Percutaneous absorption of organophosphorus insecticides in pigs - the influence of different vehicles N. GYRD-HANSEN, L. BRIMER & F. RASMUSSEN

Department of Pharmacology and Pathobiology, Royal Veterinary and Agricultural University, Bulowsvej 13,1870 Frederiksberg C, Denmark

Gyrd-Hansen, N., Brinier, L., Rasmussen, F. Percutaneous absorption of‘ organophosphorus insecticides in pigs - the influence of‘ different vehicles. J. i~et. I’harviiacol. Therap. 16, 174-180.

In 40 experiments on 20 pigs three different organophosphor~~s insecticides (Oh) , parathion (n = (i), phoxim (11 = 7) and phosmet ( n = 7), were administered both intravenously (i.v.) and dermally (d.) ;IS ‘pour-ons’ i n a cross- over design in order to determine the dermal bioavailability of‘ the OPs. As percutaneous absorption of drugs may be affected by the vehicle used, three chemically different vehicles - DMSO, I-octanol and macrogol 400 - were used for the dermal administration of each o f the OPs. The pliarniacoki~ietic parameters measured showed that 15-30% of‘ dernially applied parathion is absorbed when administered in DMSO or octanol, hut only 4-58 when administered in macrogol. Absorption was fastest with DMSO and slowest wi th niacrogol. For the two ectoparasiticides, phoxim and phosniet, only between 0.5 and 3% of the dernial dose was absorbed with little difference i n the absorption rate between the three vehicles. On the basis of the very limited dermal bioavailability for these two OPs it seenu doubtful whether sufficient concentra- tioris can reach the ectoparasites through the systemic route.

N . Cyrd-Hunsen, Uepurlmeiit of I’/iurinurology and Palhobiology, Roycil Vetcririuiy und AgrTzculturul University, Coperihagepn, Denmark.

I N T R O D U C T I O N

Organophosphorus insecticides (OPs) are used in veterinary medicinal products for treatment of ectoparasites and parasitic skin conditions, e.g. mange, in domestic animals. For such treatment OPs are normally applied to the entire surface of the animals by washing, dipping or spraying. OPs are, however, also available as so-called pour-on preparations, which are applied to only a very limited part of the surface of the animals, most often to the skin in the middle of the back. Pour-on preparations of OPs are sup- posed to have systemic effect, i.e. to reach the ectoparasites after percutaneous absorption

and distribution in the body, rather than by being spread on the surface to all parts of the skin.

Whether OPs in pour-on preparations actually act systemically or not seems not to have been demonstrated conclusively, - sys- temic action would require a certain absorp- tion of the O P after dermal application. In goats such percutaneous absorption has been demonstrated for coumaphos with at least 5% of the coumaphos applied being absorbed (Konar & Ivie, 1988), and for another OP, ethion, where Mosha el ul. (1990) showed approximately 20% of a dermally applied dose to be absorbed.

Pour-on preparations of OPs are to a large

174

Percutaneous ahorption of OPs in Pigs 175

extent used to treat for ectoparasites in pigs. As little is known about percutaneous absorp- tion of drugs in pigs it was the purpose of the present study to establish the bioavailability of three OPs, parathion, phoxim and phosmet, after dermal application in pigs. As the compositions of the vehicles used in licenced OP pour-on preparations are commercial secrets it was decided to study the influence of different vehicles on the OP bioavailability.

MATERIALS A N D METHODS

Chemicals

Three different OPs - parathion (0,O- diethyl 0-(p-nitrophenyl) phosphorothioate), phoxim (phenylglyoxylonitrile oxime 0,O- diethyl phosphorothioate) and phosmet (0,O- dimethyl S-phthalimidomethyl phosphoro- dithioate)- and three vehicles with different degrees of polarity - dimethyl sulphoxide (DMSO), I-octanol and macrogol (Carbo- wax@) 400 - were used in the experiments.

The three OPs used are all medium polar substances, i.e. they show the highest solubil- ity, above 200 g/l, in solvents like acetone and toluene, whereas they are virtually insoluble in water and have a limited solubility in for example hexane. For an OP pour-on prepara- tion the vehicle should be a good solvent for the OP, i.e. concentrations of 100-200 g/1 should be possible, and at the same time the viscosity of the preparation at room tempera- ture should be such that application is easy and run-off is avoided.

