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Basic toxicokinetics

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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiated on December 14, 1981 and completed by March 30, 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was performed in accordance with Good Laboratory Practice standards. This study was performed prior to the adoption of the Organisation for Economic Co-operation and Development's (OECD)'s Test Guideline (TG) 417 Toxicokinetics, and the U.S. Environmental Protection Agency's TGs 870.7498 Metabolism and Pharmacokinetics, 870.8223 Pharmacokinetic Test, 870.8320 Oral/Dermal Pharmacokinetics, 870.8340 Oral and Inhalation Pharmacokinetic Test, and 870.8500 Toxicokinetic Test. The data are, however, well documented and scientifically acceptable.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The two-phase study was performed consisting of a preliminary study, followed by a definitive study. For the preliminary study, rats were administered a single dose of 10 or 50 mg/kg radiolabeled 14-C-ring-labeled DETDA. Blood level determinations were made by tracing 14-C-DETDA at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours following dosing. Respiratory elimination of 14-CO2 and other volatile materials were assessed during 0-12, 12-24, and 24-48 hour intervals. Urine was sampled at 4, 8, 12, 24, 48, 72, and 96 hours. Feces was collected at 12, 24, 48, 72, and 96 hours following dosing. At sacrifice, tissues were examined for residual radioactivity. For the definitive study, rats were either administered 10 daily doses of 14-C-DETDA or 9 daily doses of vehicle, followed by a tenth dose of 14-C-DETDA. Following the last dose, blood level determinations and respiratory, urinary, and fecal elimination were evaluated at specific intervals. Animals were sacrificed at 96 hours and tissues were analyzed for radioactivity.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diethyltoluenediamine
- Physical state: liquid
- Analytical purity: >99%
- Impurities (identity and concentrations):
- Composition of test material, percentage of components: 80% 2,4-diethyltoluenediamine and 20% 2,6-diethyltoluenediamine
- Lot/batch No.: XP213997A
- Specific activity (if radiolabelling): 14.1 mCi/mmole
- Locations of the label (if radiolabelling): ring labeled
- Storage condition of test material: Stored frozen under argon
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston Facility, Stoneridge, New York
- Age at study initiation: 64-73 days old
- Weight at study initiation: males (158-228 grams), females (102-154 grams)
- Housing: Polycarbonate cages with corn cob bedding
- Individual metabolism cages: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 2 °C
- Humidity (%): 50 +/- 10%
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 ml/kg of 0.5 N hydrochloric acid
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were prepared by dissolving the appropriate amounts of the nonlabeled and 14-C-labeled DETDA in a volume of 5 ml/kg of 0.05 N hydrochloric acid. Fresh dosing solutions were prepared for the preliminary and definitive studies in order to avoid any possible decomposition. Aliquots of each solution were counted to determine the amounts of radioactivity. The dosing solutions used in this study contained between 173 and 213 microCi of the labeled compound per 5 ml of the vehicle. When not in use, the dose was stored frozen under argon. The stability of the dosing solution was checked periodically by radio-gas chromatography.

VEHICLE
- Concentration in vehicle: 173 to 213 microCi
- Amount of vehicle (if gavage): 5 mL/kg
Duration and frequency of treatment / exposure:
A single dose of either 10 or 50 mg/kg was used for the preliminary studies.

For the definitive study, one group of animals received ten daily doses of 50 mg/kg. A second group received nine daily doses of vechicle only and a dose of 50 mg/kg DETDA on the tenth day.
Doses / concentrations
Remarks:
Doses / Concentrations:
Preliminary study = 10 or 50 mg/kg

Definitive study = 50 mg/kg
No. of animals per sex per dose:
Preliminary study = 1 male and 1 female rat received a dose of 10 mg/kg; 3 male and 3 female rats received 50 mg/kg.

Definitive study = 6 male and 6 female rats received ten daily doses of 50 mg/kg; 6 male and 6 female rats received nine daily doses of vehicle and one dose of 50 mg/kg on the tenth day.
Control animals:
no
Details on study design:
1. PRELIMINARY STUDIES
The preliminary studies were performed with eight rats (four males and four females). Three males and three females were treated with single oral doses of 50 mg/kg I4-C-DETA. Blood levels were examined during a 24-hr period in one male and one female rat. Another male and female rat were used to assess respiratory elimination of radioactivity during a 48-hr period. The remaining male and female rats were used to assess urinary and fecal excretion of radioactivity during a 96-hr period. In addition, one male and one female rat were treated with a lower (10 mg/kg) dose of 14-C-DETDA and were used to assess urinary and fecal excretion of radioactivity during a 96-hr period. At sacrifice, tissues of all rats were examined for residual radioactivity (see definitive studies).

