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Toxicological information

Basic toxicokinetics

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Administrative data

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Absorption, distribution,|and excretion of 1,3-diphenylguanidine in the male F344 rat.
Author:
Ioannou YM and Matthews HB
Year:
1984
Bibliographic source:
Fundam Appl Toxicol, 4, 22-29.

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Administration iv (one dose) and oral (3 doses). Group of 3 males/dose. Radiolabeling.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-diphenylguanidine
EC Number:
203-002-1
EC Name:
1,3-diphenylguanidine
Cas Number:
102-06-7
Molecular formula:
C13H13N3
IUPAC Name:
1,3-diphenylguanidine
Details on test material:
Substance purchased from New England Nuclear (Boston).
Radiochemical purity = 99% (HPLC) - Specific activity = 1.3 mCi/mmol
Radiolabelling:
yes
Remarks:
1.3-[ring-14C(U)] DPG

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Kingston, NY)
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: 180-220 g
- Fasting period before study: no data
- Housing: individual
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): ad libitum (pelleted NIH 31 rat chow)
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS : no data

Administration / exposure

Route of administration:
other: i.v. or oral
Vehicle:
other: ethanol and emulphor EL-620 and water
Details on exposure:
For administration of DPG (intravenous or oral) the desired amount of compound was dissolved in ethanol and emulphor EL-620 and water was added to a final solvent ratio ethanol:emulphor:water 1:1:8. radiolabeled DPG was diluted as needed with unlabeled DPG such that the dose administered orally were 1,52, 15.15 and 151.5 µmol/kg (0.32 , 3.2 and 32.0 mg/kg) at 1 ml/kg and represented 0.001 , 0.01 and 0.1 of the LD50 of DPG to rats.
The radioactivity administered at each dose level was approximately 15 µCi/kg.
The iv dose of 15.15 µmol/kg was administered into a tail vein for tissue distribution studies or into an exposed femoral vein for studies of biliary excretion.
Duration and frequency of treatment / exposure:
One administration. Animals were treated between 9 and 10 AM, then were killed at time points from 15 min to 3 days after DPG administration.
Multiple administrations : animals were administered daily oral doses of 15.15 µmol/kg DPG by intubations for a total of 1, 3 or 9 days.
Doses / concentrations
Remarks:
Doses / Concentrations:
One administration : IV = 15.15 µmol/kg ; Oral = 1.52, 15.15 or 151.5 µmol/kg
Multiple administrations : Oral = 15.15 µmol/kg
No. of animals per sex per dose / concentration:
One administration : 9 males / dose
Multiple administrations : 3 males/ dose
Control animals:
no
Positive control reference chemical:
No
Details on study design:
No
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled : urine, feces, blood, muscle, liver, adipose, skin, kidney, lung, spleen, heart, brain, thymus, testes, adrenals, intestinal, bile
- Time and frequency of sampling: 15 min to 3 days after DPG administration.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled : urine, feces, bile and some tissues (liver, muscle, kidney, lung, skin, adipose)
- Time and frequency of sampling: 0.75 h, 2h, 6h, 24h
- From how many animals: 3 males at each time point
- Method type(s) for identification : HPLC
Samples were collected for 45 min in 0.5 ml fractions and the radioactivity in each fraction was quantitated by liquid scintillation counting. A radioactivity monitor was used in connection with the HPLC for instant detection of radioactive peaks.
- Limits of detection and quantification: no data

TREATMENT FOR CLEAVAGE OF CONJUGATES:
Radioactivity excreted in urine and bile was subjected to enzymatic and/or chemical hydrolysis to free possible conjugates of DPG matabolites. For enzymatic digestion the incubation mixture contained 50 nmol of DPG-derived radioactivity, 2000 units of beta-glucuronidase, or 20 units of aryl sulfatase in a total volume of 600 µl of sodium acetate buffer. Similar incubations minus enzyme served as controls.
Statistics:
Data from tissue distribution and excretion of DPG-derived radioactivity were analyzed by a nonlinear regression analysis computer program based on decay curves following an exponential pattern. The number of exponential terms was determined by the best fit. In all cases the data are expressed as the mean +/- standard deviation.

