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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Augst 2007 - 29 January 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
NON-RADIOLABELLED
Product name: TL 10 ST
Supplier: Albemarle

RADIOLABELLED
Position radiolabel: chloroethyl-14C(U)
Specific activity: 24.3 mCi/mmol
Supplier: Moravek
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 9 - 10 weeks
- Weight variation at study initiation: did not exceed +/-20% of average weight
- Fasting period before study:
- Housing: in groups of 4-5 during acclimatisation, individually during study
- Individual metabolism cages: yes
- Diet: ad libitum (commercial rat diet)
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
other: oral and intravenous
Vehicle:
other: oral: 0.5% hydroxypropyl-methyl cellulose in water; i.v. Cremophor ELP/ethanol 49.7: 50.3 (v/v)
Details on exposure:
DOSE PREPARATION
- Oral: test substance in 0.5% hydroxyproyl-methylcellulose in water, prepared freshly on day before dosing
- Intravenous: test substance mixed with 430 microL of Cremophor ELP/ethanol (49.7:50.3 v/v) and diluted with saline to 10 mL just before dosing
- Dose formulation consisted of radiolabelled material diluted with non-radiolabelled test substance to required concentrations
Duration and frequency of treatment / exposure:
single oral (group A+B) or intravenous (group C) dose
Doses / concentrations
Remarks:
Doses / Concentrations:
- Group A: 15 mg test substance/kg bw - oral
- Group B: 600 mg test substance/kg bw - oral
- Group C: 15 mg test substance/kg bw - intravenous
No. of animals per sex per dose:
4 male and 4 female animals/group
Control animals:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY
- Tissues and body fluids sampled: urine, faeces, blood kinetics, tissues, carcass (same for all 3 groups, except tissues only for group A+B) expired air (CO2 and volatiles) was collected at 24 h intervals for 48 h.
- Time and frequency of sampling: blood (100 microL) at 30 min, 1h, 2h, 3h, 4h, 6h, 8h, 24h, 48h, 96h after dosing and at sacrifice (7 days); urine and feaces collected daily; expired air sampled on day 1 and 2 after dosing

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine (in selected samples to cover > 90% of excreted dose)
- Time and frequency of sampling: twice, 0-24h and 24-48h after dosing
- From how many animals: pooled by group, sex and time point
- Method types for identification HPLC, Liquid scintillation counting, LC-MS/MS
Statistics:
Pharmacokinetic parameters calculated by EXcel program developed by TNO (2-compartment model for intravenous dose and 1-compartment model for oral dose)

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Following oral administration of [14C]-2,2-bis(chloromethyl) trimethylene bis(bis(2-chloroethyl) phosphate), highest concentrations of radioactivity in the blood were found at 8 hours post dosing in both sexes and both concentrations. The Cmax values for males and females of the low dose were comparable (1.73 and 1.5 µg/g, respectively), but were higher for females in the high dose (18.9 and 29.5 µg/g, for males and females respectively). The bioavailability derived from the area under the curve after oral dosing in comparison with that after i.v. dosing was 142 and 143% for males and females of the high dose group respectively and 47 and 55% for males and females of the low dose group respectively.
In the oral low dose and IV dose groups, the AUC0-168hr and AUC0-infinity values were comparable for males and females. However, in the oral high dose group, the AUC values were higher in females than males. The higher AUC values following the oral dose when compared with the IV dose are probably due to differences in metabolism, since the elimination half-lives for the two routes are comparable. The greater than 100% bioavailability seen is due to the slightly lower AUC0-infinity values observed for IV dose when compared with the oral dose (approximately 114 and 168 µg/g/hr, respectively). iIt should be noted that in calculating the bioavailability in the high dose, the reference IV AUC was taken from a lower dose IV administration. Also, after oral high dose administration only very little radioactivity attributable to the intact parent compound was found in the faeces (<1%) indicating practically complete absorption from the gastro-intestinal tract.
Total retention of radioactivity was around 2.5% after oral low dose and about 0.8% after high oral dose
Details on distribution in tissues:
The total recovered radioactivity was around 80%, regardless of the dose, route of administration or sex. Following oral dosing, the total retention of radioactivity was around 2.5% after the low dose and 0.8% after the high dose, with most of the radioactivity excreted within 3 days.
At 168 hours post dose, the highest concentrations of radioactivity were found in the liver, kidney, adrenals and abdominal skin in both sexes, and in uterus of both low and high dose females. The lowest radioactivity was found in brain, plasma and fat, the latter indicating no bioaccumulation of the test substance. Therefore, [14C]-2,2-bis(chloromethyl) trimethylene bis(bis(2-chloroethyl) phosphate), or its metabolites, was distributed all over the body, but no specific target organs, other than the organs of elimination, were identified.

