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Basic toxicokinetics

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basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well described, reliable with restrictions

Data source

Reference Type:
The Fate of Inhaled Azodicarbonamide in Rats
Mewhinney JA, Ayres PH, Bechtold WE
Bibliographic source:
Fundamental and Applied Toxicology 8, 372-381

Materials and methods

Objective of study:
Test guideline
equivalent or similar to guideline
OECD Guideline 417 (Toxicokinetics)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
(E)-(carbamoylimino)urea; (Z)-(carbamoylimino)urea
Details on test material:
- Name of test material (as cited in study report): 14C-labeled ADCA (in the report, cited as ADA)
- Analytical purity: higher than 97% determined by HPLC
- Impurities (identity and concentrations):no data
- Lot/batch No.: no data

- Radiochemical purity (if radiolabelling):higher than 97% determined by HPLC
- Specific activity (if radiolabelling):ranges from 0.29 to 26mCi/g. Labeled ADCA was diluted with unlabeled ADCA to obtain the specific activity required.
- Locations of the label (if radiolabelling):14C

- Source:
labeled ADCA: Pathfinder Laboratories (St Louis, MO);
unlabeled ADCA: Midwest Research Institute (Kansas City, MO)
14C labelled ADCA

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
- Source: Institute 's barrier maintained colony
- Age at study initiation:11-15weeks
- Weight at study initiation:200-250g
- Fasting period before study:
- Housing:2 rats in polycarbonate cage (20 x 25 x 48cm), with hardwood chip bedding, and filter caps.
- Individual metabolism cages: yes
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:no data

- Temperature (°C):21±1
- Humidity (%):20-50
- Air changes (per hr):no data
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:
other: comparison of different routes: oral, intratracheal and inhalation
physiological saline
Details on exposure:
A saline suspension of the precipitated [14C]ADCA (0.1 mg ADA/0.5 ml saline) containing 5 µCi [14C]ADCA was administered to each rat by either gavage or by intratracheal instillation..

GAVAGE: Gavage of ADCA in rats was accomplished using a curved, bulb-ended cannula mounted on a syringe.

INTRATRACHEAL: Intratracheal instillation was done by anesthetizing the rat with halothane (5% in O2), placing the animal in a vertical position, and inserting into the trachea a bluntended needle attached to a syringe.

- Exposure apparatus:Wright dust feeder (BGI Incorporated, Waltham, MA)
- Method of holding animals in test chamber:no data
- Source and rate of air:air flow of 20 liters/min
- Method of conditioning air:no data
- System of generating particulates/aerosols: The desired aerosol concentrations within the exposure chamber were obtained by regulation of the speed at which the material reservoir cup rotated in relation to the scraper blade of the dust feeder.
- Method of particle size determination:periodic sampling using cascade impactors
- Treatment of exhaust air:no data

For each exposure except the one of 6 hr duration, five rats were exposed in specially designed exposure tubes which served as whole-body plethysmographs. This allowed measurement of the breathing rate and minute volumes of these rats throughout the exposure period and subsequent calculation of the percentage of the aerosol deposited in the rats.

During each inhalation exposure of rats, the aerosol concentration and particle size were determined. The relative concentration was monitored continuously by a real-time aerosol monitor (GCA Corp., Bedford, MA, Model RAM-S). The aerosol concentration was also measured twice per hour throughout the exposure period by collection on glass fiber filters (Gelman Instrument Co., Ann Arbor, MI, Type A/E) for periods of 10 min.
Aerosol size was determined by periodic sampling using cascade impactors.
Quantitation of 14C-labeled ACDA on filter samples or cascade impactor stages was by liquid scintillation spectroscopy (Packard Instrument Co., Downers Grove, IL, Model 460).
Duration and frequency of treatment / exposure:
Doses / concentrations
Doses / Concentrations:
gavage: 0.1mg/animal containing 5 µCi [14C]ADCA
intratracheal: 0.1mg/animal containing 5 µCi [14C]ADCA
inhalation: 1.5 (3h-low dose), 150 (3h-high dose), 20 (5.5 and 6h) mg/m3
No. of animals per sex per dose / concentration:
gavage 3
intratracheal 3
inhalation 4/group (see table)
Control animals:
Positive control reference chemical:
Details on study design:
All rats in these studies were sacrificed by intraperitoneal injection of 1 ml of T61 euthanasia solution (Taylor Pharmacal Co., Decatur, IL) and tissues were dissected and weighed immediately. Blood samples were obtained by heart puncture using heparinized syringes and needles.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, exhaled air, lungs
- Time and frequency of sampling:
Rats in the gavage and intratracheal instillation studies were dosed and placed in metabolism cages for a period of 72 hr, then sacrificed.
Rats in the 3-hr inhalation studies were either sacrificed immediately upon cessation of the exposure (3 rats in normal exposure tubes and 5 rats in plethysmograph tubes) or placed into metabolism cages for a 72-hr period, then sacrificed (4 rats).
For the 5.5-hr inhalation exposure, groups of 3 rats were withdrawn from the exposure apparatus at times of 20, 40, 60, 90, 120, 180, 240, 300, or 330min after the beginning of the exposure to determine the rate of build-up of 14C in blood and lung.
Additionally, 8 rats were sacrificed immediately upon cessation of the exposure (3 from normal exposure tubes and 5 from plethysmograph tubes) and 4 rats were placed into metabolism cages for 96 hr, and then sacrificed. After the 6-hr exposure, 3 rats were sacrificed immediately after cessation of the exposure, 4 rats were placed into metabolism cages for a 96-hr period, then sacrificed, and 36 rats were scheduled for sacrifice in groups of 3 at 0.02, 0.17, 0.33, 0.66, 1, 2, 4, 8, 12, 16, 24, and 102days after cessation of the exposure to determine the time course of tissue distribution of the [14C]ADCA or its metabolites.

