Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not applicable
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study did not list guidelines but was conducted according to GLPs and the report contains sufficient data for interpretation of study results.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report date:
1981

Materials and methods

Objective of study:
metabolism
toxicokinetics
Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
An attempt was made to conduct the DETA metabolism and pharmacokinetic studies in a manner consistent with that of the EDA studies of Yang, R. S. H,, K. A. Pittman, D, R. Rourke and V. B, Stein (1978). "Pharmacokinetics and Metabolism of Hexachlorobenzene in the Rat and Rhesus Monkey," J. Agric. Food Chem. 26:1076-1083 and Yang, R. S. H. (1978). "Pharmacokinetics and Metabolism of Ethylenediamine - Interim Report No, 1: Material Balance Studies of Ethylenediamine in the Rat Following Single Oral, Endotracheal and Intravenous Dosing." BRRC Report 941-144.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-iminodi(ethylamine)
EC Number:
203-865-4
EC Name:
2,2'-iminodi(ethylamine)
Cas Number:
111-40-0
Molecular formula:
C4H13N3
IUPAC Name:
bis(2-aminoethyl)amine
Details on test material:
Diethylenetriamine*3HCl [1,2 E-1 4 C ] ( DETA-E14C)was prepared by the Midwest Research Institute, Kansas City, MO. The material was prepared in a seven step sequence of synthetic reactions ending with the catalytic decomposition of bis(2-phthalimidoethyl)amine to the final product. The overall radioactive yield from the starting material , Ba14 C03, to DETA -14 C was a disappointingly low 0.73% (2.2 millicuries of final yield) ; however, this was sufficient to complete the disposition and pharmacokinetic studies. MRI judged the purity of the final product to be greater than or equal to 98% using a thin layer chromatography procedure. Cation exchange chromatography of this material gave a minimum purity of 92%. The radiochemical was assigned BRRC sample number 43-121.

High purity DETA was obtained from P. R. Umberger, Union Carbide Corporation under reference 1PRU-72 and assigned a BRRC sample number of 42-186. This chemical has been assigned CAS number 111-40-0.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
Male Fischer 344 rats obtained from the Charles River Breeding Laboratories, Portage, Michigan facility were used exclusively in these studies. The animals were 36 days of age upon receipt. They were held in wire-bottom and -front stainless steel cages, three animals per cage, for 9 to 14 days prior
to the start of each study. During this time period, the animals were provided free access to ground feed (NIII-07 diet, Zeigler Brothers, Gardner, PA) and tap water supplied by the Municipal Authority of Westmoreland County. Each animal was identified using a toe-clip procedure, A randomization procedure was used to select animals from the shipment groups prior to the initiation of each study, i.e., material balance and pharmacokinetic studies. Some of the rats not selected by this procedure were later used as replacements for animals which died or were not used because of technical diffi culties. The remaining culls were sacrificed at the termination of the studies.

Administration / exposure

Route of administration:
other: oral and endotracheal
Vehicle:
physiological saline
Details on exposure:
DETA 14-C w as mixed with unlabeled DETA in physiological saline solution at concentrations sufficient to dose animals with the appropriate quantity of chemical and amount of radioactivity. The dosing solutions were adjusted to pH 7.4 using hydrochloric acid or ammonium hydroxide, as required. For the material balance study, two dosing solutions were prepared, one for dosing animals at the 50 mg per kg level and the other for the 500 mg per kg group. For the pharmacokinetic study, two solutions were also prepared, one for dosing rats by the oral and endotracheal routes and the second for dosing them intravenously (iv). All solutions were formulated so that each animal would receive approximately 0.25 ml total volume. Standardization was accomplished by weighing samples of the solutions, approximately 10 µl in volume, into 5 ml volumetric flasks , bringing to volume with methanol and counting aliquots of these dilutions for radioactivity .
Duration and frequency of treatment / exposure:
Refer to the Details and Sampling section
Doses / concentrations
Remarks:
Doses / Concentrations:
The dosage level goals for this study were 50 and 500 mg per kg body weight.
No. of animals per sex per dose / concentration:
Four
Control animals:
no
Positive control reference chemical:
Not applicable.
Details on study design:
Urine and feces were collected after 24 and 48 hrs. Blood samples were collected at 48 hours just before sacrifice. At sacrifice, selected tissues were removed.

Male Fischer rats were administered radiolabeled diethylene triamine-3HCl [1,2 -14C] (DETA-14C) via the oral route or endotracheally. Radiolabled DETA was mixed with unlabeled DETA in physiological saline and pH adjusted to 7.4 to deliver a 50 mg/kg or 500 mg/kg dose. All solutions formulated to deliver a total volume of .25 mls.