For blood level determinations in the preliminary studies, samples were collected by tail bleeding at 0.25, 0.5, 1, 2, 4, 6, 8. 12, and 24 hr following dosing. During the experiment the rats were housed in individual stainless steel metabolism cages. Urine was collected at 4, 8, 12, and 24 hr, and feces were sampled at 12 and 24 hr, and then the rats were sacrificed.

Respiratory elimination of 14-CO2 or other volatile materials was assessed during 0-12, 12-24, and 24-48 hr intervals following dosing. The rats were housed individually in Delmar-Roth type glass metabolism cages. The C02 trapping solution was 5 M ethanolamine in 2-methoxyethanol. For collection of other volatile products, methanol:water (50:50) was used. Urine and feces were sampled during the same intervals, and the animals were sacrificed at 48 hr.

Urinary and fecal excretion were assessed during a 96-hr period in one male and one female rat treated with a 50 mg/kg dose, and one male and female treated with a 10 mg/kg dose of 14-C-DETDA. The rats were placed in stainless steel metabolism cages and urine was sampled at 4, 8, 12, 24, 48, 72, and 96 hr; feces were collected at 12, 24, 48, 72, and 96 hr following dosing, and then the rats were sacrificed.

2. DEFINITIVE STUDIES
A total of 24 rats (12 males and 12 females) were divided into two groups of 12 rats (6 males and 6 females) each. The rats in each group received either 10 daily doses of 14-C-DETDA or 9 daily doses of the vehicle followed by a tenth dose of 14C-DETDA. Daily dosing was performed between 8 and 9 a.m. After the last dose, the rats were placed in individual stainless steel metabolism cages. Three males and three females from each group were used for blood level determinations. Blood samples were collected by tail bleeding at 0.5, 1, 2, 4, 8, 12, and 24 hr. To assess total recovery, urine and feces were also collected. The rats were sacrificed at 24 hr for tissue sampling.

The remaining three rats/sex/group were used for assessment of urinary and fecal excretion of radioactivity. Urine was collected at 4, 8, 12, 24, 48, 72, and 96 hr. Feces were sampled at 12, 24, 48, 72, and 96 hr following the last dose. After each collection, the cages were rinsed and the cage washings sampled for radioactivity. The animals were sacrificed at 96 hr for analysis of radioactivity in tissues.

For the preliminary and definitive studies, rats were sacrificed by withdrawal of blood from the abdominal aorta under light ether anesthesia and the following tissues and organs were removed, weighed, and analyzed for radioactivity: Liver, Kidneys, Lungs, Heart, Spleen, Pancreas, Brain, Thyroids, Adrenals, Gonads, Bone marrow, Urinary bladder, Skeletal muscle, Retroperitoneal fat, Skin, and GI tract plus contents.

Portions of blood were centrifuged; plasma and red blood cells (RBCs) were separated for radioactivity determinations. Prior to sampling, the RBCs were resuspended in 0.9% NaCl and recentrifuged. The urinary bladder contents were removed and the bladders were washed thoroughly with 10% ethanol before analysis.
Details on dosing and sampling:
SAMPLE PREPARATION AND ANALYSIS
Aliquots (50 to 250 microliters) of whole blood, plasma, and RBCs were analyzed for total radioactivity. Volumes of urine and cage rinse were measured and samples (250 microliters) were analyzed. The trapping solutions for expired radioactivity were also measured, and samples (2 ml) were analyzed. Feces, GI tract, and tissues larger than 0.25 g were weighed and homogenized in five volumes of ethanol: water (10:90). Samples (60 to 120 mg) of other organs and tissues were weighed and assayed for 14-C content. Blood components, tissues, and fecal samples were cornbusted in a Packard Tricarb Sample Oxidizer (Model C306). Permafluor V in combination with Carbo-Sorb (Packard Instrument Company) was used as the scintillation cocktail. Urine and cage rinses were counted directly in Phase Counting Solution (PCS, hersham).

MEASUREMENT OF RADIOACTIVITY
Vials were cooled for a minimum of 24 hr before counting in a liquid scintillation counter (Packard Tricarb Model 3255). Correction for background was carried out automaticelly on the counter. Background deter minations were obtained from the average of natural counts of several tissue homogenates from nontreated animals. The counting efficiency was determined using the automa tic external standard (AES) method. An AES versus efficiency curve was prepared by processing a quench curve set through the counter under the conditions used throughout the experiment. Assays not within +/- 10% of the mean of the duplicates were reassayed in duplicate except when the sample was no longer available or when radioactivity counts were low and nonsignificant, i.e., less than two times the background, 30 cpm (counts per minute).
Statistics:
The significance of the data was determined by the two-tailed Student's t test. Significant differences were indicated when p < 0.05.

