closely ma tched for age and sex. There was a statistically signif icant excess of chromosomal damage (mainly chromatic breaks ~ in women wi th dyed ha ir when age-matched women were regrouped according to whether their hair was dyed or not. Men (mean age 22 .9 years) with dyed hair had signif icantly less cheomosomal damage than did men (mean age 31.5 years) whose hair was not dyed. Possible confounding factors in these findings are that most tinters wear gloves when applying hair dyes and, even without gloves, percutaneous absorption of ha ir dye constituents may be effectively impeded by the horny surface of the bands and by the lack of sebaceous glands in the palms ; ha ir dye constituents are readily absorbed through the scalp, which contains numerous sebaceous glands; and the lower frequency of chromosomal aberrations in young men with dyed hair (compared with the frequency in slightly older men without dyed hair) is 217

probably due to an age effect. This preliminary evidence on the genotoxic effects of hair dyes in humans warrants further study in view of the known mutagenicity and carcinogenicity in animals of several hair dye constituents. C NCLUSIONS 2,4-DT is carcinogenic in rats and mice after oral administration; the produces amine produces liver and mammary gland tumors. There is some evidence that it also induces sarcomas at the site of subcutaneous injection in rats. Published data on the carcinogenicity of 2,4-DT after application to mouse skin are not adequate for evaluation. 2,4-DT is a potent mutagen in microbial test systems and causes germ cell mutation in D. melanogaster. The substance may cause chromosome! damage to (chromatic and chromosome gaps and breaks) in humans. It also induces cell transformation in an _ vitro mammalian carcinogenesis test system. However, there are no data, such as case reports or epidemiologic studies, on the carcinogenicity of 2,4-DT in humans. RECOMMENDATIONS There is absolutely no information available on the 218

mechanism by which 2,4-DT is activated in susceptible species, including rats and mice. However , given the demonstration of carcinogenicity in two species of animals and the data on the genotoxic effects in In vitro systems, it is prudent to assume that humans are under some increased rink from exposure to 2, 4-DT. Recommendations for future research with 2,4-DT include studies of the mechanism by which the chemical is activated in rats, mice and humans, testing for carcinogenicity in additional species to obtain more data on the relationship between metabolism and carcinogenicity, and examination of the in vitro metabolism of 2,4-DT in human tissues to supplement the preliminary observations of Glinsukon et al. {19757. This kind of additional data would make it easier to estimate the extent of risk to humans exposed to 2,4-DT 219 .

REFERENCES Production, Uses, Exposure Chemical Economics Handbook. 1980. Stanford Research Institute International, Menlo Park, Calif . Code of Federal Regulations. food and additives: Polymers. 1980. Title 21, Part 177. Indirect Office of the Federal Register, National Archives and Records Service, General Services Administration, Washington, D.C. Colour Index. 1971. Third edition, volume 4. m e Society of Dyers and Colourists, Bradford, Yorkshire. International Agency for Research on Cancer. 1978. 2,4-Diaminotoluene. Pp. 83-95 in IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Volume 16. International Agency for Research on Cancer, Lyon. Milligan, B., and K.E. Gilbert. 1978. ~nines, aromatic--Diaminotoluenes. Pp. 321-329 in Kirk-Othmer Encyclopedia of Chemical Technology, Third edition, volume 2. John Wiley & Sons, New York . SRI International. 1979. 1979 Directory of Chemical Producers: United States of America. Stanford Research Institute International, Menlo Park, Calif. 1122 p. 220

V. S. International Trade Commission. 1977. Synthetic Organic Chemicals . Un ited States Production and Sales, 1976 . USED Publication 833. U.S. Government Printing Office, Washington, D.C. 357 pp. UPS. International Trade Commission. 1978. Synthetic Organic Chemicals. United States Production and Sales, 1977. USTTC Publication 920. U.S. Government Printing Office, Washington, r).c. 417 pp. U. S. International Trade Commission . 1979. Synthetic Organic Chemicals. United States Production and Sales, 1978. USITC Publication 1001. U.S. Government Printing Office, Washington, D.C. 369 pp. 221

Analytic Methods Bc~danova, T.M., P.V. Mulyanov, N.M. Monabarzh, and M.L. Piroshkova. 1976. Ultrasonic method for analysis of amine solutions. Zavod. Lab. 42:1486-1487. [Chem. Abs. 86:199421s, 1977.] Boufford, C.E. 1968. Determination of isomeric diaminotoluenes by direct gas-liquid chromatography. J. Gas. Chromatogr. 6:438-440. Guthrie, J.L., and R.W. McKinney. 1977. Determination of 2,4- and 2, 6-diaminotoluene in flexible urethane foams. Anal. Chem. 49: 1676-1680 . Jones, A.D., D. Hoar, and S.G. Sellings. 1978. Separation of non-dye components of Brown FK by high-performance liquid chromatography. J. Chromatogr. 166:619-622. Lepri, L., P.G. Desideri, and V. Coas. 1976. Separation and identification of colouring agents in the oxidation-type hair dyes by ion exchange thin-layer chromatography. Ann. Chim. (Rome) 66:451-460. Lesiak, T., and H. Orlikowska. 1978. ~In situ~ determination of toluenediamine and methylenedianiline isomers in the products of hydrolyltic degradation of polyurethanes after separation by thin-layer chromatography. Chem. Anal. (Warsaw) 23:469-475. [Chem. AbS. 89: 216094u, 1978.] 222