The study was conducted with 16 rats: 4 per dose level and each dose level by the 2 routes of exposure. Urine and feces were collected after 24 and 48 hrs. Blood samples were collected at 48 hours just before sacrifice. At sacrifice, selected tissues were removed. The tissues and the remaining carcass were analyzed for radioactivity.

The 6 rats were also implanted with a venous cannula one day prior to administration of an intravenous dose. Four rats were selected for the intravenous part of this study based on the condition of the cannula. Approximately, 0.2 mls of blood was taken via the cannula at 5, 15, 30, 45 minutes and at 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, and 24 hours post-dose.




Details on dosing and sampling:
Dose Administration: All rats were dosed using the weighed syringe procedure. A one ml tuberculin syringe was filled to the 0.25 ml mark with the
appropriate dosing solution. The syringe and contents were weighed, the animal dosed and the syringe reweighed. The administered dose was calculated from the weight difference. Animals were orally dosed using a stainless steel animal feeding needle. Endotracheal instillation was accomplished after lightly anesthetizing the rats with Metofanem. The dose was injected into the trachea through a small polyethylene tube. Intravenous administration was carried out via tail vein injection.

The material balance study was conducted with 16 rats: four per dosage level and each dosage level administered by two routes of administration. Immediately after dosing, the animals were placed in all-glass Roth metabolism cages. Air was drawn into the cages through DrieriteB (W. A. Hammond Drierite Co., Xenia, OH) and Ascaritea (Arthur H. Thomas Co., Philadelphia, PA) traps to remove moisture and C02. Air was drawn from the cages through two consecutive traps, each trap containing 2-methoxyethanol and ethanolamine (7:3 v/v) to remove expired CO2. The flow rate through the system was approximately 250 ml/min. The C02 trapping agent was collected and sampled for radioactivity determination at 6, 24 and 48 hours after dose administration.

Urine and feces were obtained separately, the urine chilled with dry ice upon collection. Both were collected at 24 and 48 hours after dose administration with samples of urine taken immediately for radiometric analyses. The feces were frozen until analyses could be conducted. Also, at these same time intervals, 24 and 48 hours postdose, the cages were washed with a solution of 50% acetone in water and samples of the wash solution were also taken for radiometric analyses.

Forty-eight hours after dose administration the rats were anesthetized with Metofane® and blood samples were obtained from the abdominal aorta. After exsanguination, selected tissues were removed, sampled and analyzed for radioactivity. The remaining carcasses were solubilized in 10N NaOH and samples of t h i s solution were also analyzed for radioactivity.

For the pharmacokinetic study, at least six animals per dosage route were surgically implanted with venous cannulae in the right external jugular vein one day prior to dose administration. The surgery was carried out under Metofanem anesthesia. Following an overnight resting period, four rat s were selected on the basis of the condition of the cannula and administered DETA. The rats were placed in stainless steel wire metabolism cages and blood samples (approximately 0.2 ml) were taken via the cannulae at 5, 15, 30 and 45 minutes and at 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16 and 24 hours. For each blood sample collected, the following operations were conducted: The plasma was separated from the blood cells in microhematocrit capillary tubes (usually two or more capillary tubes/sample) by centrifugation. Part of the plasma was transferred to a tared liquid scintillation vial . The weight of the plasma was obtained and 10 ml of Aquasol II (New England Nuclear) were added to each vial for determination of radioactivity by standard liquid scintillation counting technique.
Statistics:
Where possible a two way analysis of variance was used to analyze data using dosage route and dosage level as the two main effects . In the case of pharmacokinetics, a one-way analysis of variance (dosage routes being the main effect) was used to analyze the data . The Minitab program was
utilized for the two way analyses while the Statistical Package for Social Sciences (SPSS) was used for the one-way analyses (Nie et al., 1975). Bartlett's test for homogeneity of variance was used to determine the validity of these approaches. If the variances were heterogeneous (using the fiducial limit of 0.01) Cochran's t-test was employed to test differences between treatment (dosage route, dosage level) means. Multiple regression analysis (Minitab) was used to analyze radioactivity concentrations over all tissue types. Dosage route, dosage level and tissue type were used as factors in this analysis.

