N,N-dimethyl-p-toluidine

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data is from peer reviewed journals

Objective of study:

excretion

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

The objective of the study was to assess the rates and routes of excretion and tissue distribution of [14C]test chemical-derived radioactivity in F344 rats.

GLP compliance:

not specified

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories; Raleigh, NC
- Age at study initiation: approx 10-12 weeks of age
- Weight at study initiation: Body weight ranges were 180–267 g (male rats), 157–178 g (female rats)
- Housing:From 1 d prior to dosing until sacrifice animals were housed individually in all-glass metabolism cages
- Diet (e.g. ad libitum): Animals were fed Teklad certified rodent diet (W) 8728C (Harlan Teklad, Madison, WI) ad libitum,
- Water (e.g. ad libitum): Muncipal Water supplu, ad libitum

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Oral dose formulations contained [14C]test chemical and non-radiolabeled test chemical in 10% aqueous Alkamuls EL-620 (Rhodia, Cranbury, NJ). Alkamuls EL-620 (also known as PEG-30 castor oil) was used as an emulsifier. The target dose levels were 2.5, 25, and 250 mg/kg body weight administered in a dose volume of 5 ml/kg for rats. Animals received approximately 5–25 μCi of radioactivity.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

Duration and frequency of treatment / exposure:

daily

Remarks:

2.5,25,250 mg/kgbw - for male rats
25 mg/kgbw - female rats

No. of animals per sex per dose / concentration:

4 male rats/dose group - 2.5, 25,250 mg/kgbw
4 female rats - 25 mg/kgbw

Control animals:

not specified

Positive control reference chemical:

no data available

Details on study design:

no data available

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, blood, plasma, serum or other tissues, cage washes, bile : The following tissues were excised and weighed: samples of blood, adipose, muscle, and skin, as well as the following organs and glands in their entirety: brain, cecum (with contents), heart, kidneys, large intestine (with contents), liver, lung, small intestine (with contents), spleen, stomach (with contents), testes, thyroid, urinary bladder, and uterus. Blood and tissues were solubilized in Soluene- 350, neutralized, bleached as necessary, and analyzed for total radioactivity by LSC. The stomach with contents,
cecum with contents, small and large intestines with contents, tail (iv studies only), and residual carcass were solubilized in ethanolic sodium hydroxide. Weighed aliquots of each digestate were neutralized, bleached, and analyzed for total radioactivity
- Time and frequency of sampling:
- Other: Urine and feces were collected separately from each animal into receptacles cooled on dry ice. Urine was collected at 6, 12, and 24 h; feces were collected at 12 and 24 h. Bladder urine was also collected from the euthanized animals and added to the last urine collection. At the end of each collection interval, the metabolism cages were rinsed with water or ethanol (after the terminal collection only), and the rinsates were analyzed for total 14C content as described for urine. Recovery of radioactivity in the rinsate was added to the recovery of radioactivity in urine at the corresponding time point. Weighed aliquots of urine were analyzed directly for total radioactivity. Aliquots of feces were mixed with an approximately equal mass of water, homogenized, and solubilized in Soluene-350. The solubilized samples were neutralized and decolorized as described earlier before analysis for total radioactivity by LSC.

Statistics:

Means (reported to three significant figures) and standard deviations are presented. Recovery of radioactivity in a given matrix (i.e., urine, feces, VOCs, 14CO2, and tissues), expressed as percent of administered dose, were analyzed using analysis of variance with the Tukey post test for pairwise multiple comparisons (SigmaStat, version 3.1; Systat Software, Inc., Point Richmond, CA). A p value of <0.05 was considered significant. For male–female comparisons at the mid oral dose within a species, recovery of radioactivity in a given matrix was analyzed using a t-test.

Type:

excretion

Results:

[14C]test chemical-derived radioactivity was rapidly absorbed and excreted at oral doses up to 25 mg/kg. Excretion in urine accounted for approximately 88–94% in rats in the 2.5- and 25-mg/kg dose groups.

Details on absorption:

[14C]test chemical-derived radioactivity was rapidly absorbed and excreted at oral doses up to 25 mg/kg. Excretion in urine accounted for approximately 88–94% in rats in the 2.5- and 25-mg/kg dose groups. The balance of the administered dose was recovered in feces and tissues, with little excreted as exhaled VOCs.

Details on distribution in tissues:

For all doses in disposition studies, approximately 1–2% of the administered dose was present in liver 24 h after dosing

Details on excretion:

[14C]test chemical-derived radioactivity was rapidly absorbed and excreted at oral doses up to 25 mg/kg. Excretion in urine accounted for approximately 88–94% in rats in the 2.5- and 25-mg/kg dose groups. The balance of the administered dose was recovered in feces and tissues, with little excreted as exhaled VOCs.