- Quantification of 14C in Excreta and Tissue samples
Quantification of 14C in urine or exhaled air was accomplished by addition of duplicate aliquots of 0.5 ml of urine or trapping solution directly to scintillation cocktail and counting in the liquid scintillation spectrometer (Packard Instrument Co.). Feces and large tissue samples were prepared for counting by addition of water and formation ofa slurry (feces) or homogenization in a blender.
Duplicate aliquots of a slurry or homogenate were placed in cellulose cones and the 14C content was oxidized to 14CO2 using a tissue oxidizer (Packard Instrument Co., Model 306B). The 14CO2 formed upon oxidation was trapped in liquid scintillation cocktail (Packard, Permaflcur V) and the 14C content was determined by liquid scintillation counting. Small tissue samples < 0.2 g) were oxidized in toto and counted in the same fashion.

- Tissues and body fluids sampled: urine, faeces, exhaled air
- Time and frequency of sampling:
Immediately after administration of 14C-labeled ADCA, randomly selected rats were placed in glass metabolism cages (Stanford Glass, Palo Alto, CA) for separate collection of urine, feces, and exhaled metabolites. A vacuum pump was used to pull 0.5 liter/min of filtered room air through the cages and two bubblers containing 0.5 M KOH, in series. Urine and feces were collected in glass vials placed on dry ice to prevent microbial activity. The collection vials and the trapping solutions were changed at 3, 6, 9, 18, 24, 48, 72 and 96hr after administration of the compound.

- Method type(s) for identification: HPLC
- Metabolite identification
A high-performance liquid chromatography method for separating ADCA from its major metabolite, biurea, in urine and feces was developed. The conditions were: silica gel column (Alltech Chemical Co., Deerfield, IL, 600 SIB, 25 cm by 4.6 mm, 10µm); 80:20 ethyl acetate:methanol (Burdick and Johnson, Muskegon MI, distilled in glass); 1 ml/min flow rate; and a refractive index or radioactive flow monitor/detector.
Retention times of compounds under these conditions were 7.1 min (ADA); 11.9 min (urea), and 19.0 min (biurea). For spiked urine, an aliquot was dried under nitrogen and the residue taken up in DMSO and injected onto the column. Eluant from the HPLC was collected and assayed for 14C. Recovery for this procedure was 90%.
For feces, approximately 0.5 g of feces was homogenized with 2.5 g silica gel and DMSO. This slurry was added to a small glass column and 10 ml DMSO was used to elute 14Cfrom the sample. The eluant was collected and counted for l4Cand the remainder lyophilized to dryness. The residue was then re-dissolved in a small volume of DMSO and analyzed by HPLC, as described for urine. Recovery of radioactivity as eluted from the silica gel was 89%.
Only one peak eluted from the HPLC with a retention time equivalent to biurea.
To establish whether ADCA or biurea was present in rat blood after inhalation exposure, an ancillary experiment was conducted to determine the stability of ADCA in rat whole blood. Duplicate 1 ml fresh blood samples were spiked with a known amount of [14C]ADCA/DMSO solution (about 5 µg ADCA), mixed, and immediately frozen in liquid nitrogen. The samples were lyophilized to dryness. One aliquot was reconstituted in I ml of DMSO; the second was reconstituted with 1 ml of a solution containing 5 mg/ml unlabeled-ADA dissolved in DMSO.
The initial body burden (IBB) for rats in each inhalation exposure was determined by sacrifice at the end of the exposure of three rats exposed in standard exposure tubes and sacrifice of five rats exposed in plethysmograph tubes. The 14C content of all tissues, excluding the pelt, was summed to estimate the IBB. For rats maintained in metabolism cages, the tissue content at sacrifice (excluding the pelt) was summed with the 14C content measured in all excreta for that animal to estimate the IBB. The ADCA content in any animal or tissue was calculated from the 14C content of the sample and the specific activity was measured for the: bulk material in the Wright dust feeder reservoir (mean of triplicate determinations).
Fitting of retention curves to data sets was accomplished using a nonlinear least-squares method. The function fitted to excreta and tissue data was either a one-, two- or three-component negative exponential.
Data for tissue content were expressed as percentages of the IBB. Excretion data were expressed as percentages of the IBB excreted per hour. The likelihood ratio statistic was used to determine if the addition of a second or third term of the typical negative exponential function was appropriate. An F statistic was used to determine if the data subsets were from a single population. When data sets were combined, the mean values for each collection period were obtained and subsequent fitting of such data was accomplished using a weighted nonlinear routine where the weights were the reciprocal of the variance of each mean value. Student's t distribution was used in estimating 95% confidence intervals. Data values for excretory products in the urine, feces, or exhaled air were analyzed and plotted using the midpoint of the collection interval as the time axis.