Results and discussion

Preliminary studies:
No data

Toxicokinetic / pharmacokinetic studies

Details on absorption:
A comparison of DPG tissue distribution and excretion following oral vs iv administration of 15.15 µmol/kg indicates that gastrointestinal absorption of DPG was near complete and that tissue distribution and excretion were not significantly affected by the route of administration (Table 1). A comparison of tissue distribution and excretion over the 100-fold dose range studied indicates that absorption and disposition of DPG are not significantly affected by dose in the range studied (Table 1).
Details on distribution in tissues:
Major organ and tissue volumes were sampled for radioactive content at various time points following iv administration of a 15.15-µmol/kg DPG dose. Initially the highest concentration (% total dose/g tissue) of DPG-derived radioactivity was observed in liver followed by kidney and lung (Table 2). The peak concentration in liver was reached in 45 min after administration whereas the DPG-derived radioactivity in other tissues with the possible exception of testes and adipose tissues showed a decline. The concentration of DPG-derived radioactivity in liver was higher than in other tissues at every time point examined. At 24 hr post-exposure the concentration of DPG in liver was 5-10 times higher than in most other tissues. Interestingly, the brain and most lean tissues contained similar concentrations of DPG-derived radioactivity at comparable time points.
The distribution of radioactivity in rat tissues at various time points following a single iv dose of DPG of 15.15 µmol/kg is presented in Table 3. DPG-derived radioactivity was readily cleared from all tissues so that within 24 hr after exposure the total tissue burden was approximately 10-fold lower than that observed at the earliest time point, 15 min (Table 3).
Clearance of DPG-derived radio-activity from the tissues followed a biphasic curve. The initial phase of the curve was rapid and accounted for a major portion of the dose. The second component was much slower. The rapid and relatively nonspecific distribution of DPG front blood to the other tissues is well illustrated by the amount of DPG in muscle. Muscle accounts for approximately 50% of the tissue volume of the rat, has no apparent affinity for DPG, and contained approximately 40%, of the DPG dose within 15 min after an iv administration. This radioactivity was rapidly cleared from muscle so that by 24 hr after injection only 1.2% of the total injected dose was still present (Table 3). The data in Table 2 indicate that clearance of DPG-derived radioactivity front most lean tissues occurred at rates similar to those described for muscle. Skin and adipose tissue receive a lower portion of the blood supply than do the lean tissues and thus received less DPG following the iv dose. The lower perfusion rate of adipose tissue may account for the fact that the peak concentration of DPG-derived radioactivity in this tissue was observed at 45 rather than 15 min. The initial higher concentrations of DPG-derived radioactivity in liver and kidney may be accounted for by the higher perfusion of these tissues with blood. In kidney, this concentration decreases sharply from 6 to 24 hr which might suggest that DPG elimination through urine is near complete by 24 hr. On the other hand, DPG appears to have an affinity for liver as evidenced by the higher DPG concentrations in liver at all time points examined (Table 2). Clearance of most of the DPG-derived radioactivity from liver appears to be similar to that of other tissues. However, the concentration of DPG-derived radioactivity in liver remained high relative to other tissues because of the higher concentrations initially sequestered by liver. The initial higher concentration of DPG-derived radioactivity in kidney was cleared more rapidly than from other tissues.
Transfer into organs
Transfer type:
blood/brain barrier
Observation:
slight transfer
Remarks:
< 1% of total dose
Details on excretion:
Total excretion of [14C]DPG-derived radioactivity was determined by daily collection of urine and feces from each animal held from 1 to 3 days. Approximately equal amounts of radioactivity were excreted in both urine and feces and the relative amounts of excretion by these routes were not affected by the dose in the range studied or the route of administration (Table 1). Approximately 80% of the radioactivity is excreted in urine and feces 24 hr after an iv dose and total excretion approaches 100% in 3 days. Total clearance followed a single component exponential decay with a half-life of approximately 9.6 hr. These results indicate that DPG is not at all persistent in the rat. The importance of the feces as a route of DPG elimination indicated that much of the DPG-derived radioactivity might be eliminated in bile. The elimination of DPG in bile was studied by cannulating the common bile duct of anesthetized rats. Approximately 50% of the injected dose was excreted within 2 hr and up to 75% of the total dose excreted in 6 hr. These results indicate that DPG excretion in feces (55% in 3 days) accounts for only a portion of the DPG excreted in bile. That portion of the DPG-derived radioactivity exereted in bile and not excreted in feces most probably undergoes extensive enterohepatic recirculation and is subsequently excreted in urine. while in muscle and kidney this peak accounted for 50 and 60% of the radioactivity, respectively. In liver a major metabolite (Peak III) appeared 24 hr after DPG administration and accounted for approximately 60% of the extracted radioactivity (Table 4). Radioactivity extracted from lung, skin, and adipose tissue at the 45-min and 2-hr time points was present only in the form of the parent compound. The radioactivity extracted from other tissues at the 24-hr time point was insufficient for accurate metabolite determination.

