4-methyl-m-phenylenediamine

Endpoint summary

Additional information

Absorption:

A good absorption of 2,4-TDA via the gastro-intestinal tract of animals can be assumed according to the quantity of excretion via the urine and the faeces in animal experiments (Sauerhoff et al. 1977; Timchalk et al. 1994).

No data are available concerning absorption via the respiratory tract.

 

Dermal penetration:

2,4-TDA is well absorbed via the skin. The dermal absorption rate of [¹⁴C]2,4-TDA*2HCl (4 μg/cm² in acetone) in human skin was determined to be 24% of the dose during a 24 h period, compared to 54% in monkeys. Since in this study no tissue levels were determined in order to recover the total radioactivity applied, the dermal absorption rate was extrapolated by the amount of radiotracer (¹⁴C) found in the urine over a 5-day period. This amount was 23.5% in human and 43% in monkeys, respectively (Marzulli et al., 1981).

 

Distribution:

In rats the highest tissue concentrations were measured in liver and kidney after oral (DuPont, 1974) or intraperitoneal administration (Grantham et al., 1979) of [¹⁴C]2,4-TDA. Concentrations in heart, lungs, spleen, and testes were significantly lower. Following i.p. injection in rats, the concentration in blood was maximal after 1 to 8 h. It decreased within 6 to 24 h to a low residual level (Grantham et al., 1979). The peak blood levels after oral dosing were reached within 2 hours (DuPont, 1974).

No species-related differences in tissue distribution in mice and rats were observed (Unger et al., 1980; Unger et al., 1978; Grantham et al., 1979), though biphasic tissue elimination kinetics were observed (Sauerhoff et al., 1977; Unger et al., 1980; Unger et al.,1978). The half-lives of elimination for the liver, kidney and blood ranged between 0.43-1.51 h in the fast phase (up to 6h following dosage) and 9.1-12.6 h in the slow phase.

 

Elimination:

The excretion of the 2,4-TDA metabolites predominantly occurs via urine in rats and mice (Grantham et al., 1979; Timchalk et al., 1994; Sauerhoff et al., 1977).

Similar excretion patterns were observed in mice (Unger, 1978) and rats (Grantham et al., 1979) following i.p. administration of radiolabelled 2,4-TDA. Fast urinary excretion of more than 50% of the applied dose occurred within the first hours after application. Then the predominant route shifts to fecal elimination, most likely through bilary excretion. 24 hours after i.p. administration the male rats had excreted 73% of the [¹⁴C]-2,4-TDA dose in the urine, but during the succeeding four days, only an additional 3.4% was excreted in the urine. Fecal excretion accounted for 8% of the dose by 24 hr, increasing to 22% by five days (Grantham et al., 1979). 

The renal elimination after i.p. injection in mice is more rapid and more complete than in rats and is essentially completed in both species after 48 h. Rats excrete up to 74% of the activity via urine within 48 h whereas 92% of the dose was excreted in mice after 48 h (Grantham et al., 1979).

Additionally biphasic clearance kinetics of [¹⁴C]2,4-TDA-derived radioactivity from the plasma were described, with peak levels observed one hour after dosing, followed by rapid clearance during the next six hours, and more gradual clearance thereafter (Unger, 1978; Grantham et al., 1979). 

After a single oral dose (0.5, 50 or 60 mg [¹⁴C]-TDA/kg bw) the rat eliminated 60 to 65% of the dose within 48 hours in the urine (Sauerhoff et al., 1977; Timchalk et al., 1994).

After a single i.v. dose (3 or 50mg/kg bw) 72% of the applied activity were eliminated in the urine within 48h following application (Timchalk et al., 1994; Jeffcoat et al., 1988).

The excretion of the activity in the feces of rats after i.v. or i.p. injection was 20 - 30% after 2 to 5 days. Since these studies show very similar fecal excretion rates it can be assumed that the absorption of 2,4-TDA in rats after oral dosing is almost complete and the fecal elimination of orally administered 2,4-TDA occurs via bile. Within 2 days mice excreted only 3% of the total radioactivity in the feces after i.p. injection (Grantham et al., 1979).

A study in rats with oral administration or i.v. injection of 3 mg/kg bw resulted in an elimination half-life time in urine of 4.6 h. An oral dose of 60 mg/kg bw increased the elimination half-life to 8 hours (Timchalk et al., 1994).

 

 

Metabolism

 

In rats, rabbits, and guinea pigs only small amounts of unchanged 2,4-TDA were detected in 24- or 12 h-urine samples following oral, i.v. or i.p. application (Timchalk et al.,1994; Waring & Pheasant, 1976; Jeffcoat, 1988). After oral doses of 50 or 60 mg/kg, only traces of 0.1 to 1.3% of the applied dose were recovered in urine (Waring & Pheasant, 1976). Therefore a nearly complete biotransformation can be anticipated.

 

Additional to the elimination of small amounts of the unchanged parent compound, many metabolites and conjugates could be identified in the urine with differences in rats and mice.