Results and discussion

Metabolite characterisation studies

Metabolites identified:

Any other information on results incl. tables

Metabolite Identification

In the ancillary studies designed to determine the form of the 14C in urine, feces, and blood samples following gavage or intratracheal instillation of 14C-Iabeled, the 14C label was always associated with the metabolite biurea. When 14C-Iabeled was added to fresh blood samples taken from control rats, the conversion occurred within a period of 5 min, the time required for sample preparation for analysis by HPLC. No evidence was found for other potential metabolites. Only when fresh blood samples were pre-treated with relatively massive quantities of 5 mg/ml prior to addition of 14C-labeled was the 14C associated with the parent compound. These results indicate that the conversion fromto biurea in blood can be saturated, but only at unrealistic levels ofcontent in blood.


Gavage and Intratracheal Administration

The actual percentages of the administered absorbed (sum of total urinary excretion, amount exhaled in respired air, and content in tissues at sacrifice) may be slightly underestimated by this method because it does not account for the potential role of biliary excretion. However, in rats biliary excretion would be expected to result in fecal excretion of at most 5%of the administered dose for compounds with molecular weights of less than 120 (Smith, 1973). Within this limitation, absorption ofwas 33% of the administered dose following gavage and 90% following intratracheal installation.

Urinary excretion rate of [14C]equivalents expressed as a percentage of IBB per hour was much greater for intratracheal instillation compared to gavage.

Fecal excretion rates were not different for these two modes of administration (by gavage: 30±2.8% in urine and 67±2.5% in the feces; by intratracheal: 88±2.0% in urine and 9.8±2.4% in feces, with compared to the IBB). Only a small percentage (less than 2%) of the IBB was converted to 14CO2 and exhaled, regardless of the mode of administraion. At 72 hr after gavage, the gastrointestinal tract and carcass contained 0.06 and 0.6% of the IBB, respectively. After intratracheal instillation these values were 0.2 and 1.0%, respectively.

The lung contained 0.5% of the IBB at 72 hr after intratracheal instillation.


Inhalation Exposures

The particle size ranged from 1.8 to 3.4 µm AMAD. The particle sizes measured during the two exposures conducted to determine the potential role of the level of IBB upon subsequent retention and clearance of [14C]-equivalents were not different (1.8 and 2.1 µm AMAD-Activity Median Average Diameter).

The particle size for the exposures conducted to determine the rate of build-up of 14C in blood or the tissue retention of [14C]ADCA equivalents were also not different from each other (3.1 and 3.4 µm).


The measured geometric standard deviation for all four exposures was either 1.6 or 1.7. Deposition, expressed as the mean (±SD,n=5) percentage of the inhaled material deposited in the respiratory tract, was 24±8, 38±12 and 33±13 % after exposure to 1.8, 2.1 and 3.1 µm AMAD aerosols, respectively.


The rate of urinary excretion of [14C]equivalents by rats for each exposure was not different for the 72-hr period after exposure when the three data sets were compared using theFstatistic. Similarly, the rate of fecal excretion was not different among the four exposures.

The rate of build-up of [14C]equivalents in blood of rats withdrawn from the exposure apparatus at selected times during the 5.5-hr exposure was a linear function. The rate of build-up of [14C]equivalentsinlung of these same rats was also linear. Consequently, the ratio of [14C]in total lung to the [14C]mlof blood was a linear function with a slope indistinguishable from zero.

The [14C]equivalents (as %IBB)present in tissues of rats at 72 hr after exposure to aerosols ofwere comparable for all four exposures.

At sacrifice, the gastrointestinal tract contained<1.1%, the carcass <2.7%, all other tissues <0.06%, and the lung <0.2% of the IBB.

Applicant's summary and conclusion

Executive summary:

Groups of male F344/N rats were administered ADCA by gavage, by intratracheal instillation, and by inhalation exposure to determine the disposition and modes of excretion-of ADCA and its metabolites. At 72 hr following gavage, 30% of-the administered ADCA was absorbed whereas following intratracheal instillation, absorption was 90%. Comparison between groups of rats exposed by inhalation to ADCA to achieve body burdens of 24 or 1230µg showed no significant differences in modes or rates of excretion of [14C]ADCA equivalents. ADCA was readily converted to biurea under physiological conditions and biurea was the only 14C labeled compound present in excreta. [14C]ADCA equivalents were present in all examined tissues immediately after inhalation exposure, and clearance

half-times on the order of 1 day were evident for all tissues investigated. Storage depots for [14C]ADCAequivalents were not observed. The rate of buildup of [14C]ADCA equivalents in blood was linearly related to the lung content as measured from rats withdrawn at selected times during a 6-hr inhalation exposure at an aerosol concentration of 25 µg ADCA/liter.