Metabolites identified:

not specified

At the 250-mg/kg oral dose (aqueous), all rats showed signs of decreased activity, piloerection, blinking eyes, and hunched posture within 10–15 min of dosing.Decreased food and water intake also were observed, but all animals were clinically normal by 24 h.

Conclusions:

At the 250-mg/kg oral dose (aqueous), all rats showed signs of decreased activity, piloerection, blinking eyes, and hunched posture within 10–15 min of dosing.Decreased food and water intake also were observed, but all animals were clinically normal by 24 h.[14C]test chemical-derived radioactivity was rapidly absorbed and excreted at oral doses up to 25 mg/kg. Excretion in urine accounted for approximately 88–94% in rats in the 2.5- and 25-mg/kg dose groups. The balance of the administered dose was recovered in feces and tissues, with little excreted as exhaled VOCs. Recovery of radioactivity in the various matrices, including feces and tissues, was similar for the 2.5-mg/kg dose regardless of route of administration.

Executive summary:

The objective of the study was to assess the rates and routes of excretion and tissue distribution of [14C]test chemical-derived radioactivity in F344 rats. Oral dose formulations contained [14C]test chemical and non-radiolabeled test chemical in 10% aqueous Alkamuls EL-620 (Rhodia, Cranbury, NJ). Alkamuls EL-620 (also known as PEG-30 castor oil) was used as an emulsifier. The target dose levels were 2.5, 25, and 250 mg/kg body weight administered in a dose volume of 5 ml/kg for rats. Animals received approximately 5–25 μCi of radioactivity. A single oral (2.5, 25, or 250 mg/kg) dose of [14C]DMPT (1–25 mCi) was administered in an aqueous vehicle to male and female Fischer 344 rats. The following tissues were excised and weighed: samples of blood, adipose, muscle, and skin, as well as the following organs and glands in their entirety: brain, cecum (with contents), heart, kidneys, large intestine (with contents), liver, lung, small intestine (with contents), spleen, stomach (with contents), testes, thyroid, urinary bladder, and uterus. Blood and tissues were solubilized in Soluene- 350, neutralized, bleached as necessary, and analyzed for total radioactivity by LSC. The stomach with contents, cecum with contents, small and large intestines with contents, tail (iv studies only), and residual carcass were solubilized in ethanolic sodium hydroxide. Weighed aliquots of each digestate were neutralized, bleached, and analyzed for total radioactivity.Urine and feces were collected separately from each animal into receptacles cooled on dry ice. Urine was collected at 6, 12, and 24 h; feces were collected at 12 and 24 h. Bladder urine was also collected from the euthanized animals and added to the last urine collection. At the end of each collection interval, the metabolism cages were rinsed with water or ethanol (after the terminal collection only), and the rinsates were analyzed for total 14C content as described for urine. Recovery of radioactivity in the rinsate was added to the recovery of radioactivity in urine at the corresponding time point. Weighed aliquots of urine were analyzed directly for total radioactivity. Aliquots of feces were mixed with an approximately equal mass of water, homogenized, and solubilized in Soluene-350. The solubilized samples were neutralized and decolorized as described earlier before analysis for total radioactivity by LSC. At the 250-mg/kg oral dose (aqueous), all rats showed signs of decreased activity, piloerection, blinking eyes, and hunched posture within 10–15 min of dosing.Decreased food and water intake also were observed, but all animals were clinically normal by 24 h.[14C]test chemical-derived radioactivity was rapidly absorbed and excreted at oral doses up to 25 mg/kg. Excretion in urine accounted for approximately 88–94% in rats in the 2.5- and 25-mg/kg dose groups. The balance of the administered dose was recovered in feces and tissues, with little excreted as exhaled VOCs. Recovery of radioactivity in the various matrices, including feces and tissues, was similar for the 2.5-mg/kg dose regardless of route of administration.

Description of key information

At the 250-mg/kg oral dose (aqueous), all rats showed signs of decreased activity, piloerection, blinking eyes, and hunched posture within 10–15 min of dosing.Decreased food and water intake also were observed, but all animals were clinically normal by 24 h.[14C]test chemical-derived radioactivity was rapidly absorbed and excreted at oral doses up to 25 mg/kg. Excretion in urine accounted for approximately 88–94% in rats in the 2.5- and 25-mg/kg dose groups. The balance of the administered dose was recovered in feces and tissues, with little excreted as exhaled VOCs. Recovery of radioactivity in the various matrices, including feces and tissues, was similar for the 2.5-mg/kg dose regardless of route of administration.