Results and discussion

Preliminary studies:
Radioactivitv appeared in blood at a slow rate, and peak levels were not reached until 6to 8 hr after dosing. 14-C disappearance from blood was slow after this time. At peak levels, only 1.4% of the administered doses were recovered in blood of both rats. A faster rate of 14-C appearance in blood was demonstrated in the female rat, but disappearance occurred at the same rate in both sexes. At 24 hr, 14-C levels in blood were essentially equal in both rats.

Only 0.04 and 0.07% of the administered doses were expired by the male and female rats, respectively, during a 48-hr period following 14-C-DETDA treatment. During this period, most of the administered doses were excreted in urine: 68 and 62% in the male and female rats, respectively. Major amounts of radioactivity were also eliminated in feces: 27 and 37% in the male and female rats, resp'ectively.

Following administration of either 10 or 50 mg/kg 14-C-DETDA, the administered radioactivity was eliminated primarily in urine and to a lesser extent in feces. Urinary excretion of 14-C was rapid. The male and female rats treated with the 50 mg/kg dose eliminated 50% of the administered radioactivity in urine by 24 hr and approximately 7% between 24 and 96 hr. Rats treated with the 10 mg/kg dose demonstrated faster rates of urinary elimination; 24 to 33% eliminated in the first 4 hr as compared to 14 to 15% following the 50 mg/kg dose. In addition, greater amounts (% of dose) of 14-C were recovered in urine of these animals than in rats treated with the 50 mg/kg dose. Elimination of 14-C in feces was less rapid. The male rat receiving the 50 mg/kg dose eliminated approximately 25% of the dose in feces by 24 hr and approximately 18% between 24 and 96 hr. The female excreted approximately 19% in feces by 24 hr and only 7% after this time. Fecal excretion of 14-C following the 10 mg/kg dose was equal in the male and female rats; approximately 27% of the dose was eliminated by 24 hr and approximately 5% between 24 and 96 hr.

At 24 hr, considerable amounts of radioactivity were recovered in most tissues examined. Tissue levels appeared to decline slowly thereafter (at 48 and 96 hr). 14-C concentrations at 24 hr were highest in the liver, thyroids, adrenals, bone marrow, and kidneys of the male rat, and liver, adrenals, bone narrow, and kidneys of the female rat. The male rat demonstrated higher levels of 14-C in the liver and thyroid than the female; other tissue levels were similar.

At 48 hr, both the male and female showed the highest concentrations in liver and thyroid, but the male demonstrated greater than 2-fold higher levels in these tissues than the female. Levels in other tissues were similar. At 96 hr, the liver, kidneys, and adrenals contained high levels of radioactivity but the highest levels were recovered in the thyroid (especially in the male).

At 24 hr, the major portion of blood radioactivity was recovered in plasma. However, disappearance of 14-C from plasma was rapid, and by 96 hr, most of the blood radioactivity was recovered in the RBCs . As indicated by slow disappearance of 14-C, some retention was apparent in the spleen, but there was no indication of 14C retention in fat. The skin showed higher levels than fat and muscle throughout the 96-hr sampling period.

Tissue levels in rats receiving the 10 mg/kg dose were examined at 96 hr only. As seen following the 50 mg/kg dose, the thyroid showed the 14-C concentrations, the levels being 2-fold greater in the male than the female rat. Concentrations in other tissues were similar in both rats. The percentages of the administered doses recovered in tissue at 96 hr did not differ substantially in rats treated with the low or high doses of 14-C-DETDA.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Slow rates of 14-C appearance and disappearance from blood were demonstrated in all rats receiving 1 or 10 doses of 14-C-DEDTA. Significantly higher levels of radioactivity were present in the blood of rats treated with the 10 doses. The concentrations were 2.1 to 3.2 times (males) or 1.6 to 3.6 times (females) higher than those of rats receiving one dose of 14-C-DETDA. The rates of appearance and disappearance of 14-C in blood were, however, parallel in rats of either treatment group up to 24 hr. Between 24 and 96 hr, disappearance of radioactivity from blood of rats treated repeatedly with 14-C-DEDTA was slower. Rats receiving the 10 doses reached peak concentrations at 1 to 2 hr following the last dose, while rats receiving a single dose had the highest concentrations between 2 and 12 hr. Female rats of both treatment groups displayed higher levels of radioactivity than their male counterparts throughout the 24-hr sampling period, but the rates of appearance and disappearance of radioactivity appeared similar in both sexes.
Details on distribution in tissues:
At 24 hr following 1 or 10 doses of 14-C-DETDA, concentration of radioactivity in tissues (expressed as microg equivalent/g tissue) of male and female rats was highest in thyroid followed by liver, kidneys, and adrenals; higher levels of radioactivity were found in skin than in muscle or fat. Rats treated with 10 doses showed significantly higher levels of 14-C in most tissues examined. In male rats, the greatest differences were demonstrated in the RBCs, spleen, pancreas, and epididymides, which were 3.5 to 4.8 times higher in rats treated with 10 doses. Females treated with 10 doses displayed the greatest increases in RBCs, spleen, and thyroids, which were 2.9 to 3.3 times higher than in females treated with one dose.