- Distribution in tissues irrespective of dose administered (group A+B, oral dosing)
- Highest concentration of radioactivity found in liver, kidney, adrenals and abdominal skin
- Lowest concentration of radioactivity found in brain, plasma and fat
Details on excretion:
Excretion occurred mainly by the biliary route (approx. 60%), with excretion in urine approximately 20% and a small amount of radioactivity exhaled as 14CO2. Volatile radioactivity could not be detected; however the study report states that it is possible that part of the radioactivity was exhaled as ethylchloride or more likely 2-chloroethanol, as all major metabolites were missing an ethylchloride group. These compounds are very volatile and could not be trapped in the conditions of the experimental design. The study director also comments in the study report that this could explain the low recovery of total radioactivity (of 80%) since one ethylchloride group attributes 25% of the radioactivity of the molecule.
Total recovered excreted radioactivity was around 80% independently of dose route and sex
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
Cmax: 1.50-1.73 microg/g at 8h - oral, low dose
Test no.:
#2
Toxicokinetic parameters:
Cmax: 18.9-29.5 microg/g at 8h - oral, high dose
Test no.:
#3
Toxicokinetic parameters:
Cmax: 5.07-5.25 mircog/g at 0h - intravenous, low dose
Toxicokinetic parameters:
Tmax: oral all doses: 8 h
Toxicokinetic parameters:
half-life 1st: 102-111 h -elimination oral
Toxicokinetic parameters:
half-life 1st: 99-113h - elimination, intravenous
Toxicokinetic parameters:
AUC: 0-168 h low dose (m+f): 115.4 microg/g h
Toxicokinetic parameters:
AUC: 0-168 h high dose m: 1450 f: 1822 microg/g h
Toxicokinetic parameters:
AUC: 0-168 h i.v. (m+f) 81.3 microg/g h
Toxicokinetic parameters:
AUC: 0-infinity oral low dose m: 166.9, f: 168.3 microg/g h
Toxicokinetic parameters:
AUC: 0-infinity oral high dose: m: 2177, f: 2632 microg/g h
Toxicokinetic parameters:
AUC: 0-infinity i.v.: m: 113.9, f: 115.4 microg/g h
Toxicokinetic parameters:
other: Bioavailability: 142% - oral, low dose (male+female)
Toxicokinetic parameters:
other: Bioavailability: 47% (male), 55% (female) - oral high dose

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
- Small amount of intact radiolabelled test substance detected in feaces (<1%), only first 2 days, indicating almost complete absorption from gastro-intestinal tract
- At least 12 metabolites detected in urine, at least 14 in feaces
- 4 major metabolites (more than 5% of administered dose (4 in feaces, 1 also in urine)
- Metabolite 1: present up to 30% of administered dose (all groups), C11H22O8Cl5P2
- Metabolite 2: present up to 20% of administered dose (lower in low oral dose group), C13H24O10Cl5P2
- Metabolite 3 + 4: present up to 9% of administered dose (lower in high oral dose group), C11H23O9Cl4P2 and C13H25O11Cl4P2

Any other information on results incl. tables

Table 7.1.1.1 Elemental composition of metabolites 1-4 determined by LC-MS

Elemental composition

Difference from Parent

Test substance – V6

C13H25O8Cl6P2

-

Metabolite 1

C11H22O8Cl5P2

-C2H3Cl

Metabolite 2

C13H24O10Cl5P2

-HCl + 2O

Metabolite 3

C11H23O9Cl4P2

-C2H3Cl –Cl + OH

Metabolite 4

C13H25O11Cl4P2

-HCl +2O –Cl +OH

Metabolites 1-4 are either missing a chloroethyl moiety or the chlorine was replaced by an OH group, and further oxidised to a carboxyl group. The likely major metabolic pathways are the cleavage of one phosphate ester bond (metabolite 1) and the oxidation (substitution) of one chloroethyl sidechain to the corresponding hydroxyl and further oxidation to the carboxy group (metabolites 4 and 2, respectively). It is the opinion of the study director that metabolite 3 is likely to be a secondary metabolite of metabolite 1, undergoing a further hydroxylation on a second chloroethyl group.

- Administered dose was close to inteded dose for all animals

- No study or test substance related signs of toxicity or unusual behaviour were observed

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
The test substance was rapidly and completely absorbed from the gastrointestinal tract. The test substance and its metabolites were distributed all over the body. No target organs except the organs of elimination were found. Low levels of the test substance were found in muscle and fat, indicating no bioaccumulation. Most of the radioactivity was excreted in faeces and to a minor extend in urine within 3 days after administration of the test substance. The test substance is metabolised almost completely to a relatively high number of different metabolites. The likely major metabolic pathways are the cleavage of one phosphate ester bond (metabolite 1) and the oxidation (substitution) of one chloroethyl sidechain to the corresponding hydroxyl and further oxidation to the carboxy group (metabolites 4 and 2, respectively).
Executive summary:

In a toxicokinetic study 2,2-bis(chloromethyl)trimethylene bis(bis(2-chloroethyl)phosphate) (chloroethyl-14C) was administered to 4 male and 4 female Wistar rats per group at dose levels of 15 and 600 mg/kg bw by oral administration and at a dose level of 15 mg/kg bw by intravenous dosing.

Elimination half life of the test substance was 102 -111h after oral dose and 99 -113 after intravenous dose. Bioavalability was around 142% after low oral dose and 47-55% after high oral dose. Absorption from the gastrointestinal tract is almost complete. Recovery of total radioactivity was low (around 80%, independently of dosing route and sex), possibly due to exhalation of small volatile compounds that could not be trapped in this study.

The test substance and its metabolites were distributed all over the body. No target organs except the organs of elimination were found. Low levels of the test substance were found in muscle and fat, indicating no bioaccumulation. The substance is completely metabolised to a number of different metabolites. The likely major metabolic pathways are the cleavage of one phosphate ester bond (metabolite 1) and the oxidation (substitution) of one chloroethyl sidechain to the corresponding hydroxyl and further oxidation to the carboxy group (metabolites 4 and 2, respectively). The majority of the radioactivity is excreted within 3 days in the faeces and to a minor extent in the urine in the form of metabolites. Volatile metabolites are likely exhaled, but could not be trapped in the study due to the high volatility.

 

This GLP metabolism study in the Wistar rat is classified valid without restrictions and satisfies the guideline requirement for a metabolism study according to OECD Guideline No.417.