The toxicokinetics of deltamethrin in rats after intravenous administration of a toxic dose

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

18,

205-215 (1982)

The Toxicokinetics of Deltamethrin in Rats after Intravenous Administration of a Toxic Dose A.J.GRAY’ MRC

Toxicology

Unit.

Medical

AND J. RICKARD

Research Council Surrey SMS 4EF,

Laboratories, Woodrnansterne United Kingdom

Road,

Curshalton.

Received March 31, 1982: accepted June 23, 1982 The distribution of W-acid-, 14C-alcohol-, and 14C-cyano-labeled deltamethrin and selected metabolites were followed in the liver, blood, cerebrum, cerebellum, and spinal cord after iv administration of a toxic, but nonlethal dose (1.75 mg/kg) to rats. Approximately 50% of the dose was cleared from the blood within 0.7-0.8 min, after which the rate of clearance decreased. 3Phenoxybenzoic acid (PBacid) was isolated from the blood in rfvo, and was also the major metabolite when L4C-alcohol-labeled deltamethrin was incubated with blood in vitro. Deltamethrin levels in the liver peaked at 7- 10 nmol/g at 5 min and then decreased to 1 nmolig by 30 min. In contrast, peak central nervous system levels of deltamethrin were achieved within 1 min (0.5 nmoYg), decreasing to 0.2 nmol/g at 15 min. and remaining stable until 60 min. Peak levels of deltamethrin did not correspond to the severity of toxicity, although the levels of non-pentane-soluble radiolabel did appear to correlate with motor signs of toxicity. Experiments with brain homogenates, using in viva concentrations of deltamethrin, failed to reproduce the pentane-unextractable radioactivity in vitro nor was any metabolism demonstrated. INTRODUCTION

The pyrethroid deltamethrin [S-a-cyano(lR-cis)-3-phenoxybenzyl-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-carboxylate] is one of the most potent insecticides ever used on a commercial scale. Recent studies (1) have demonstrated that, in general, pyrethroids such as deltamethrin that contained an cr-cyano-3-phenoxybenzyl alcohol and a halogen group in the acid moiety produced a writhing type of toxicity in rats (choreoathetosis) (2) usually associated with salivation (1). These signs of toxicity are quite distinct from the fine muscular tremors produced by pyrethroids not containing such groups (1, 3, 4), e.g., cismethrin [$benzyl-3-furylmethyl-( lRcis)-chrysanthemic acid]. The distribution and clearance of cismethrin after iv administration to rats indicated that this pyrethroid was rapidly metabolized in viva and that ‘To whom correspondence should be addressed: Division of Toxicology and Physiology, Department of Entomology, University of California, Riverside, Calif. 92521.

there was a threshold brain concentration at which the tremors occurred (4, 5). Studies on the distribution of radiolabel after iv administration of a toxic dose of deltamethrin showed that the label distribution pattern in the majority of tissues was similar to cismethrin (6). However, the concentration of deltamethrin radiolabel in the central nervous system (CNS)2 associated with toxicity was approximately 10% of that required for cismethrin (6). This difference in CNS threshold level for cismethrin and deltamethrin was confirmed by direct administration of these pyrethroids into the CNS (7). This paper reports the findings of continuing studies on the distribution, clearance, and metabolism of a toxic dose of deltamethrin in viva in an effort to relate tisp Abbreviations used: CNS, central nervous system; PBalc, PBald, and PBacid, 3-phenoxybenzyl alcohol and its aldehyde and acid derivatives; glycerinformal, 75% 5-hydroxy-1,3-dioxan + 25% 4 hydroxymethyl 1.3-dioxolan; TLC. thin-layer chromatography; W-MS, gas chromatography-mass spectrometry: LSC, liquid scintillation counting. 205 0048-3575/82/050205-l 1$02.00/O Copyright All rights

@ 1982 by Academic Press, Inc. of reproduction in any form reserved.

206 sue levels of parent compound ity produced in rats.