All three vehicles mentioned above fulfill these demands although they differ consider- ably in physico-chemical characteristics, as shown in Table I.

Animal exfleriments

Forty experiments were performed on 20 clinically healthy pigs weighing between 18 and 37 kg. The animals were kept individually in pens and fed a commercial feed mixture twice daily according to weight and had free access to water.

Each pig was exposed to an OP first by the intravenous route (Lv.) and a week later by the dermal route (d.). The pigs were treated as shown in Table 11. The dermal doses for phoxim and phosmet are three and five times higher t.han those recommended for the corresponding commercial products (SebaciP pour-on vet. and Porect@ pour-on vet.), re- spectively.

The i.v. injection of the OPs was done through a catheter placed in an ear vein. The OPs were dissolved in glycerol-formal. Appro- ximately 20 blood samples were collected over the following 72 h through a catheter in a vein on the opposite ear.

Dermal application of the OPs was per- formed by dissolving the OP in 10 ml of the vehicle and applying the solution along the midline of the back of the pig taking care that run-off was avoided. The pigs were restrained on special tables for at least 3 h after dosing. Over the following 10 days around 25 blood samples were collected, at first through a

T A B L E I . Characteristics of the three OP vehicles used*

Property DMSO Octanol Macrogol

Miscible with water Yes insoluble Yes Miscible with ethanol Yes Yes Yes

Melting point, "C 18 - 16 0-10

Miscible with diethyl ether Yes Yes insoluble Molecular weight 78 130 400t

Skin absorption Yes Yes poor Skin irritant medium weak no Known absorption enhancer Yes no no

*Reynolds (1989); Budavari (1989); Sax (1984); ?Average MW.

176 N . Gyrd-Harw-en et al.

. l 'AHLE I I . Treatment with OPs in 20 pigs - dose, route and vehicle

Dose i.v. Dose d. Pig no. OP irigikg ingikg Vehicle d

25.26 27,2x 29,30 50, 5 1 33.54.55 52,53 45,46 48,49 47,56,57

Parathion Parat hion Parathion Phoxim Phoxim Phoxim Phosmet Phosn~et Phosrnet

1 1 1

10 I 0 1 0 10 10 10

50 50 50

100 100 100 100 100 I00

DMSO Octanol Macrogol DMSO Octanol Macrogol DMSO Ocr./GF* Macrogol

*C>ctanol:Glycerol-fornial (1 : 1). used because phosmet is slightly soluble in octanol.

catheter in an ear vein, and later from the bijugular trunk by means of a cannula.

Blood samples were cooled in ice-water immediately after collection. Plasma was sepa- rated from the heparinized blood and stored at -20°C until analysed.

Analytical methods

The quantification of OPs in plasma was done by gas chromatography. The OPs were extracted from plasma by equal amounts of hexane containing ethion as an internal stand- ard. Some hexane extracts were concen- trated in order to determine low OP levels. The hexane extracts were analysed .on a Hewlet-Packard 5890A gas chromatograph with a nitrogen phosphorus detector (NPD). Chromatographic conditions were as follows: Column - 5 m X 530 Fm HP-1. Carrier gas - Helium, flow rate 15 ml/min. Tempera- tures - Injector 185°C for parathion and phoxim and 240°C for phosmet; column 165-195°C for parathion and phoxim and 210°C for phosmet; detector 250°C for para- thion and phoxim and 290°C for phosmet. Injection volume 4 PI. Retention times - Parathion 2.0 min, phoxim 2.0 min, phosmet 3.1 min and ethion 4.1 min (parathion and phoxim) and 2.0 min (phosmet). Detection limits - Parathion 1 ppb, phoxim 3 ppb and phosmet 3 ppb. All solvents used were of analytical stanclarti.

Pharmacokinetic analysis

Non-compartmental pharmacokinetic para- meters were calculated (Gibaldi & Perrier, 1982) including area under the plasma con- centration vs time curve (AUC), mean resi- dence time (MRT), mean absorption time (MAT), body clearance ( C I B ) , volume of distribution at steady-state (Vss) and the half- life for the terminal elimination phase (t,12k). The bioavailability (%) of an OP after der- mal application was calculated as the ratio between the AUCs after dermal and i.v. admi- nistration multiplied by 100.