Results and discussion

Preliminary studies:
Not applicable

Toxicokinetic / pharmacokinetic studies

Details on absorption:
DETA is mostly absorbed into the general circulation when administered either orally or endotracheally, 95% and 90% of the dose. The terminal plasma half-lives were calculated for oral, endotracheal and intravenous administration which were 13.9, 7.1 and 5.7 hours, respectively.
Details on distribution in tissues:
Fecal excretion was the major route of elimination (40% to 46 %) followed closely by urinary excretion (31% to 43%). Only a small percentage of the radioactivity was recovered as CO2 or found in the tissues and carcass (1.8 to 3.1%). There were no significant differences in the percentages of radioactivity excreted in the urine, feces, or CO2. Although, the percentage of radioactivity retained in the carcass of endotracheally exposed animals is greater that those dosed orally, the difference is not biologically significant.
Details on excretion:
Fecal excretion was the major route of elimination (40% to 46 %) followed closely by urinary excretion (31% to 43%). Only a small percentage of the radioactivity was recovered as CO2 or found in the tissues and carcass (1.8 to 3.1%). There were no significant differences in the percentages of radioactivity excreted in the urine, feces, or CO2. Although, the percentage of radioactivity retained in the carcass of endotracheally exposed animals is greater that those dosed orally, the difference is not biologically significant.

Any other information on results incl. tables

The route of administration, oral or endotracheal, had little effect on the distribution within the body or the elimination of radioactivity from the rat. Feces and urine were the primary routes of excretion with less than 2% of the admininstered dose being expired as 14CO2. More than 96% of the recovered dose was eliminated within 48 hours after dosing. In comparing results from animals receiving the compound at 500 mg/kg with those receiving it at 50 mg/kg there was a significant increase in the percentage of radioactivity excreted in the urine and a significant decrease in that eliminated as 14CO2 at the higher dose level. The route of administration did not affect the following pharmacokinetic parameters at the 50 mg/kg level.

Applicant's summary and conclusion

Conclusions:
When DETA was administered to rats either orally or endotracheally, it was readily absorbed into the general circulatory system. The primary routes of elimination of radioactivity from the compound were feces and urine with smaller amounts leaving by way of 14CO2. Forty-eight hours after dosing more than 96% of the recovered radioactivity had been eliminated from the animals.

The concentration of radioactivity in tissues of rats receiving DETA at the 500 mg per kg level were 4 to 10 times higher than in rats receiving the compound at 50 mg per kg. There were no significant differences in the concentrations in tissues of rats receiving the compound by different routes of administration. The higher level of dose administration did produce a shift in the pattern of radioactivity in urinary chromatograms with a greater percentage associated with a fraction having characteristics similar to those of the unchanged amine.

There were no apparent biologically important differences in material balance parameters (excretion pattern, tissue distribution patterns or urinary chromatographic profiles) or i n the pharmacokinetic parameters among animals receiving DETA by different routes of administration. These observations would give metabolic support for predicting risks from an inhalation exposure based upon data from a chronic feeding toxicity study.

Although DETA and EDA both appear to be readily absorbed, the former is excreted more readily in the feces, retained to a lesser degree in the carcass and not as readily degraded as the latter. At similar dosage levels, tissues had one-fifth to one-twentieth the concentration of DETA than that of EDA. These facts may have important ramifications in the toxicities of the two chemicals.
Executive summary:

The metabolism and disposition of diethylenetriamine (DETA) in rats was studies with regard to the route of administration and the dosage level. The pharmacokinetics of the compound was studies only with respect to the route of administration. Finally, a comparison of the pharmacokinetics and metabolism was made between DETA and ethylenediamine (EDA).

The route of administration, oral or endotracheal, had little effect on the distribution within the body or the elimination of radioactivity from the rat. Feces and urine were the primary routes of excretion with less than 2% of the administered dose being expired as 14CO2. More than 96% of the recovered dose was eliminated within 48 hours after dosing. In comparing results from animals receiving the compound at 500 mg per kg with those receiving it at 50 mg per kg there was a significant increase in the persentage of redioactivity excreted in the urine and a significant decrease in that eliminated at 14 CO2 at the higher dosage level. The route of dose administered did not affect the following pharmacokinetic parameters at the 50 mg/kg level: bioavailability, total clearance or terminal half-life.

A number of major differences were elucidated in a comparison of the metabolism and pharmacokinetics of DETA and EDA. DETA was excreted through the feces to a greater extent than EDA and degraded to 14CO2 to a lesser extent. Tissues contained one-fifth to one-twentieth the concentration of radioactivity from DETA than from EDA when administered at comparable levels. These facts may have important implication in the comparative toxicities of the two chemicals.