GRAY

AND

to the toxic-

RICKARD

identical to that used for cismethrin and bioresmethrin (5). The concentrated extracts were analyzed by TLC (Merck, silica MATERIALS AND METHODS gel 60, UVZs4, 0.2 mm, aluminum-backed plates), developed in benzene, and the Chemicals spots visualized under uv light. The spots Both unlabeled and 14C-acid-, 14C- corresponding to the authentic deltamethrin alcohol-, and 14C-cyano-radiolabeled del- standard (R, 0.43) were cut out and counted tamethrin were gifts from Roussel Uclaf, in 10 ml of Insta-gel (Packard). The blood Romainville, France [see (6) for specific residue remaining after extraction was solactivities and labeling positions]. Deltamethubilized with 1 ml Soluene 100 (Packrin metabolite standards were prepared in ard):isopropanol (1: 1, v/v) overnight at the Department of Entomology, University 50°C. The sample was decolorized with of California, and PBalc was obtained from 0.5 ml of hydrogen peroxide (30%) and its the McLaughlin Gormley King Company, radioactive content determined in 10 ml of Minneapolis, Minnesota. Glycerinformal Insta-gel containing 1% (v/v) glacial acetic was purchased from Fluka, A. G., and all acid. The efficiency of extraction of delother solvents of analytical grade from Fi- tamethrin added to blood prior to extracsons, Ltd., and BDH Ltd. tion was 95.3 t 1.0%. Of the added label, 6.6 ? 0.6% was extracted but not deterAnimals and Dosing mined as deltamethrin, and 3.5 ? 0.4% reFemale LAC:Porton rats (140-165 g), mained in the blood residue (n = 5). given free access to MRC 41B cubed diet Deltamethrin metabolites in blood were and water, were used throughout these ex- analyzed using ethyl acetate as the experiments. Deltamethrin dosing solutions tracting solvent as described previously (5), were prepared at 3.5 mg/ml in glycerinforexcept that authentic deltamethrin, PBalc, ma1 (8) with specific activities of 8.80 (al- PBald, and PBacid (10 pg of each) were cohol labeled), 9.46 (acid labeled), and 6.34 added to each sample prior to TLC. Plates mCi/mmol (cyan0 labeled). Dosing solu- were developed twice in benzene (saturated tions were stored at -40°C and warmed to with formic acid):ether (10:3) (BFE) and the room temperature prior to use. The radiospots were visualized under uv light. The chemical purity of the stock deltamethblood residue remaining after extraction rin and the dosing solutions were checked was solubilized as described above after the and repurified to >98% by TLC in benzene addition of 100 ~1 water. as necessary. Brain. Animals were killed by decapitation at selected time intervals between 1 Estimation of Deltamethrin in Tissues min and 4 hr after dosing. The extraction Blood. The animals were anesthetized and analysis methods used to estimate the with ether and the lateral tail vein and ven- brain levels of deltamethrin were identical tral tail artery cannulated. The wound was to those used for cismethrin and bioresmethsprayed with local anesthetic (xylocaine) tin (5). The extraction efficiency of 14Cand the rat placed in a restraining cage and alcohol-labeled deltamethrin added to brain allowed to recover from the general prior to sonication was 86.2 +- 0.9%. A anesthetic for at least 1 hr. One blood sam- further 7.2 rt 1.0% was extracted as nonple was taken prior to iv administration of deltamethrin label with 3.8 2 0.6% re14C-acid-, 14C-alcohol-, or 14C-cyanomaining in the blood residue (n = 5). The labeled deltamethrin at 1.75 mg/kg. A level of deltamethrin in spinal cord of some further 12-15 blood samples (approxianimals was also estimated by this method. mately 100 ~1) were taken at selected times Liver. The method of analysis was similar after dosing. The method of extraction was to that described previously (5) except that