RESULTS

The pigs were not affected by the treatment with the three OPs although slight salivation was observed in a few cases after i.v. adminis- tration. None of the dermal preparations caused skin irritation at the site of application. Pharmacokinetic parameters for the three OPs used are shown in Tables 111, IV and V.

From Table I11 is seen that parathion is absorbed through the skin, but at different rates and to different degrees depending 011

the vehicle used. With both DMSO and octa- no1 15-30% of the dermally applied dose is absorbed, but the absorption rate is twice as fast with DMSO as with octanol. When macro- gel was used as vehicle the absorption fraction

Percutaneous absorption of’ OPs in Pigs 177

T A B L E I 1 I . Pharrnacokinetic parameters of parathion in pigs after i.v. and dermal (d) administration in three different vehicles

d. - 50 mg/kg i .v. - 1 rng/kg DMSO Octanol Macrogol

Pigs (n) Weight (kg) AUC (pm g.h/l) MRT(h) MAT (h) T,,, (h) cmax (CLg4

V,, Wkg) tM1 (h)

CtB (Yhikg)

Bioavailability %

6 26.3 f 2.6 249 f 50 1.7 k 0.2 - -

4.2 f 0.9 7.1 f 0.6 2.9 ? 0.5

100

2 32

1628-395 1 9.7- 1 4.5 8.2-12.8

3 203-385 - - -

18.6-27.5

2 33-37

2007-3301 22.1-30.6 20.4-28.8

8 222-608 - - -

14.5-29.0

2 28.5

596-599 53.6-60.3 51.5-58.7

6-15 9.8-10.7 - - -

3.9-5.4

was as low as 4-595 and the rate of.absorption only half of that for octanol.

For both phoxim (Table IV) and phosmet (Table V) the rate of percutaneous absorption shows considerable individual variations, but does not seem to be much influenced by the vehicle used. With all three vehicles the bioavailability is very low compared to para- thion. Again macrogol seems to be responsible for the lowest absorption.

From the tables it is furthermore seen that after i.v. administration parathion is elimin- ated at a faster rate than phosmet and phox- im, while the volume of distribution is nearly the same for all three OPs. For all three OPs dermal application resulted in relatively low blood levels maintained over a prolonged period of time varying from 2 to 10 days.

DISCUSSION

Of the three OPs used parathion was chosen because it is the prototype of an OP, and because it is known to produce poisoning after dermal exposure in man and animals, i.e. it should be readily absorbed through the skin. For the same reason parathion has never been used as an ectoparasiticide. Phoxim and phos- met on the other hand were selected because they are licenced as ectoparasiticides to be used on domestic animals in pour-on prepara- tions.

The difference in toxicity between para- thion on the one side and phoxim and phosmet on the other was demonstrated in a preliminary study on the cholinesterase in- hibitory effect of these three OPs in pigs (Table VI). The cholinesterase activity in blood was measured by the Warburg techni- que (Augustinsson, 1948).

The pharmacokinetic parameters for para- thion presented in Table 111 correspond to previous findings in pigs (Gyrd-Hansen et al., 1991). Nielsen et al. (1991) have studied the pharmacokinetics of parathion in neonatal and young pigs and reported that both vo- lume of distribution and rate of elimination increase markedly with age. At 8 weeks the volume of distribution was 4.9 k 2.0 I/kg and thus only slightly lower than the 7.1 k 0.6 l/kg found in the older pigs (8-13 weeks) used in the present study. Body clearance in the 8- week-old piglets was 7.3 f 1.4 I/h/kg (Nielsen et al., 1991) and thus higher than the 4.2 k 0.9 ni l/h/kg seen in Table 111. The explanation for this difference is probably that parathion in plasma could be detected only for approxi- mately 2 h by Nielsen et al. (detection limit 5 ppb), while in the present study measurable amounts of parathion were present in plasma for at least 8 h resulting in a higher AUC and thereby a lower ClJj (C~B = Dose/AUC).

Based on the low bioavailabilities found for the three OPs - especially for phoxim and phosmet - it would seem that they have

I78 N . Gyrd-Hanwri et al.