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
The nature of the [14C]DPG derived radioactivity excreted in urine and bile was examined by direct HPLC analysis (Table 4). Bile contained only small amounts of parent compound at all time points examined. Most of the radioactivity in bile (95%) was in the form of a major metabolite (Peak II) of DPG with traces of another metabolite (Peak I). The major metabolite (Peak II) excreted in bile was resistant to hydrolysis by arylsulfatase, by strong acid, or by strong base. However, incubation of this metabolite with b-glucuronidase resulted in near complete hydrolysis to yield metabolite V. It is believe that this metabolite (Peak II) is in the form of a glucuronide, the position of glucuronidation has not been determined. DPG-derived radioactivity excreted in feces was primarily (94%) in the form of metabolite V. Therefore, it appears that the glucuronide present in bile (Peak II) was subsequently hydrolysed in the intestine, most probably by intestinal flora, to release metabolite V which accounted for most of the radioactivity excreted in feces. HPLC analysis of urine indicated that around 28% of the radioactivity excreted in urine was in the form of parent compound. The major metabolite (Peak II) in urine accounted for approximately 37% of the total radioactivity. Treatment of this metabolite with b-glucuronidase resulted in its hydrolysis to yield metabolite V. Comparison of excretion in bile versus feces indicates that as much as 30% of the total dose is reabsorbed from the intestine after excretion in bile. Since most of this material is metabolite V, reabsorption from the intestine and reconjugation may account for most of the metabolite II excreted in urine. Two other metabolites were detected in urine. Metabolite III which eluted from the column shortly after peak II accounted for approximately 32% of the radioactivity while the unconjugated metabolite V accounted only for 3% of the radioactivity. The nature of radioactivity retained in some of the major tissue volumes was determined at several time points after an iv injection of 15.15 µmol/kg DPG. Tissues were extracted and the extracts analysed HPLC. With the exception of liver, all tissues examined at 45 min after DPG administration contained only the parent compound. In the liver, approximately 88% of the radioactivity was present as parent compound while the rest was present as a single metabolite, Peak II (Table 4). At the 2-hr time point, Peak II was present only in liver and muscle and accounted for approximately 18 and 10% of the radioactivity, respectively. At the 6-hr time point, Peak II increased slightly in liver,

Any other information on results incl. tables

Table 1: Distribution and excretion of radioactivity 1 day
after administration of [14C]-DPG to F344 male rats

-------------------------------------------------------
                        Percentage total dose
        -----------------------------------------------
        Intravenous             oral
        ---------  ------------------------------------
-------------------------------------------------------
Tissue  15.15         1.52        15.15     151.5 mmol/kg
-------------------------------------------------------
Liver   1.37±0.08  1.31±0.09   1.23±0.11  0.92±0.09
Muscle  1.18±0.08  1.08±0.02   1.08±0.01  1.09±0.08
Adipose 0.56±0.07  0.62±0.03   0.47±0.03  0.49±0.03
Skin    0.52±0.07  0.40±0.41   0.41±0.05  0.39±0.02
Blood   0.24±0.01  0.27±0.01   0.23±0.01  0.24±0.02

Total excreted
Urine   35.50±3.38 31.76±2.68  29.12±1.72 43.61±2.83
Feces   45.67±9.01 48.25±4.49  45.26±2.94 39.39±1.84

Total*  81.17±6.12 80.01±6.24  74.38±1.27 83.00±2.41
-------------------------------------------------------
* DPG-derived radioactivity excreted in urine and feces in
24 hr. The remainder is still present in tissues and
intestinal contents.