Additional information

Basic Toxicokinetics

The objective of the study was to assess the rates and routes of excretion and tissue distribution of [14C]test chemical-derived radioactivity in F344 rats. Oral dose formulations contained [14C]test chemical and non-radiolabeled test chemical in 10% aqueous Alkamuls EL-620 (Rhodia, Cranbury, NJ). Alkamuls EL-620 (also known as PEG-30 castor oil) was used as an emulsifier. The target dose levels were 2.5, 25, and 250 mg/kg body weight administered in a dose volume of 5 ml/kg for rats. Animals received approximately 5–25 μCi of radioactivity. A single oral (2.5, 25, or 250 mg/kg) dose of [14C]DMPT (1–25 mCi) was administered in an aqueous vehicle to male and female Fischer 344 rats. The following tissues were excised and weighed: samples of blood, adipose, muscle, and skin, as well as the following organs and glands in their entirety: brain, cecum (with contents), heart, kidneys, large intestine (with contents), liver, lung, small intestine (with contents), spleen, stomach (with contents), testes, thyroid, urinary bladder, and uterus. Blood and tissues were solubilized in Soluene- 350, neutralized, bleached as necessary, and analyzed for total radioactivity by LSC. The stomach with contents, cecum with contents, small and large intestines with contents, tail (iv studies only), and residual carcass were solubilized in ethanolic sodium hydroxide. Weighed aliquots of each digestate were neutralized, bleached, and analyzed for total radioactivity.Urine and feces were collected separately from each animal into receptacles cooled on dry ice. Urine was collected at 6, 12, and 24 h; feces were collected at 12 and 24 h. Bladder urine was also collected from the euthanized animals and added to the last urine collection. At the end of each collection interval, the metabolism cages were rinsed with water or ethanol (after the terminal collection only), and the rinsates were analyzed for total 14C content as described for urine. Recovery of radioactivity in the rinsate was added to the recovery of radioactivity in urine at the corresponding time point. Weighed aliquots of urine were analyzed directly for total radioactivity. Aliquots of feces were mixed with an approximately equal mass of water, homogenized, and solubilized in Soluene-350. The solubilized samples were neutralized and decolorized as described earlier before analysis for total radioactivity by LSC. At the 250-mg/kg oral dose (aqueous), all rats showed signs of decreased activity, piloerection, blinking eyes, and hunched posture within 10–15 min of dosing.Decreased food and water intake also were observed, but all animals were clinically normal by 24 h.[14C]test chemical-derived radioactivity was rapidly absorbed and excreted at oral doses up to 25 mg/kg. Excretion in urine accounted for approximately 88–94% in rats in the 2.5- and 25-mg/kg dose groups. The balance of the administered dose was recovered in feces and tissues, with little excreted as exhaled VOCs. Recovery of radioactivity in the various matrices, including feces and tissues, was similar for the 2.5-mg/kg dose regardless of route of administration.

This result is supported by a similar study performed on B6C3F1 mice. Oral dose formulations contained [14C]test chemical and non-radiolabeled test chemical in 10% aqueous Alkamuls EL-620 (Rhodia, Cranbury, NJ). Alkamuls EL-620 (also known as PEG-30 castor oil) was used as an emulsifier. The target dose levels were 2.5, 25, and 250 mg/kg body weight administered in a dose volume of 10ml/kg for mice. Animals received approximately 5–25 μCi of radioactivity. The following tissues were excised and weighed: samples of blood, adipose, muscle, and skin, as well as the following organs and glands in their entirety: brain, cecum (with contents), heart, kidneys, large intestine (with contents), liver, lung, small intestine (with contents), spleen, stomach (with contents), testes, thyroid, urinary bladder, and uterus. Blood and tissues were solubilized in Soluene- 350, neutralized, bleached as necessary, and analyzed for total radioactivity by LSC. The stomach with contents, cecum with contents, small and large intestines with contents, tail (iv studies only), and residual carcass were solubilized in ethanolic sodium hydroxide. Weighed aliquots of each digestate were neutralized, bleached, and analyzed for total radioactivity.Urine and feces were collected separately from each animal into receptacles cooled on dry ice. Urine was collected at 6, 12, and 24 h; feces were collected at 12 and 24 h. Bladder urine was also collected from the euthanized animals and added to the last urine collection. At the end of each collection interval, the metabolism cages were rinsed with water or ethanol (after the terminal collection only), and the rinsates were analyzed for total 14C content as described for urine. Recovery of radioactivity in the rinsate was added to the recovery of radioactivity in urine at the corresponding time point. Weighed aliquots of urine were analyzed directly for total radioactivity. Aliquots of feces were mixed with an approximately equal mass of water, homogenized, and solubilized in Soluene-350. The solubilized samples were neutralized and decolorized as described earlier before analysis for total radioactivity by LSC. Toxicity at the 250-mg/kg oral dose to male mice was consistent with acute renal failure.[14C]test chemical-derived radioactivity was rapidly absorbed and excreted at oral doses up to 25 mg/kg. Excretion in urine accounted for approximately 88–94% in rats in the 2.5- and 25-mg/kg dose groups. The balance of the administered dose was recovered in feces and tissues, with little excreted as exhaled VOCs. Recovery of radioactivity in the various matrices, including feces and tissues, was similar for the 2.5-mg/kg dose regardless of route of administration. For all doses in disposition studies, approximately 1–2% of the administered dose was present in liver 24 h after dosing.