TABLE I V . Pharmacokinetic parameters of phoxim in pigs after i.v. and dermal (d) administration in three different vehicles

d. - 100 mg/kg i.v. - 10 mglkg DMSO Octanol Macrogol

Pigs (n) Weight (kg) AUC (pg.h/l) MRT (h) MAT (h) Tm,, (h) Cm,, (wg/l) Cl, (I/h/kg) V,, (I/kg) th* (h) Bioavailability %

7 23.5 f 2.5

5848 * 1546 6.1 f 2.4

2 24

1344-1738

30.4-49.3 8-48

2 3.8-64.3

32.5-5 1 .0

2 23-32

887-936 54.743.5 47.2-59.4

4 4 8 12.5- 1 5.7

2 23-27

885-1 178 34.7-40.5 30.5-34.0

16.6-29.4 8-1 5

- 1.8 f 0.4

10.7 f 5.0 9.7 f 3.6

100 2.9 1.6-2.2 1.2-1.5

TABLE V . Pharmacokinetic parameters of phosmet in pigs after i.v. and dermal (d) administration in three different vehicles

d. - 100 mglkg i.v. - 10 mg/kg DMSO Octanol GF* Macrogol

7 24.0 f 4.4

4033 f 811 2.9 2 0.7

2 28-33

630-964 27.9-42.4 26.3-39.6

5 28.1-30.7

2 22-25

351-840 15.1-31.3 12.1-28.2

2-30 18.1-25.6

2 31-33

141-387 22.439.1 19.9-36.0

15-30 7.9-9.1 -

2.6 f 0.6 7.4 f 2.1 5.0 _+ 1.1

100 1.3-3.0 1.1-2.2 0.4-0.8

*See Table I1

T A B L E V1. Blood cholinesterase inhibition by three OPs in pigs

Maximal Route Dose (mg/kg) inhibition*

1.V. 1 60 d. 50 60 1.v. 10 60 d. 100 30 i.v. 10 60 d. 100 15

OP

Parathion Parathion Phoxim Phoxim Phosmet Phosmet

*as 70 of the control activity.

Percularieous absoipiori of OPs i n Pig5 179

Considerable species differences in percuta- neous absorption have been demonstrated for a number of compounds, including parathion (Maibach & Wester, 1989). In these studies parathion was shown to be most efficiently absorbed by rabbits, while penetration in pigs and humans was much more difficult. An ex- planation for such differences could be differ- ences in the thickness of epidermis and espe- cially of stratum corneum - both of which are quite thick in pigs compared to other animal species (Bronaugh et al., 1982).

The very low bioavailabilities seen when macrogol 400 was used as vehicle can be explained by the fact that macrogol itself is poorly absorbed through the skin (Table I). It was on the other hand surprising to see that DMSO hardly increased the bioavailabilities compared to octanol, as DMSO is known to be a potent absorption enhancer for many chem- ical compounds (Karzel & Liedtke, 1989).

On basis of the results obtained in the present experiments it is not possible to make any clear conclusions concerning the mode of action for OP-containing pour-on prepara- tions. With the three vehicles used it seems, however, unlikely, the low bioavailability for phoxim and phosmet taken into account, that sufficiently high concentrations can be attained at the site of the ectoparasites through percutaneous absorption followed by systemic distribution. In order to reach a final conclusion, however, it would be necessary to establish tissue concentrations of the OPs active against the relevant estoparasites and to measure the tissue levels of OPs that can be obtained through dermal administration of the recommended doses.

difficulties in passing the barriers of the skin. Absorption of chemicals through skin is generally assumed to be a passive diffusion process where the difficult part to pass is the stratum corneum - at least for compounds with limited lipid solubility (Bronaugh et al., 1989; Maibach & Wester, 1989). All three OPs used have a low water solubility of 7-25 mg/l, but are easily soluble in most organic solvents. It seems therefore difficult to explain why the bioavailability is so low, especially for phoxim and phosmet. One possible explanation would be that the OPs are metabolized in the skin itself during the passage. That the skin has drug metabolizing abilities has been demon- strated on several occasions (Martin et al., 1987; Kao & Carver, 1990; Storm et al., 1990), however, the enzymatic activity of the skin is in most cases only a small fraction of the corresponding activity in the liver (Bronaugh et al., 1989; Storm et al., 1990). Nevertheless, Carver et al. (1990) found that perfused porcine skin was very efficient in transform- ing parathion to paraoxon. However, in the in vitro model used the perfusion medium was continuously recirculated through the skin preparation for 8-10 h, while under in vivo circumstances the OP will pass the skin only once.