Table 2: Concentration of DPG-derived radioactivity in male
F-344 rats vs time
----------------------------------------------------------
                Percentage total dose#/g tissue
        --------------------------------------------------
Tissue    15 min    45 min     2 hr    6 hr         24 hr
----------------------------------------------------------

Liver   2.20±0.23 2.44±0.41 1.21±0.33 0.42 0.09  0.17±0.02
Kidney  1.82±0.30 1.35±0.35 0.38±0.14 0.19±0.06  0.02±0.004
Muscle  0.44±0.04 0.26±0.08 0.08±0.04 0.02±0.01  0.01±0.009
Blood   0.24±0.02 0.18±0.03 0.07±0.02 0.03±0.006 0.02±0.001
Skin    0.20±0.05 0.18±0.02 0.08±0.02 0.03±0.01  0.02±0.002
Adipose 0.10±0.01 0.11±0.04 0.04±0.02 0.02±0.01  0.03±0.003
Lungs   1.05±0.16 0.35±0.12 0.19±0.06 0.06±0.03  <0.01
Spleen  0.49±0.02 0.27±0.04 0.09±0.03 0.04±0.02  <0.01
Heart   0.41±0.05 0.23±0.05 0.07±0.02 0.02±0.01  <0.0l
Brain   0.39±0.03 0.32±0.03 0.09±0.02 0.02±0.01  <0.01
Thymus  0.34±0.01 0.27±0.03 0.07±0.01 0.02±0.01  <0.01
Testes  0.10±0.02 0.19±0.05 0.14±0.02 0.06±0.002 <0.01
Adrenals                    0.14±0.03 0.06±0.03         <0.01
----------------------------------------------------------
#15.15 mmol/kg, iv.



Table 3: DPG-DERIVED RADIOACTIVITY IN MAJOR F344 RAT TISSUES
VS TIME
----------------------------------------------------------
                        Percentage total dose
        --------------------------------------------------
Tissue   15 min      45 min     2 hr       6 hr      24 hr        
----------------------------------------------------------
Blood   3.52±0.33  2.60±0.41  1.18±0.15  0.55±0.05 0.24±0.01
Liver   17.69±1.73 20.04±2.65 10.17±1.13 3.27±0.51 1.37±0.08        
Kidney  3.05±0.61  2.24±0.72  0.62±0.21  0.30±0.08 0.03±0.01
Thymus  0.18±0.03  0.11±0.03  0.03±0.01  <0.01     <0.01
Skin    5.73±1.26  5.05±0.52  2.86±0.39  0.87±0.37 0.52±0.07
Adipose 1.89±0.13  2.08±0.87  0.80±0.12  0.44±0.34 0.56±0.07        

Muscle  40.01±2.28 22.88±7.73 8.33±2.56  1.99±1.31 1.18±0.08
Brain   0.64±0.06  0.55±0.03  0.15±0.03  0.02±0.01 <0.01
Spleen  0.21±0.03  0.11±0.01  0.04±0.02  0.01±0.01 <0.01
Testes  0.24±0.05  0.43±0.11  0.32±0.04  0.15±0.01 <0.01
Lungs   0.96±0.10  0.39±0.12  0.22±0.08  0.06±0.03 <0.01
Heart   0.27±0.04  0.15±0.03  0.05±0.01  <0.01     <0.01
Small 
intest. 0.82±0.22  1.28±0.48  1.90±0.46  0.50±0.15 0.05±0.05        
Small intest. cont.        
        1.31±0.46  7.15±2.15  11.43±2.17 3.57±1.89 0.17±0.10
Large intest.        
        0.40±0.21  0.70±0.30  0.32±0.39  0.51±0.25 0.15±0.12
large intest. cont.
        0.08±0.03  0.10±<0.01 0.10±0.05  7.74±3.71 1.44±0.29
----------------------------------------------------------
#iv dose of 15.15 µmol/kg.
 
