2,6-xylidine

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented publications, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of the investigation was to determine whether species differences in the oxidative metabolism of 2,4-DMA and 2,6-DMA could be related to species specific hepatotoxicities in rat and dog.

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,6-dimethylaniline
- Analytical purity: >99 %
- Supplier Aldrich Chemical Co., Inc.

Radiolabelling:

no

Species:

dog

Strain:

Beagle

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: of Veterinary Medicine breeding program
- Age at study initiation: 2 years
- Fasting period before study: none
- Housing: individual
- Individual metabolism cages: yes
- Diet: Purina Field and Farm Dog Chow ad libitum
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- standardized

Route of administration:

oral: capsule

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
test substance was applied as gelatine capsules

Duration and frequency of treatment / exposure:

10 days

Dose / conc.:

25 mg/kg bw/day

Remarks:

2,4-DMA (group 1)

Dose / conc.:

25 mg/kg bw/day

Remarks:

2,6-DMA (group 2)

No. of animals per sex per dose / concentration:

5 males per group

Control animals:

no

Positive control reference chemical:

not done

Details on study design:

The test substance was administered orally in gelatin capsules with no vehicle. Dogs were weighed every 5 days and doses were adjusted in order to maintain a constant weight/weight (mg/kg) dose.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: days 1 and 10 of treatment, 24-h urine samples were collected
- From how many animals: 5 per group, samples were not pooled
- Method type(s) for identification GC-MS

Statistics:

Analysis of variance and Dunnett's test were used to compare the effects of PB, 3MC and SKF-525A on urinary excretory products of 2,4- and 2,6-DMA on Days 1 and 10 of treatment. A paired t-test was used to compare the effect of length of treatment (Day 1 vs. Day 10) on urinary excretory products within each rat and dog treatment group (10) . Differences were accepted at the (P < 0.05) level.

Metabolites identified:

yes

Details on metabolites:

2,4-DMA was excreted in the urine of treated dogs largely as the parent compound, 6-hydroxy-2,4-DMA (6-HDMA), and 4-amino-3-methylbenzoic acid (4-AMBA). A trace level of N,2,4-trimethylamine was occasionally detected, but concentrations were too low for quantitation. 6-Hydroxylation was the major pathway in the dog, with a relatively much smaller amount of 4-AMBA, and no AAMBA, occurring as urinary metabolites. As with rats, 10 days of treatment with 2,4-DMA did not alter the urine content of 2,4-DMA or metabolites significantly. Dogs excreted approximately the same amount of 2,4-DMA/24-h period as rats, but total metabolite excretion was several-fold higher than for rats.

2,6-DMA was excreted in the urine of dogs as the parent compound, 4-HDMA and 2-amino-3-methylbenzoic acid (2-AMBA). A compound with an apparent molecular weight of 135 was also observed in the urine of dogs treated with 2,6-DMA. The mass spectrum of the unknown did not correlate with any standard on hand. Four possible structures of molecular weight 135 were proposed: 4-imino-3,5-dimethylquinone, its isomer 3-imino-2,4-dimethylquinone,
2,6-dimethyl-nitrosobenzene and 2-amino-3-methylbenzaldehyde, 2,6-Dimethylnitrosobenzene and 2-amino-3-methyl-benzaldehyde were eliminated on the basis of retention times and mass spectra . The formula 4-imino-2,6-dimethyl-quinone would appear to be the best choice for the unknown. 4-Hydroxy-2,6-DMA is a major metabolite of 2,6-DMA in the dog; hydroxylation occurring at the proposed location of the quinone's oxygen. Quinone formation could proceed in vivo or in vitro from the hydroxy compound. The possibility that the oxygen may be located meta to the nitrogen cannot be ruled out, but appears unlikely considering the metabolism of related compounds. N,2,6-Trimethylalanine, 2,6-dimethyl-nitrosobenzene and the glycine conjugate of 2-AMBA were also detected. These latter 4 compounds were observed in trace levels only and were not quantitated. The major excretory product of 2,6-DMA detected in dog urine after 1 or 10 days of treatment was 4-HDMA. Although the mean concentration of the metabolite recovered in urine decreased 3-fold with treatment, the difference between Day 1 and 10 means was not significant. The concentration of the 2-AMBA, however, did decrease significantly to about one-third that on Day 1.

Conclusions:

Metabolism of 2,4-DMA in the dog was considerably different than in the rat, with 6-hydroxylation being the major pathway. Previous reports have demonstrated that the dog and other carnivores oxidized aniline preferentially in the ortho rather than in the para position. Rodents on the other hand, have shown a preference for hydroxylation in the para position, although o-hydroxylation does occur. This relationship appears to also hold true for bridged dianilines [i.e., 4,4'-methylene-bis-dianiline (MDA)]. Nevertheless, the dog did form a smaller amount of 4-AMBA.