These results are supported by a study where the metabolism of orally administered test chemical was studied in Fischer 344 rats. Adult male F344 rats were purchased and housed two to a cage during a 2 week quarantine. For 1 day prior to and following dosing, animals were housed individually in all-glass metabolism chambers. Animals received a single oral dose of [14C]DMPT (2.5 – 250 mg/kg) in 10% aqueous Alkamuls. Urine was collected separately from each animal into receivers cooled over dry ice. Samples were stored in tightly capped containers in the dark at −22 °C until analysis. The collection- interval composites of rat urine from the 250-mg/kg dose group were prepared by combining 10% by weight of the total urinary output from 4 rats to yield composites in 5 different hourly collections (6, 12, 24, 48, and 72 hr). Samples were analyzed for total radioactivity using a Packard Model 2500 TR Liquid Scintillation Analyzer (Packard Instrument Co. Inc., Meridien, CT). Samples were assayed for 14C by directly dissolving them in Ultima Gold scintillation cocktail (Packard Instrument Co. Inc.).Prior to HPLC analysis, urine samples were cleaned using an Eppendorf 5417C microcentrifuge for 10 min at 10,000 RCF (relative centrifugal force) to pellet any particulate material. Prior to semi-preparative HPLC, solid-phase extraction (SPE) was performed for crude purification of metabolites using Sep-Pak reversed-phase (C18) cartridges (3 mL, 500 mg sorbent; Waters, Milford, MA). The C18 SPE cartridge was pre-conditioned with 3 mL of H2O prior to sample loading. Urine (3 mL) from rats was loaded onto the conditioned cartridge.Urine was washed through the cartridge with no elution solvent and collected as fraction 1. The cartridge was then washed with 3 mL of purified water (fraction 2) followed by 3 mL of acetonitrile (fraction 3). Fraction 1 contained M1 and M2 with M1 as the major peak. Fraction 2 also contained M1 and M2 but with M2 as the major peak. Fraction 3 contained M3 and what appeared to be parent test chemical based on chromatographic retention time. Analytical HPLC was performed using an Agilent Model 1100 HPLC system (Agilent Technologies, Palo Alto, CA) coupled with a β-RAM-Model 3 radioactivity detector (IN/ US Systems, Tampa, FL) attached to a Phenomenex (Torrance, CA) Luna C-18 column (5 μm, 150 × 4.6 mm internal diameter [i.d.]). ChemStation (version A.09.01 or A.09.03, Agilent Technologies) was used for system control and data acquisition. Signals were monitored at 254 nm and the column was maintained at 40°C. Preparative HPLC was carried out on the same HPLC system with the exception of a Phenomenex Luna semi-preparative column (5 μm, 250 × 10 mm i.d.). The HPLC mobile phase was 5% acetonitrile in H2O for 5 min, then linearly increasing acetonitrile content to 95% over 30 min. The flow rate was 1 mL/min for the analytical HPLC and 5 mL/min for semi-preparative HPLC. The rat urinary metabolite profile was determined by analytical reverse-phase high performance liquid chromatography (HPLC). Four radiolabeled peaks were observed, isolated, and purified by solid-phase extraction (SPE) and preparative HPLC methods. The 4 peaks were identified as p-(N-acetylhydroxyamino) hippuric acid (M1), DMPT N-oxide (M2), N-methyl-p-toluidine (M3), and parent DMPT. Metabolites M1 and M2 were identified by spectrometric and spectroscopic methods, including mass fragmentation pattern identification from both liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry, and from chemical analysis of nuclear magnetic resonance spectra. Structural confirmation of metabolite M2 was accomplished by comparison with a synthetic standard. Peaks M3 and the peak suspected to be DMPT were identified by comparison of their HPLC retention times and mass fragmentation patterns with authentic standards of N-methyl-p-toluidine and DMPT, respectively. DMPT metabolism is similar to that reported for N,N-dimethylaniline.