TOXICOKINETICS

OF

DELTAMETHRIN

IN

RATS

207

clearance Teflon pestle to fit a 20-mmdiameter glass homogenizer (9, 10). The final volume of the homogenate was adjusted to give a 25% (w/v) homogenate by the addition of Hepes buffer. Samples (250 ~1) of the homogenate were taken for extraction and also LSC in Insta-gel (10 ml containing 1% glacial acetic acid) after solubilization in 1 ml Soluene 350. A further 6-ml sample was transferred to a 25-ml Erlenmeyer flask in a 37°C shaking water bath. After 5 min, either 1 ~1 of the alcohol-labeled deltamethrin dosing solution was added to give a final concentration of 3.73 nmoYg brain, or 5 ~1 of alcoholradiolabeled deltamethrin solution (59.3 mCi/mmol) in glycerinformal was added to give a final concentration of 0.5 nmol/g brain. Triplicate samples (250 ~1) were taken at 0, 10, 20, 30, and 60 min and extracted 3 times with ethyl acetate. Concentrated extracts were applied to TLC plates, developed twice in the BFE solvent system and exposed to X-ray film for 28 days. All In Vitro Metabolism radioactive spots were cut out and their Blood. The rate of ester cleavage by radiolabel content determined. Water blood was determined in vitro by incubating (100 ~1) was added to the brain residue be2 ml heparinized blood with 10 ,ul of 14C- fore solubilization in 1 ml Soluene 350. alcohol-labeled deltamethrin dosing solu- Insta-gel (10 ml), containing 1% glacial acetion, resulting in a concentration of ap- tic acid was then added for LSC. proximately 25 nmol/g blood, similar to that measured approximately 1 min after iv in- Identification of Metabolites in Blood jection of deltamethrin at 1.75 mg/kg. The blood was incubated at 37°C in a shaking Alcohol- and acid-labeled deltamethrin water bath and allowed to equilibrate for 5 metabolites extracted from blood with ethyl min. Triplicate samples (100 ~1) were taken acetate during the kinetic studies were prior to addition of the pyrethroid and at 0, purified by TLC developed twice in solvent 10, 20, and 30 min. The samples were ex- system BFE. They were tentatively identracted with ethyl acetate and processed as tified by cochromatography against the were the in vivo blood samples. The TLC known deltamethrin metabolite standards plates were developed twice in the BFE and attempts were made to confirm these solvent system and exposed to X-ray film findings by GC-MS. A 70-70 VG double(Ilfex 90) for 28 days before cutting out the focusing mass spectrometer linked with a radioactive areas for LSC in Insta-gel. VG 2035 Data System was used, interfaced Brain. After decapitation, the brain was to a Pye-Unicam Series 204 gas chroremoved from a rat, rinsed in saline, and matograph. Electron impact mass spectra blotted dry to remove surface blood. The were obtained from solid injection of the brain was weighed and then homogenized metabolite standards and TLC extracts in 5 ml of 0.1 M Hepes buffer, pH 7.4 onto a 20-m, fused-silica, capillary column (Sigma), with two strokes of a 0.5-mm coated with SE 52. acetonitrile replaced n-pentane as extracting solvent and no water was added prior to homogenization. Three or four samples of each rat liver (200-400 mg) were taken and extracted 3 times. After each extraction samples were centrifuged at 2000g prior to removal of the acetronitrile. The combined extracts were evaporated under a flow of nitrogen gas in a 45°C water bath and the residue resuspended in acetonitrile (200 ~1) for TLC analysis. Water (100 ~1) was added to the liver residue before solubilization in 1.5 ml Soluene 350 (Packard) at 50°C overnight. The samples were decolorized with 0.5 ml of hydrogen peroxide (30%) prior to adding 10 ml of Insta-gel containing 1% (v/v) glacial acetic acid. The recovery of 14C-alcohol-labeled deltamethrin added to liver prior to homogenization was 91.9 + 1.3%. Of the added label, 3.1 2 1.1% was extracted but not determined as deltamethrin, and 0.6 & 0.1% remained in the liver residue (n = 5).