Additionally, the major metabolite of 2,6-DMA in both the rat and dog was the p-hydroxy-product. The lack of N-acetylation of 4-AMBA, which appeared to be complete in the rat, is not an unexpected finding in the dog, a species known to be deficient in N-acetyltransferase activity. The lack of a significant effect of repeated 2,4-DMA or 2,6-DMA administration on the appearance of AAMBA or 4-HDMA indicates that neither xylidine induced its own metabolism .

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented publications, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of the investigation was to determine whether species differences in the oxidative metabolism of 2,4-DMA and 2,6-DMA could be related to species specific hepatotoxicities in rat and dog.

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,6-dimethylaniline
- Analytical purity: >99 %
- Supplier Aldrich Chemical Co., Inc.

Radiolabelling:

no

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hilltop Lab Animals, Inc. (Scottsdale, PA)
- Age at study initiation: 12 weeks
- Individual metabolism cages: 2 rats per metabolism cage
- Diet: Purina Rat Chow ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- standard laboratory conditions

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

2,4-DMA (group 1-4);
2,6-DMA (group 5-8)

Duration and frequency of treatment / exposure:

10 days

Dose / conc.:

117 mg/kg bw/day

Remarks:

2,4-DMA (group 1-4)

Dose / conc.:

262.5 mg/kg bw/day

Remarks:

2,6-DMA (group 5-8)

No. of animals per sex per dose / concentration:

16 rats in total, to each of 8 groups

Control animals:

no

Positive control reference chemical:

not done

Details on study design:

- Dose selection rationale: test substances were equal to 25% of their respective LD50 values (LD50, oral 2,4-DMA = 467 mg/kg ; LD50, oral 2,6-DMA = 1050 mg/kg)

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: day 1 and 10
- From how many animals: 2, samples pooled
- Method type(s) for identification: GC-MS

Statistics:

Analysis of variance and Dunnett's test were used to compare the effects of PB, 3MC and SKF-525A on urinary excretory products of 2,4- and 2,6-DMA on Days 1 and 10 of treatment. A paired t-test was used to compare the effect of length of treatment (Day 1 vs. Day 10) on urinary excretory products within each rat and dog treatment group (10) . Differences were accepted at the (P < 0.05) level.

Metabolites identified:

yes

Details on metabolites:

2,4-DMA was excreted in the urine of rats as the parent compound, N-acetyl-4-amino-3-methylenzoic acid (AAMBA) or as the sulfate or glucuronide conjugates of these compounds. Trace levels of N,2,4-trimethylaniline were also detected, but at levels too low to permit quantitation.There was no significant difference in the total excretion of parent compound or AAMBA between Days 1 and 10 in rats treated with 2,4-DMA only.
Phenobarbital treatment produced inappetence, marked weight loss, an unthrifty appearance, chromodacryorrhea, and death in 50 % of the rats by was Day 5. Therefore no data was available for this group except on Day 1. Histopathological examination of the livers from animals of this group sacrificed on Day 5 revealed no remarkable lesions. Treatment with 3-MC caused an increase in the metabolite to parent (M/P) ratio on Day 1 but the comparison of this ratio between Group 3 and Group 1 on Day 10 was not a significant. However, there was a decrease in the M/P ratio when comparing Day 10 to Day 1. Treatment with SKF-525A did not alter the amount of AAMBA compared to Group 1 on Day 1 or after 10 days of treatment. None of the treatments caused any other metabolite to be detected at any time.


2,6-DMA was excreted in the urine of rats as the parent compound, 4-hydroxy-2,6-DMA, (4-HDMA) and a trace level of N,2,6-trimethylaniline was occasionally detected. In rats receiving 2,6-DMA only, 4-HDMA was the major excretory product on both Days 1 and 10 .
Phenobarbital treatment reduced the amount of 2,6-DMA excreted on Day 1 but did not alter the amount of 4-HDMA appearing in urine. Combined treatment with PB and 2,6-DMA did cause weight loss and an unthrifty appearance, but signs of toxicity took longer to develop and were less severe than for the PB: 2,4-DMA group. Treatment with 3-MC caused an increase in the M/P ratio on Day 1, and by Day 10 it had markedly decreased the excretion of parent compound and increased the M/P ratio. Treatment with SKF-525A did not affect 2,6-DMA excretion on either the first or tenth days. There was no effect on 4-HDMA excretion on Day 1, but 10 days of treatment caused a slight decrease, compared to 2,6-DMA excretion.