The fate of parathion after dermal adminis- tration has been studied using ''C-labelled parathion in order to see whether first-pass metabolism was involved in the percutaneous absorption (Gyrd-Hansen et al., 1991 ; Brimer et al., unpublished). The experiments showed that the bioavailabilities for parathion and 14C were almost the same i.e. that dermal metabol- ism of parathion is of little importance for its bioavailability, which is in accordance with what Bronaugh et al. (1989) found for a number of other compounds.

The two studies on parathion just men- tioned also showed that a relatively large proportion of the dose was trapped in the skin and stayed there for a considerable period of time. Such an epidermal reservoir where the chemical compounds are stored in the stratum corneum, depending on their physico- chemical characteristics and the lipid content of the stratum corneum, has been demon- strated previously (Maibach & Wester, 1989), and could be part of the explanation for the low bioavailability seen in the present study.

ACKNOWLEDGMENTS

The authors are grateful to Ms Kitty Petersen for excellent technical assistance. The study was supported by the Danish Agricultural and Veterinary Research Council, grant no. 13- 4386.

REFERENCES

Augustinsson, K.-B. (1948) Cholinesterases. A study in comparative enzymology. Acta Physiolup'- cn Scandiriovica, 15, suppl. 52, 3-5.

180 N . C;yrrl-Han.trn et al.

Bronaugh, R.L., Stewart, R.F. & Congdon, E.R. (1982) Methods for in vitro percutaneous absorp- tion studies 11. Animal models for human skin. Toxicology and Applied Pharmacology, 62, 48 1-488.

Bronaugh, R.L., Stewart, R.F. & Storm, J.E. (1989) Extent of cutaneous metabolism during percuta- neous absorption of xenobiotics. Toxicology and Applied Pharmacology, 99, 534-543.

Budavari, S. (ed.) (1989) The Merck index. 1 Ith ed. pp. 3247-48, 6674-75, 7542. Merck & Co. Inc., Ra h way.

Carver, M.P., Levi, P.E. Bc Kiviere, J.E. (1990) Parathion metabolism during percutaneous absorption in perfused porcine skin. Prsticide tliocluvnzstry and P/iy.riology, 38, 245-254.

Gibaldi, M. & Perrier, D. (1982) Pharmacokinetics. 2nd ed. New York. Marcel Dekker Inc.

Gyrd-Hansen, N., Brimer, L. & Rasmussen, F. (1991) Percutaneous absorption and recovery of parathion in pigs. Acla Veterinaria Scandinauica, suppl. 87, 410-412.

Kao, J. & Carver, M.P. (1990) Cutaneous metabol- ism of xenobiotics. Drug Metabolism Review, 24, 363-4 10.

Karzel, K. & Liedtke, R.K. (1989) Mechanismen transkutaner Kesorption. Arz,irirniltrl-Fo,.schunR, 39, 1487-1401.

Konar, A. & Ivie, G.W. (1988) Fate of 14-C- coumaphos after dermal application to lactating

goats as a pour-on formulation. American Journal of Veterinary Research, 49, 488-492.

Maibach, H.I. & Wester, R.C. (1989) Percutaneous absorption: In vivo methods in human and animals. Journal of American College of Toxicology, 8, 803-813.

Martin, R.J., Denyer, S.P. & Hadgraft, J. (1987) Skin metabolism of topically applied compounds. International Journal of Pharmaceuticals, 39, 23-32.

Mosha, R.D., Gyrd-Hansen, N. & Nielsen, P. (1990) Fate of ethion in goats after intravenous, oral and dermal administration. Phanacology and Toxico- logy, 67, 24tb251.

Nielsen, P., Friis, C., Gyrd-Hansen, N. & Kraul, I. (1991) Disposition of parathion in neonatal and young pigs. Pharmacology and Toxicology, 68, 233- 237.

Reynolds, J.E.F. (ed.) (1989) Martindale. The extra pharmacopoeia. 29th ed. pp. 1124-30, 142W27. T h e Pharmaceutical Press, London.

Sax, N.I. (1984) Dangerous properties of industrial materials. 6th ed. pp. 1201-02, 2084. Van Nos- trand Reinhold, New York.

Storm, J.E., Collier, S.W., Stewart, R.F. & Bronaugh, R.L. (1990) Metabolism of xenobiotics during percutaneous penetration: Role of absorption rate and cutaneous enzyme activity. Fundamentals of Applied Toxicology, 15, 132-14 1.