Anger, P.D., and M.A. Friedman. 1979. High-performance liquid chromatography of 2,6- and 2,4-diaminotoluene and its application to the determination of 2,4-diaminotoluene in urine and plasma. J. Chromatogr. 174:379-384. Willeboordse, F., Q. Quick, and E.T. Bishop. 1968. Direct gas chromatographic analysis of i~omeric diaminotoluenes. Anal. Chem. 40:1455-1458. 223

Health Effects Ames, B.N., H.O. Kammen, and E. Yamasaki. 1975. Hair dye. are mutagenic: ingredients. Identification of a variety of mutagenic Proc. Natl. Acad. Sci. U.S.A. 7~:2423-2427. Aune, T., S.D. Nelson, and E. Dybing. 1979. Mutagenicity and irreversible binding of the hepatocarcinogen 2,4-diaminotoluene. Chem. Biol. Interact. 25:23-33. Blij~even, W.G.H. 1977. Mutagenicity of four hair dyes in Drosophila melanogaster. Mutat. Res. 48:181-186. Burnett, C., B. Lanman, R. Giovacchini, G. Walcott, R. Scala, and H. Keplinger. Oxidation of ha ir dyes. Fahmy, M.J., and O.G. Fahmy. 1975. Long-term toxicity studies on Food Cosmet. Toxicol. 13: 353-357 . 1977. Mutagenicity of hair dye components relative to the carcinogen benzidine in Drosophila melanogaster. Mutat. Res. 56: 31-38. Giles, A.L., Jr., C.W. Chung, and C. Kommineni. 1976. Dermal carcinogenicity study by mouse-skin painting with 2,4-toluenediamine alone or in representative hair dye formulations. J. Toxicol. Environ. Health 1:433-440. 224

Glinsukon, T., T. Benjamin, P. Granthem, E. Weiaburger, and Poller. 1975. Enzy~na tic N-acetylation of 2,4-toluenediamine by liver cytosole from various species. Xenobiotica 5 (87: 475-483. Gosselin, R.E., H.C. Hodge, R.P. Smith, and M.N. Gleason. 1976. Section II, p. 141 in Clinical Toxicology of Commercial Products: Acute Poisoning. E our th ed. Williams and Wilkins, Baltimore. Grantham, P.H., L. Mohan, T. Ben jamin, P.P. Poller, J.R. Miller, and E.K. Weisburger. 1979. Comparison of the metabolism of 2,4-toluenediamine in rats and mice. J. Environ . Pathol. Toxicol. 3 :149-166. Hossack, D.J.N., and J.C. Richardson. 1977. Examination of the potential mutagenicity of hair dye constituents using the micronucleus test. Exper ientia 33:377-378. International Agency for Research on Cancer. 1978. 2,4-Diaminotoluene. Pp. 83-95 in lARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Volume 16. International Agency for Research on Cancer, Lyon. Ito, N., Y. Hiasa, Y. Konishi, and M. Marugami. 1969. The development of carcinoma in liver of rats treated with m-toluylenediamine and the synergistic and antagonistic effects with other chemicals. Cancer Res. 29 :1137-1145. 225

Kirkland, D.J., S.D. Lawler, and S. Venitt. 1978. Chromosomal damage and hair dyes. Lancet 2:124-128. National Cancer Institute. 1979. Bioassay of 2,4-Diaminotoluene for Possible Carcinogenicity. CAS No. 95-80-7. ITS Carcinogenesis Technical Report Series No. 162. DHEW Publication No. (NIB) 79-1718. U.S. Dept. of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, Md. 122 pp. National Institute for Occupational Safety and Health. 1978. Pp. 1233 in Registry of Toxic Effects of Chemical Substances, 1978, Lewis, R.J., Sr., ed. DHEW (NIOSH) Publication No. 79-100. U.S. Department of Health, Education, and Welfare, Public Health Service, Center for Disease Control, National Institute for Occupational Safety and Health, Cincinnati, Ohio. Ong, T. 1978. Use of the spot, plate and suspension test systems for the detection of the mutagenicity of environmental agents and chemical carcinogens in Neurospora crassa. Mutat. Res. 53:297-308. Pienta, R.J., M.J. Shah, W.B. Lebherz III, and A.W. Andrews. 1977. Correlation of bacterial mutagenicity and hamster cell transformation with tumorigenicity induced by 2,4-toluenediamine. Cancer Lett. 3:45-52. 226

thneda, M. 1955. Production of rat sarcoma by injections of propylene glycol solution of m-toloylenediamine. Gann. 46: 597-604 . Unger, P.D., A.Je Salerno, W.C. Hess, and M.A. Friedman. 1980 . Tissue distr ibution and excretion of 2, 4- ~ 14C] Toluenediamine in the mouse. J. Box. Environ. Health. 6: 107-114 . venitt, S. 1978. Mutagenicity of hair dyes: Some more evidence and the problems of its interpretation. Mutat. Res. 53: 278-279 . (Abstract No. 214 ~ . Waring, R.H., and A.E. Pheasant. 1976. Some phenolic metabolites of 2, 4-diam~notoluene in the rabbit, rat and guinea-pig . Xenobiotica 6: 257-262. Weisburger , E.K., A.B. Russf ield, F. Hombur'3er , J.H. Weisburger, E. Boger, C.G. Van Dongen, and K.C. Chu. 1978. Testing of twenty-one environmental aromatic amines or der i~ratives for long-term toxicity or carcinogen icity. J. Environ . Pathol . 0~xicol. 2: 3 25-356 . 227

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