Dimethyl methylphosphonate

Basic toxicokinetics

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:

other: well documented publication

Data source

Reference

Reference Type:

publication

Title:

Biotransformation and Male Rat-Specific Renal Toxicity of Diethyl Ethyl- and Dimethyl Methylphosphonate

Author:

Blumbach K, Pähler A, Deger HM and and Dekant W

Year:

2000

Bibliographic source:

Toxicological Sciences 53: 24-32

Materials and methods

Objective of study:

metabolism

Principles of method if other than guideline:

Evaluation of the biotransformation of both Dimethyl-methylphosphonate (DMMP) and Diethyl-ethylphosphonate (DEEP) in male and female rats after single oral doses of 50 and 100 mg/kg bw. Alpha2u-globulin in renal enal cytosol fractions was also analyzed

GLP compliance:

no

Remarks:

well documented study

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl methylphosphonate

EC Number:

212-052-3

EC Name:

Dimethyl methylphosphonate

Cas Number:

756-79-6

Molecular formula:

C3H9O3P

IUPAC Name:

dimethyl methylphosphonate

Details on test material:

- Name of test material (as cited in study report): dimethyl methylphosphonate (DMMP) obtained from Aldrich Chemical Company (Deisenhofen, Germany)
No additional details provided; all chemicals used were reagent, electrophoresis, or gradient grade as commercially available.

Radiolabelling:

no

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: adult Fischer F344 rats were obtained from Harlan Winkelmann (Borehen, Germany).
- Age at study initiation: 125-136 days
- Weight at study initiation: 282-317 g
- Acclimation period: before the metabolism studies, the animals were allowed to become accustomed to the metabolism cages for 3 days and control urine was collected for 12 h before the exposures.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Photoperiod (hrs dark / hrs light): 12-hour light/dark eyde

No additional data provided

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

To study alkylphosphonate metabolism, male and female rats (4 per dose per sex) were orally administered the alkylphosphonates dissolved in corn oil (1 ml/kg bw). Dose levels for the metabolism studies were lower than those for the alpha2u-globulin-accumulation studies for DMMP, in order to be able to compare DMMP and DEEP biotransformation without reaching doses saturating biotransformation pathways.
No additional details provided.

Duration and frequency of treatment / exposure:

the animals were treated once for biotransformation study and daily for 5 days for alpha2u-globulin content study

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50 and 100 mg/kg bw per day for biotransformation study and 0, 500 and 1000 mg/kg bw per day for alpha2u-globulin content study

No. of animals per sex per dose / concentration:

4 per dose for biotransformation study and 5 per dose for alpha2u-globulin content study

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

positive controls animals received five doses of 500 mg/kg bw trimethylpeotane (TMP) in the alpha2u-globulin content study

Details on study design:

- Dose selection rationale: DMMP doses were selected to compare with those used in the bioassay, lower doses of DEEP bad to be used, due to its toxicity. Animals were sacrificed by cervical dislocation 4 h after the last dosing, and kidney cytosol was prepared.

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, feces
- Time and frequency of sampling: After dosing, animals were immediately transferred into metabolic cages (Macrolon-) and urine and feces were collected at 12-h intervals for 72 h
- From how many animals: all
- Method type(s) for identification: metabolites were identified by [13]P-NMR; metabolites were quantified by GC/MS
- Limits of detection and quantification: not specified
- Other: during collection, samples were kept at 4°C. Samples were stored at -20°C until analysis

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

not evaluated

Details on distribution in tissues:

not evaluated

Details on excretion:

Methyl methylphosphonate was the major product recovered in urine of male rats. In female rats, unchanged DMMP was the major product excreted with urine.

The major pathway of excretion for orally administered DMMP and DEEP is urine, in part both compounds are excreted as parent compound (due to having the same water solubility) and as metabolites (Tables 1 and 2). Excretion is rapid with elimination half-lives between 3 and 6 h in rats. The concentrations of metabolites and parent compounds were below the limit of detection at 36 h after oral administration. After administration of both compounds, most of the administered doses were recovered in urine of female rats within 24 h after administration. In male rats, for both compounds, the recovery of metabolites in urine was significantly lower and accounted for only 58 to 74% of the administered doses within 24 h after administration (Tables 1 and 2).