Rats, rabbits, and guinea pigs metabolize 2,4-TDA to aminophenols as a major pathway (about 40% of an oral dose) (Waring & Pheasant, 1976). 2,4-TDA and its metabolites are also excreted as acid-labile conjugates (Timchalk et al.,1994; Waring & Pheasant, 1976). Originating from para-hydroxylation, the main component was 5-hydroxy-2,4-diaminotoluene in all species (Waring & Pheasant, 1976). Rats additionally excreted the 6-hydroxy isomer (the m-aminophenol) and greater amounts of the acetylated phenols than rabbits and guinea-pigs.

Though, no hydroxylamines and aminobenzoic acids were detected following an i.p. injection of the test substance. Additionally in this study the levels of methaemoglobin correlated well with total urinary aminophenol excretion. Rabbits which produced the largest amounts of free aminophenols, also exhibited the highest level of methaemoglobin.

 

In addition to oxidation on the aromatic ring of 2,4-TDA, oxidation on the benzylic methyl group to form phenolic and benzoic acid metabolites was identified as a predominant biotransformation following i.p. application in mice. In rats the 4-acetyl derivative and the 2,4-diacetyl compound were determined to be the main excretion products (Grantham et al., 1979).

 

Acetylation was determined to be a major pathway of biotransformation in rats (Timchalk et al., 1994; Jeffcoat, 1988). Following oral or i.v. application of¹⁴C-TDA (3mg/kg bw) in rats major parts (>80%) of the applied radioactivity were identified as mono- or diacatylated metabolites (Timchalk et al., 1994; The fraction of acetylated metabolites in relation to total radioactivity decreased markedly with increasing doses applied.

Acetylation of the 4-amino group of 2,4-diaminotoluene was reported to be the major acetylated amine metabolite in the rat (Grantham et al., 1975,Waring & Pheasant, 1976) and rabbits and guinea pigs (Waring & Pheasant, 1976).

The acetylation pathway represented about 15% of an intraperitoneal dose in rats and mono- as well as di-acetyl-derivates of 2,4-TDA were observed in different quantities in the urine of rats (15% of the dose), rabbits (8%), guinea pigs (8%), and dogs (about < 1%) (Waring & Pheasant 1976; Burroughs, 1975).

 

Additionally, Grantham et al. (1979) found an elimination of sulfate conjugates (10 % of the dose in the 24h urine in rats and mice), whereas glucuronic acid conjugates were found at higher levels in mice (17.4% of the dose) than in rats (7.5% of the dose). An additional 30 to 35% of the dose were unidentified water stable metabolites in this study.

In vitro studies in the presence of cytosols of different organs and species show that the N-acetylation giving 4-mono-acetyl- and 2,4-diacetyl-derivates most effectively occurs in the liver. Selective N-acetylation at the p-amino group was demonstrated N-acetylation of the o-amino group was not detected.

There are differences evident in species and sex (Glinsukon et al., 1975; Glinsukon et al., 1976). In comparing the species, the N-acetylation in hamster liver cytosol was the strongest followed by guinea pigs, rabbits, mice, and rats. In contrast, human liver cytosol formed only trace amounts of N-acetyl-derivates, and with cytosol of dogs no acetylation could be found.

The acetylation with liver cytosol of female mice or male rats was greater than in each case in the other sex (Glinsukon et al., 1975). By virtue of the lower acetylation in humans and dogs one can assume different metabolic pathways in rodents.

N-deacetylation was examined by detecting the products formed during the incubation of 4-acetylamino-2-aminotoluene and 2,4-diacetylaminotoluene with the liver cytosol fraction from male rats. The cytosol catalyzed the deacetylations to produce 2,4-TDA and 2-

acetylamino-4-aminotoluene (Sayama et al., 2002).

During incubation of 2,6-TDA with rat liver-S9-Mix from Aroclor-pretreated animals a dimethyl-diamino-dihydro-hydroxyphenazine was observed as the main metabolite (no data on the concentration used) (Cunningham et al., 1988).

 

Binding to macromolecules:

After a single oral dose of 0.25 mmol/kg (30.5 mg/kg bw) or 10 multiple doses of 0.1 mmol/kg (12 mg/kg bw) over a time range of 19 days in rats hemoglobin adducts were not detectable whereas for 2,6-TDA a hemoglobin binding index of 0.2 mmole/mole Hb/dose (single oral dose) and 0.4 mmole/mole Hb/dose (multiple oral dose) were measured, respectively (Neumann et al. 1993).

A dose-dependent formation of hemoglobin adducts for 2,4-TDA and 2,6-TDA was reported by Wilson et al. (1996) for doses from 0.5 to 250 mg/kg bw. The maximum Hb adduct content amounted to 0.36 nmol/g Hb at 24h following application for both isomers after i.p. application of 250 mg/kg bw. A linear relationship of hemoglobin versus hepatic DNA adducts was described for the 2,4-isomer. No DNA- adducts were detected for the 2,6-isomer.

3 distinct DNA-adduct isoforms were described in the livers of rats treated intraperitoneally with doses of 4.1-2046µmol/kg 2,4-TDA (La and Froines, 1992).