Following treatment with one dose, female rats demonstrated higher 14-C levels than the male rats in all tissues examined excepr the liver. After 10 doses, the differences between tissue levels in the males and females were less apparent. The liver and kidneys were significantly higher in the male rats, while the RBCs, spleen, pancreas, and brain were higher in the females. Contrary to the finding in the preliminary studies, the thyroids of the female rats sacrificed at 24 hr contained equal amounts (1 dose) or slightly higher (10 doses) 14-C activity than the thyroids of the males. Sex differences in 14-C uptake by the thyroids was, however, demonstrated at 96 hr.

At 96 hr, 14-C concentration remained highest in the thyroids, followed by the liver, kidneys, adrenals, and lung. In all tissues examined, levels of radioactivity were significantly higher following the 10 doses than after 1 dose. In male rats, the greatest differences were found in the RBCs, spleen, and fat, which were 4.6 to 5.5 times higher than those of rats receiving one dose. Other tissues contained at least 2.4 times higher 14-C levels following 10 doses. Female rats treated with 10 doses demonstrated the greatest differences in the thyroids, fat, and RBCs, which were 5.7 to 6.1 times higher than those of rats treated with one dose. Other tissues contained at least 2.6 times higher 14-C levels following 10 doses. At 96 hr following treatment with one dose of 14-C-DEDTA, levels of 14-C in the liver and thyroid of male rats were significantly higher than in females. Following 10 doses, the females demonstrated higher 14-C levels in certain tissues including adrenals, but 14-C levels in liver and kidneys were significantly higher in the male rats. Disappearance of radioactivity from blood and tissue generally occurred at a faster rate in rats treated with 1 dose as compared to 10 doses of 14-C-DETDA.
Details on excretion:
Urinary excretion of radioactivity was rapid. Rats receiving one dose of 14-C-DETDA eliminated 48% (males) and 56% (females) of the administered doses during the first 24-hr period. Only small portions (5-7%) were eliminated in urine between 24 and 96 hr. Fecal excretion was slower and occurred primarily between 12 and 48 hr. The radioactivity recovered in feces (42 and 41% in the males and females, respectively) probably represents a balance between incomplete absorption and biliary elimination. The delay in fecal elimination suggests that biliary excretion of DETDA and/or its metabolites is significant.

Following treatment with 10 daily doses of 14-C-DETDA, excretion of radioactivity in urine following the last dose was similar to or slightly more rapid than in the rats receiving one dose. By 24 hr, the males and females treated with 10 doses eliminated 52 and 62%, respectively, in urine. Only 7 to 8% of the last doses were eliminated in urine between 24 and 96 hr. Fecal excretion rates were similar (females) or slightly higher (males) than in rats receiving one dose of 14-C-DEDTA. By 96 hr, total fecal excretion in males and females receiving 10 daily doses of 14-C-DEDTA reached 58 and 45%, respectively, of the last dose.

Although urinary excretion rates in the males and females treated with one dose of 14-C-DETDA were initially similar, the total amounts eliminated in urine of female rats at 24 and 96 hr following dosing were higher than in males. This may, however, reflect the higher total recovery in female rats. Following 10 daily doses of 14-C-DETDA, the rates of urinary excretion were generally higher in the female rats and the rates of fecal excretion were higher in the males. Total recoveries in both sexes were similar.

Metabolite characterisation studies

Metabolites identified:
no

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
DETDA is slowly absorbed and distributed to various tissues when administered via the oral route. The highest concentrations of 14-C-DETDA were found in the thyroids, followed by the liver, kidneys, and adrenals at 24 and 96 hrs post dosing. DETDA was eliminated primarily in urine and to a lesser extent in feces.