Toxicokinetic parameters

Toxicokinetic parameters:

other: see Tables 1&2

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

When urine samples were subjected to [31]P-NMR to detect phosphorous-containing metabolites, urine samples from DMMP-treated rats showed two signals not present in control urine. The two signals had identical chemical shifts as authentic DMMP (δ 38.5) and methyl methylphosphonate (δ 28.3) when measured using control urine as matrix, suggesting that DMMP and methyl methylphosphonate are excreted in urine. The structure of the products was also confirmed by GC/MS of chloroform extracts (DMMP) or dried urine sampies subjected to silylation (data not shown). Thus, DMMP and methyl Methylphosphonate are identified as urinary excretion products in DMMP-treated rats.

Any other information on results incl. tables

Table 1: Biotransformation of DMMP to urinary metabolites in rats after oral administration

Sex	Dose level (mg/kg bw)	Metabolite recovered in urine (μmol) [Elimination half-life]			Percentage of administered dose(a)
		DMMP [3.3 h]	MMP [3.4 h]	Σ of metabolites
Male	50	31.7±2.2	52.4±5.2	84.1±3.9	68.1
	100	78.3±5.1	70.4±4.8	148.7±4.6	58.5
Female	50	68.9±4.2**	31.8±2.0**	100.7±3.2*	92.8**
	100	127.4±6.8**	67.1±8.2*	194.5±7.5**	87.8**
Note: half-life of urinary elimination of parent compound and metabolite are included. Results are mean ± SD from 4 rats per dose level and 2 analytical determinations per sample; (a); % of administered dose recovered as parent compound and metabolites in urine. Half-life of elimination was not significantly different between male and female rats; *: P<0.05, statistically significant differences between male and female rats in excretion; **: P<0.01

 

DEEP

Analysis of urine samples from DEEP-treated rats by [31]P-NMR showed three new NMR-signals identical in chemical shift to DEEP (δ 30.8), ethyl ethylphosphonate (δ 27.9), and ethylphosphonate (δ 25.7). The structures of these excretion products were also confirmed by GC/MS analysis of chloroform extracts (DEEP) or dried urine samples subjected to silylation (data not shown).

 

With DEEP, the monoester also was the major excretory product; however, in contrast to DMMP, further hydrolysis to ethylphosphonate occurred in low yields.

 

Table 2: Biotransformation of DEEP to urinary metabolites in rats after oral administration

Sex	Dose level (mg/kg bw)	Metabolite recovered in urine (μmol) [Elimination half-life]				Percentage of administered dose(a)
		DEEP	Ethyl ethylphosphonate		Ethylphosphonate Σ of metabolitcs
		[t1/2=6.1 h]	[t1/2=3.5 h]	[t1/2=4.1 h]
Male	50	18.9±4.2	29.1±5.0	8.3±2.4	56.1±3.9	59.4
	100	72.1±6.3	50.4±1.9	14.8±2.9	137.3±3.7	73.7
Female	50	50.7±4.8**	20.1±2.9*	7.4±2.4	78.2±3.4*	103.7**
	100	75.6±6.2	51.3±5.1	19.2±2.9*	146.1±4.7*	89.2*
Note: half-life of urinary elimination of parent compound and metabolite are included. Results are mean ± SD from 4 rats per dose level and 2 analytical determinations per sample; (a); % of administered dose recovered as parent compound and metabolites in urine. Half-life of elimination was not significantly different between male and female rats; *: P<0.05, statistically significant differences between male and female rats in excretion; **: P<0.01

RENAL EFFECTS OF DMMP AND DEEPRelative kidney weights were increased in male rats dosed with DMMP, DEEP, and TMP. Alpha2u-globulin in kidney cytosol was separated and quantified by capillary electrophoresis and by SOS-PAGE and Western blotting. In DMMP-, DEEP-, and TMP-treated rats, dose-dependent increases in the alpha2u-globulin content were observed by both methods in male, but not female rats. The increase of alpha2u-g1obulin accumulation was accompanied by the formation of protein droplets in the proximal tubules of male rats. These data demonstrate that the sex specific increase in kidney tumors by DMMP in male rats may be due to alpha2u-globulin accumulation and that similar toxic effects are to be expected from DEEP