Conclusions:

The test substance is excreted either unchanged or as the 4-hydroxy metabolite or as conjugates of both (sulfate and glucuronic acid). Phenobarbital and 3 -methylcholanthrene decreased the amount of excreted parent compound and increased the excretion of main metabolites (increase in main metabolite to parent ratio). Repeated administration of either 2,4 DMA or 2,6 DMA did not result in significant effects, indicating that xylidine does not induce its own metabolism. The fact that 3 -MC increased the urine concentration of the main metabolite 4-hydroxy-DMA suggests that 4-hydroxylation is Cyt P450 dependent.

Additional information

Toxicokinetics

2,6-Xylidine is a chemical intermediate used principally in the production of dyes. It is also a component of tobacco smoke, a degradation product of aniline-based pesticides, and a metabolite of certain drugs, particularly the xylide group of local anesthetics.

2,6-Xylidine is easily absorbed from the gastro-intestinal tract and excreted unchanged or metabolized via the urine in experimental animals. 2,6-xylidine produces only weak methaemoglobin formation.

 

 

Absorption

2,6-Xylidine is absorbed from the small intestine in rats, the half life periods for this process being 14.4 minutes (no further details; Plá Delfina, et al., 1972). Further studies dealing specifically with absorption of the xylidines are not available. Proof of absorption following oral application of 2,6-xylidine in rats and dogs, has been furnished by the detection of the xylidines and their corresponding metabolites in the urine (Hardy et al. 1986, 1987, Short et al. 1989).

Feeding male rats a diet containing 3000 ppm 2,6-Xylidine (approx. 215 mg/kg bw/day) for four weeks resulted in plasma levels of 0.36 µg/mL after 1 week and 0.20 µg/mL after 4 weeks. Plasma levels were under the detection limit of 0.02 µg/ml when 300 ppm were administered (Yasuhara et al., 2000).

Data on toxikokinetics following inhalation or dermal exposure are not available. Poisoning symptoms, which have been described following corresponding applications, suggest that absorption via the skin and the respiratory tract may take place.

Distribution

No studies or results are available for tissue distribution in animals.

 

Metabolism

The results of studies on the metabolism of the xylidines following oral application are given in shortened form in the following table. In general, investigations with different species show the same main metabolites after hydroxylation of 2,6-xylidine.

 

Species	Metabolite	Conjugate of the metabolite	Literature
rat	2,6-xylidine unchanged 4-hydroxy-2,6-xylidine 2-amino-3-methyl benzoic acid	not specified	Lindstrom et al.(1963)
rat	2,6-xylidine unchanged 4-hydroxy-2,6-xylidine N,2,5-trimethyl aniline	glucuronide, sulphate glucuronide, sulphate	Short et al.(1989)
dog	2,6-xylidine unchanged 4-hydroxy-2,6-xylidine 2-amino-3-methyl benzoic acid N,2,6-trimethyl-aniline 2,6-dimethyl-nitrosobenzene 4-imino-3,5-methylquinone	glycine	Short et al.(1989)

 

In the study by Lindstrom et al. (1963) 2,6-xylidine was given to male Osborne-Mendel rats at a dose of 20 mg/kg body weight in water by gavage. Apart from the initial substance and non-specified conjugates, the urine analysis resulted in mainly the 4-hydroxy-2,6-xylidine and, in lower concentration, the 2-amino-3-methyl benzoic acid as excretion products.

Short et al.(1989) examined the metabolism of 2,6-xylidine in rats and dogs. 16 male (12 week-old) Fischer-344 rats were treated with 2,6-xylidine (purity > 99%) in corn oil by gavage at a dose of 262.5 mg/kg body weight (1/4 of the LD50) for 10 days. 3 further groups, each consisting of 16 male Fischer-344 rats, were given intraperitoneal injections of either 80 mg phenobarbital/kg body weight, 15 mg methylcholanthrene/kg body weight or 50 mg SKF-525A/kg body weight daily for 10 days, in addition to the 2,6-xylidine treatment. Pooled 24-hour urine samples were analysed from day 1 and day 10. Analysis of the urine gave, apart from the initial substance, 4-hydroxy-2,6-xylidine and the sulphate and glucuronic conjugates of both substances. N,2,6-trimethylaniline was found in low concentration. The duration of treatment had no qualitative or significant quantitative effect on the metabolism. In the same study, following administration of 2,6-xylidine to dogs, apart from the initial substance, 4-hydroxy-2,6-xylidine was mainly found and, in lower quantities, 2-amino-3-methyl benzoic acid and its glycine conjugate, N,2,6-trimnethylaniline, 2,6-dimetbylnitrosabenzene and 4-imino-3,5-dimethylquinone. The quantity of different metabolites was subject to considerable individual variations. The longer treatment had no effect.

