Methylphosphonic acid, compound with amidinourea (1:1)

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/1995-03/1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Pre-guideline method, but data comprehensible and scientifically acceptable. (GLP: Yes) Bronaugh, R.L., R.F. Stewart and E. R. Congdon (1982) Methods for in vitro percutaneous absorption studies II. Animal Models for Human Skin Toxicology and Applied Pharmacology 62, 481-488

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

no guideline available

Guideline:

other: Bronaugh, R.L., R.F. Stewart and E. R. Congdon (1982) Methods for in vitro percutaneous absorption studies II. Animal Models for Human Skin Toxicology and Applied Pharmacology 62, 481-488

Deviations:

not specified

Principles of method if other than guideline:

Skin Permeability In vitro Absorption Through Porcine Ear Skin with FLOVAN CGN

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methylphosphonic acid, compound with amidinourea (1:1)

EC Number:

282-758-4

EC Name:

Methylphosphonic acid, compound with amidinourea (1:1)

Cas Number:

84402-58-4

Molecular formula:

C2H6N4O.CH5O3P

IUPAC Name:

Phosphonic acid, P-methyl-, compd. with N-(aminoiminomethyl)urea (1:1)

Details on test material:

technical product

Radiolabelling:

no

Test animals

Species:

other: porcine ears

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

Porcine ears were obtained from a local slaughter-house on the day of slaughter and before the pigs were steam-cleaned. The outer ear region was washed and cleaned with cold water. After carefully shaving, the skin was removed by dissection. The thickness of the dissected skin was 2-3 millimetre. The surface of the skin which was in contact with the test substance during the permeation-assay was 1.13 cm2.

For the determination of the absorption of the test article the skin was mounted in glass diffusion chambers with an area of 1.13 cm2 (area of skin) and a volume of 7 ml. These chambers are subdivided in an upper part (donor chamber) and in a lower part (receptor chamber). A volume of 7 ml physiological saline (0.9 % NaCl-solution) was placed in the receptor chamber of each diffusion cell, followed by the application of 226 pi of the test article dissolved in water (2 mg/ml) to the donor chambers. The top of the donor chamber was covered with parafilm. The diffusion chambers were placed in an incubator at 37° C. Samples (0.5 ml) were drawn from the receptor chamber after 0, 0.5, 1, 2, 4, 6, 8 and 24 hours and analysed. The volume of the fluid in the receptor chamber was kept constant by the addition of 0.5 ml of fresh receptor fluid to the receptor chamber immediately after removal of each sample.

Administration / exposure

Type of coverage:

other: pig skin inbetween donor chamber and acceptor chamber

Vehicle:

water

Duration of exposure:

Samples (0.5 ml) were drawn from the receptor chamber after 0, 0.5, 1, 2, 4, 6, 8 and 24 hours and analysed.

Doses:

226 µl of the test material dissolved in water (2 mg/ml) were applied to the donor chamber in the first and second experiment.

No. of animals per group:

2 Experiments, each using 4 glass diffusion chambers in parallel.

Control animals:

no

Details on study design:

No check of integrity of the skin preparations.
Acceptor fluid (0.9% NaCl) of sampling interval 0 hours (blank) showed no detector signal within the retention time of the substance.

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Percutaneous absorption

Percutaneous absorption 1

Dose:

2mg ai/ml

Parameter:

percentage

Absorption:

<= 0 %

Remarks on result:

other: 8 hours

Remarks:

The concentration (receptor fluid) did not exceed the lower limit of detection prior to 8 hours after start of experiments.

Percutaneous absorption 2

Dose:

2mg ai/ml

Parameter:

percentage

Absorption:

< 5 %

Remarks on result:

other: 24 hours

Remarks:

In the 24 hours speciemens (receptor fluid) the concentration of the permeated test article varied in the range of 0 to 12.7% of the amount applied and averaged to 4.6% +/- 4.2% (n=8).

Conversion factor human vs. animal skin:

In vitro dermal absorption studies are preferably carried out with human skin preparations; pig skin is considered a suitable alternative (EHC 235: Dermal Absorption, page 109). No interspecies conversion of animal data is necessary.
Percutaneous penetration refers to in vitro experiments and represents the amount of topically applied test substance that is found in the receptor fluid - this quantity is taken as systemically available (R.7C p.156). No conversion of in vitro data is necessary.

Any other information on results incl. tables

Since permeation of the test article through porcine skin was very low a graphical evaluation of

the analytical data was impossible. The concentration of the permeated test article did not

exceed the lower limit of detection prior to 8 hours after start of experiments.

The permeation rates (slope) and permeation constants (Kp) could not be determined since no

measurable penetration of the skin occurred prior to 8 hours.

Applicant's summary and conclusion

Conclusions:

The test article FLOVAN CGN was assessed for its potential to permeate through porcine skin.
The assay was performed in two independent experiments over a time scale of 0 to 24 hours. The test article was tested at the following concentration (a.i):
Experiment I and II: 0.94 mg/ml in water. Samples were drawn from the receptor chambers following a fixed schedule:
Exp. I and II: 0.0; 0.5; 1.0; 2.0; 4.0, 6.0; 8.0 and 24 hours following the application of the test article.
The samples (0.5 ml each) were labelled and transferred to the sponsor's facilities for subsequent analysis.

Specimens of a pig ear skin penetration study were separated by reversed phase HPLC followed by UV-detection at 200 nm. The separated and detected FLOVAN CGN a.i. was quantified by an external calibration function. The bottom quantification limit of the practical work amounted to 1.28 µg/mL FLOVAN CGN a.i. in aqueous solution. By means of a serial connection of two analytical HPLC-columns penetrated FLOVAN CGN a.i. in the specimens of the 24 h sampling interval were quantified. Within 24 hours less than 5 % (mean value) of the amount of FLOVAN CGN applied to the test system penetrated*. Within 8 hours no MPAAU were detected in the receptor chamber.

*) test substance applied to the test system: 212.44 µg FLOVAN CGN a.i.; 452.00 µg FLOVAN CGN
5 % of the test substance: 10.62 ug FLOVAN CGN a.i.; 22.60 ug FLOVAN CGN

Discussion: Since permeation of the test article through porcine skin was very low a graphical evaluation of the analytical data was impossible. The concentration of the permeated test article did not exceed the lower limit of detection prior to 8 hours after start of experiments.

In conclusion, it can be stated that FLOVAN CGN showed very low penetration through the skin of porcine ears. Within 8 hours no MPAAU were detected in the receptor chamber. Within 24 hours less than 5 % (mean value) of the amount of FLOVAN CGN applied to the test system penetrated.