Dimethyl methylphosphonate

Specific investigations: other studies

Administrative data

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: scientifically acceptable publication

Data source

Reference

Reference Type:

publication

Title:

Quantitation of Alpha2u-Globulin in Rat Kidney Cytosol by Capillary Electrophoresis

Author:

Pähler A, Blumbach K, Herbst J, and Dekant W

Year:

1999

Bibliographic source:

Analytical Biochemistry 267: 203–211

Materials and methods

Principles of method if other than guideline:

Alpha2u-globulin in renal enal cytosol fractions was analyzed by capillary electrophoresis as protein–SDS complexes after treatment with test substance.

GLP compliance:

no

Remarks:

welldocumented publication

Type of method:

in vivo

Endpoint addressed:

carcinogenicity

Test material

Test material information

Constituent 1

Reference substance name:

Kaolin

Cas Number:

1332-58-7

IUPAC Name:

Kaolin

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.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fischer F344/N rats were obtained from Harlan Winkelmann (Borchen, FRG)
- Age at study initiation: adult animals were used
- Acclimation period: 3 days (before the experiments, the animals were accustomed to the metabolic 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-h light/dark cycle

No additional data provided

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No details data provided

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

5 days

Frequency of treatment:

once daily

Post exposure period:

At the end of treatment, animals were sacrificed by cervical dislocation and kidney cytosolic subcellular fractions were prepared.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500 and 1000 mg/kg bw per day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

no further details provided

Examinations

Examinations:

Rat kidney cytosolic subcellular fractions of treated rats were prepared and protein concentrations in liquid samples determined. The protein separation was then performed with an anion-exchange column and a high-performance liquid chromatography system. The eluent representing a2u-globulin was collected, pooled, desalted by gel filtration chromatography, lyophilized, and stored at 280°C.
Renal cytosol fractions were analyzed by capillary electrophoresis as protein–SDS complexes. Using a2u-globulin purified from urine of male rats, the limit of detection was 10 mg/ml sample in routine analyses. Excellent run to run reproducibility in migration time (CV≤4%) and peak areas corresponding to a2u-globulin (CV≤3%) after normalization to the internal standard was observed.

Positive control:

The test substance was evaluated together with 4 other compounds, some of which also showed a positive response

Results and discussion

Details on results:

Significant increases in renal alpha2u-globulin content (up to 85% of total protein content) compared to controls (approx 15%) were observed in kidney cytosol of rats treated with alpha2u-globulin nephropathy-inducing agents such as trimethylpentane or the alkylphosphonates dimethyl methylphosphonate and diethyl ethylphosphonate, but not in kidney cytosol of male rats treated with tris-(2- chloroethyl)phosphate or the nephrotoxic agent hexachlorobutadiene (see Tables 1&2). A good correlation of the alpha2u-globulin contents determined by capillary electrophoresis and immunoblotting with an a2u-globulin-specific antibody (r2=0.997) was obtained. Capillary electrophoresis provides a simple, rapid, and highly reproducible quantitation of a2u-globulin accumulation for renal tumorigens and may assist in the risk assessment process for these chemicals.

Any other information on results incl. tables

Table 1: Alpha2u-Globulin Content (Determined by Capillary Electrophoresis) in Kidney Cytosol of Male Rats Treated with Several Nephrotoxic Compounds(a)

Treatment	Alpha2u-Globulin Content (% of total protein)	CV(%) (= SD/mean*100)
Untreated control	14.7 ± 4.8	32
TCEP, 250 mg/kg	19.4 ± 6.1	31
HCBD, 200 mg/kg	9.1 ± 7.5	82
DMMP, 500 mg/kg	68.9 ± 3.4	5
DMMP, 1000 mg/kg	78.4 ± 5.1	7
DEEP, 50 mg/kg	56.4 ± 8.3	15
DEEP, 100 mg/kg	62.4 ± 4.2	7
TMP, 50 mg/kg	85.2 ± 7.0	8
Note: data are shown as means ± SD and show interanimal variation; (a) n = 5 animals per treatment group, samples of individual animals were run as triplicates

 

Table 2: Comparison of the Intraassay Variation in Determined alpha2u-Globulin Content in Individual Kidney Cytosol Samples from Untreated Male Rats and Male Rats Treated with alpha2u-Globulin-Inducing Agents by Western Blot Analysis with an alpha2u-Globulin-Specific Antibody or by Capillary Electrophoresis

Treatment	alpha2u-Globulin Conte
	Western blot/specific antibody (intensity relative to control)	CV(%) (SD/mean*100)	CE-SDS (% of total cytosolic protein)	CV(%) (SD/mean*100)
Untreated control	1.0 ± 0.1	14	15.3 ± 0.5	3
TCEP, 250 mg/kg	1.4 ± 0.4	31	17.5 ± 1.1	6
HCBD, 200 mg/kg	0.9 ± 0.2	23	10.2 ± 0.7	7
DMMP, 500 mg/kg	4.5 ± 0.7	16	69.5 ± 0.5	1
DMMP, 1000 mg/kg	5.4 ± 1.1	22	79.8 ± 2.6	3
DEEP, 50 mg/kg	3.6 ± 0.6	16	53.3 ± 4.1	8
DEEP, 100 mg/kg	4.4 ± 0.7	17	65.2 ± 1.0	2
TMP, 50 mg/kg	4.8 ± 1.0	20	81.0 ± 4.7	6
Note: data are means ± SD, n = 3, and were determined in three samples from one animal per treatment group