Gan et al. (1999, 2000) investigated the oxidation of 2,6-xylidine in vitro by human liver microsomes (HLM) and recombinant human P450 isoforms. They found 4-hydroxy-2,6-xylidine as metabolite and CYP2A6 and CYP2E1 as major isoforms to be responsible for the production of 4-hydroxy-2,6-xylidine. Furthermore, a 2,6-xylidine hemoglobin adduct was found when hemoglobin was added into the reaction mixtures.

In a study by Lindstrom et al. (1969) the effect of xylidines on methemoglobin formation was investigated in rats in vivo. A single injection of 2,4-xylidine, 2,5-xylidine or 2,6-xylidine was conducted in 5 rats each and blood was sampled at various time points after injection. The authors stated that the xylidines were not very effective methemoglobin formers. Of this group of amines, 2,4-xylidine was the most effective; it produces maximum methemoglobin at 1 hr as compared to 3 hr for 2,5- and 2,6-xylidine. 2,4-xylidine produced about a peak value of about 3.5 % methemoglobin after 1 hour, furtheron gradually decreasing and reaching control values after about 4 hours. Data were given as xy-chart. No data on statistical significance are available.

The metabolism of xylidines occurs via N-acetylation or N-oxidation, oxidation of one methyl group, and hydroxylation of the aromatic ring. The main metabolite of 2,6-DMA in dogs and rats was 4-hydroxy-2,6-dimethylaniline (4-amino-3,5-dimethylphenol). Dogs also excreted significant quantities of 2-amino-3-methylbenzoic acid and trace amounts of the corresponding glycine conjugate, of 2,6-dimethylnitroso-benzene and of an unknown compound, possibly 3,5-dimethyl-4-imino-quinone (Lindstrom et al., 1963; Short et al., 1989a). N-Oxidation leads to N-hydroxy-2,6-DMA, which may be

further oxidised to the nitroso compound and covalently bound to haemoglobin. The corresponding haemoglobin adducts have been detected in rats after administration of 2,6-DMA (and of lidocaine) (Bryant et al., 1994).

The covalent binding of several aromatic amines was compared (Sabbioni, 1994). Alkyl substitution of aniline reduced haemoglobin binding, and two methyl groups in orthoposition to the amino group as in 2,6-DMA almost abolished haemoglobin binding.

Bioactivation of 2,6-DMA to reactive metabolites which bind to tissue was shown to occur in the liver but also in the nasal olfactory mucosa and the upper alimentary and respiratory tract of rats. The formation of protein and DNA-adducts from 2,6-DMA in vitro in the presence of microsomal preparations from various respiratory and alimentary tissues of rats revealed that binding was highest with preparations from nasal olfactory mucosa and was about 10-fold higher than with preparations from liver (Tyden et al., 2004). The differences in binding may be related to differences in the Nacetyltransferase activity between olfactory mucosa and liver: Experimental data indicate that the N-acetyltransferase activity against 2,6-DMA in the olfactory mucosa is about 10-fold higher than in liver (Genter, 2004).

 

Accumulation

In rats treated with up to 10 doses of 63 mg 14C-2,6-DMA/kg d by gavage, accumulation of the radiolabel occurred. Animals having received 10 doses had higher radioactivity levels in blood and other tissues and the radioactivity disappeared more slowly than in rats treated once with the same dose. High concentrations of radioactivity were found in red blood levels, liver and kidneys but also in nasal tissues where the concentration 24 hours after dosing was 2.5-fold higher than in the liver. High concentrations of radioactivity were also found in nasal tissues after intraperitoneal administration. Excretion of radioactivity was mainly through the kidneys with urine. The increased retention after repeated administration was not due to an impairment of excretion but due to an increased binding in blood and tissue (NTP, 1990).

Excretion

In all the investigations on metabolism of the xylidines following oral application mentioned in this section, the possibility of excretion of the xylidines or their metabolites through breathing or through the faeces was not examined. 2,6-xylidine was excreted in the urine of rats as the parent compound, 4-hydroxy-2,6-xylidine, and a trace level of N,2,6-trimethylaniline was occasionally detected (Short et al, 1989). In the urine of dogs it was excreted as the parent compound, , 4-hydroxy-2,6-xylidine and 2-amino-3-methylbenzoic acid. A compound with an apparent molecular weight of 135 was also observed in the urine of dogs treated with 2,6-xylidine.