3(or 4)-methylbenzene-1,2-diamine

Endpoint summary

Additional information

The o-TDA category consists of two individual compounds, 3,4-TDA (CAS No. 496-72-0) and 2,3-TDA (CAS No. 2687-25-4), and a commercially supplied mixture in which these isomers are the major constituents in a 60/40 ratio, respectively (CAS No. 25376-45-8 and 26966-75-6 referred to as commercial TDA mixture (2,3/3,4-TDA (40/60))). Only the isometric mixture (2,3/3,4-TDA (40/60)) is available commercially. 2,4-TDA  (CAS number 95-80-7) is a structural analogue of the sponsored substances and is used to address this endpoint.  2,4-TDA is a structural isomer of o-TDA, differing in the substitution pattern of the amine groups on the tolyl ring; having similar physical chemical properties; similar environmental fate; and similar health effects (acute toxicity and genotoxicity).  As a result, data from 2,4-TDA can be used for read across to 2,3- TDA, 3,4-TDA and commercial TDA mixture (2,3/3,4-TDA (40/60)).  A comparison of data from the commercial TDA mixture (2,3/3,4-TDA (40/60)), 2,3-TDA, 3,4-TDA and 2,4-TDA was made in the OECD SIDS Initial Assessment Report for SIAM 25 16 October 2007), for o-TDA, which supportted the use of 2,4-TDA as an appropriate structure analogue.

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. 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.

 

Distribution:

In rats the highest tissue concentrations were measured in liver and kidney after oral or intraperitoneal administration 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 . The peak blood levels after oral dosing were reached within 2 hours .

No species-related differences in tissue distribution in mice and rats were observed, though biphasic tissue elimination kinetics were observed . 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 .

Similar excretion patterns were observed in mice and rats 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 . 

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 . Additionally there was biphasic clearance kinetics of [¹⁴C]2,4 -TDA-derived radioactivity from the plasma, with peak levels observed one hour after dosing, followed by rapid clearance during the next six hours, and more gradual clearance thereafter . 

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 .

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 .

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 .

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 .

 

 

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 . 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 . 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) . 2,4-TDA and its metabolites are also excreted as acid-labile conjugates . Originating from para-hydroxylation, the main component was 5-hydroxy-2,4-diaminotoluene in all species . 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 .

 

Acetylation was determined to be a major pathway of biotransformation in rats. 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 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 and rabbits and guinea pigs.

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%).

 

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 . 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 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.

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) .

 

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 .

There was a dose-dependent formation of hemoglobin adducts for 2,4-TDA and 2,6-TDA 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.