 
Table 4: relative amounts of DPG and DPG-metabolites present
in male F344 rat liver and excreta.
----------------------------------------------------------
                                DPG metabolite (%)
                ------------------------------------------
        Time
Tissue        (hr)        I*    II     III    IV     V      DPG(%)
----------------------------------------------------------
Liver        0.75             12±1.2                          88±5.7
Liver        2.00             18±1.9                          82±4.3
Liver        6.00             30±2.1                          70±6.0
Liver        24.00             30±3.3  60±4.5                     10±1.1
Bile#        6.00   2±1.2 95±1.7                          3±0.5
Urine        24.00             37±1.6  32±1.4           3±0.8          28±0.8
Feces        24.00                       2±1.0 94±3.5  4±1.4
----------------------------------------------------------
* DPG metabolites separated by HPLC.
Percentage represents only extractable radioactivity.
# Collected continuously for 6 hr after an iv injection of
15.15 µmol/kg DPG.

The residual radioactivity present in the liver after
multiple exposures of rats to DPG was also extracted and
analysed. The metabolites present were the same and at the
same ratio as those metabolites (Peaks II and III) extracted
from liver 24 hr after a single iv dose of DPG. The nature
of the radioactivity retained in the tissues after
extraction is not known.

MULTIPLE EXPOSURES. 
One, three, or nine daily doses of DPG were administered to
groups of three rats each. Rats were sacrificed 24 hr after
the last dose. Results of these studies indicated that most
DPG-derived radioactivity was readily cleared from all
tissues assayed and that DPG concentrations in all tissues
except liver were as low or lower after nine daily doses as
compared to a single dose. However, a minor portion of the
dose in liver was cleared more slowly than observed for
other tissues and the concentration of DPG-derived
radioactivity in liver increased significantly relative to
other tissues as the number of doses increased (Table 5).
Extraction and analysis of the persistent radioactivity from
liver demonstrated that it represented metabolites II and
III (Table 4). An analysis for radioactivity covalently
bound to liver macromolecules proved negative. Therefore,
the mechanism which accounts for the slower clearance of
this minor component from liver is unknown. Likewise, the
relevance of this slower component to any toxicity which
might be associated with DPG exposure is unknown.

Table 5: Effect of single and multiple DPG doses on tissue
concentrations of DPG-derived radioactivity
------------------------------------------------------
                Concentration (nmol/g)
        ----------------------------------------------
Tissue   1 Dose*          3 Doses         9 Doses
------------------------------------------------------
Liver   4.86±0.282      7.38±0.363      11.89±0.811
sloud   0.44±0.022      0.10±0.012      0.20±0.041
Kidney  0.58±0.106      0.29±0.042      0.72±0.134
Skin    0.49±0.063      0.09±0.020      0.28±0.090
Adipose 0.77±0.091      0.03±0.011      0.10±0.024
Muscle  0.36±0.024      0.04±0.005      0.02±0.005
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* Animals were administered DPG 15.15 µmol/kg orally and
sacrificed

Applicant's summary and conclusion

Conclusions:
This study has shown that DPG is readily absorbed from the gastrointestinal tract of rats, distributed quickly to all tissues examined, metabolized to three major and two minor metabolites, and excreted in urine and feces. Slower clearance of a minor component was observed in liver, but the significance of this observation is unknown.
Executive summary:

DPG (1,3 -diphenylguanidine), a rubber accelerator, was readily absorbed from a gastrointestinal tract of the male Fischer rat and rapidly distributed throughout the body tissues. Absorption and disposition of DPG were not significantly affected by the route of administration or by the dose in the dose range studied, 1.5 to 150 µmol/kg. Most of the dose of DPG was excreted in the urine and feces at approximately equal amounts within 24 hr after oral or iv administration. Greater than 99% of the DPG dose was cleared into the urine and feces within 3 days efter administration. Approximately 30% of the DPG-derived radioactivity excreted in urine was the parent compound, DPG, while the remainder was present in the form of two major and one minor metabolite. Close to 95% of the radioactivity excreted in bile was in the form of a single major metabolite. Administration of multiple doses resulted in aproportional increase of DPG-derived radioactivity in the liver as the number of doses increased. DPG-derived radioactivity did not increase in other tissues following multiple doses.