208

GRAY

AND

RICKARD

RESULTS

Toxicity The administration of deltamethrin to rats at 1.75 mg/kg produced all the signs of toxicity typical of this type of pyrethroid (1, 2, 11) and have been described in detail elsewhere (6). At this dose level, profuse salivation was first observed at l-2 min, continued for up to 15 min, and was associated with chewing and a “bulldozing” motion of the head and neck. After 5- 10 min, coarse whole-body tremors occurred that rapidly progressed to induced, and finally spontaneous, choreoathetosis (2). These writhing motions continued for up to 1 hr, although complete recovery was not observed until 4 hr after dosing. No fatalities were recorded at this dose level. Estimation

of Deltamethrin

in Tissues

Blood. The calculated initial blood level of deltamethrin, assuming uniform distribution of the dose (520 nmoV150 g rat) in the total blood volume (9.4 g) (5), would have been 55.3 nmol/g. Approximately 50% of the dose was, therefore, cleared from the blood within the first 0.7-0.8 min (Table 1). The clearance of deltamethrin from blood after 1 min was more gradual, and significant amounts of compound were still detected at 30 min (Table 1). Using the data obtained from five animals (data in Table 1 and acid- and alcohol-dosed rats in Table 2, a total of 64 points), the triple exponential decay curve for deltamethrin calculated by the least-squares method was 21.2 exp (-8.57t) + 20.3 exp (-0.897t) + 12.6 exp (-0.097t). The amounts of radiolabel found in the non-deltamethrin, pentane-soluble fraction until at least 20 min after dosing (Table 1) were proportionately similar to those found after the addition of deltamethrin to blood. These values probably represent decomposition or loss of deltamethrin during the assay. However, after this time the amounts of radiolabel in this fraction were too large to be accounted for by deltamethrin decomposition. At all times the amounts of radiolabel left in the residue and

s s” I

$ h” % 2 z % $, 0y, 5 5 ; s h-2 4% OF %v1 g-; 4 ,E 91 .2 XI ;Q d .r: Q m ,Q <2% b Z g 2 ‘$ S .2 P .z 2s xq +9 .s ; s2 04 2= i 2 ,z 4 g 5 9< % % 2 3 2 4

0

P

m

R

19.87

0.10 2.42 1.16 23.55

25.67

0.37 0.77 1.39 28.20

22.07 1.40 3.53 2?.00

0.04 1.08 2.94 25.98

28.05

0.34 0.82 0.57 29.78

22.23 1.40 10.12 33.75

1

21.92

0.67

15.32 1.00 2.04 18.36

0.67 0.34 1.02 18.17

16.14

0.29 0.02 1.77 15.38

13.30

2

11.12 0.56 1.95 13.63

1.03 0.47 1.13 14.54

11.91

0.46 0.40 1.04 13.80

11.90

3

8.80 0.64 1.50 10.94

1.24 0.43 1.31 12.05

9.07

0.65 0.64 0.91 13.87

11.67

4

6.52 0.83 1.64 8.99

2.07 0.28 0.84 11.22

8.03

0.94 0.25 1.09 1 I .43

9.15

5

2.50 0.75 1.57 4.82

2.33 0.21 1.11 7.11

3.46

1.28 0.46 1.59 8.98

5.65

10

1.18 0.77 1.30 3.25

2.39 0.08 1.14 5.92

2.31

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3.39

15

0.12 0.29 1.14 1.55

0.32 0.26 1.57 2.15 0.55 0.60 1.66 2.81

0.45 0.89

0.87 1.98

0.95 3.31

0.26 0.03

0.15

120

0.58 0.04

0.49

2.07 0.17 2.07 4.79

0.48

60

1.34 0.09

0.93

1.70 0.20 2.05 4.90

0.95

30

Note. The data given for each radiolabeled preparation in this table were obtained from a single cannulated rat. o Rf of metabolite on silica gel TLC plates developed twice in solvent system BFE.

Alcohol-labeled deltamethrin Deltamethrin Alcohol-labeled metabolite (R, 0.74)0 Other extracted Unextracted Total Acid-labeled deltamethrin Deltamethrin Acid-labeled metabolite (I?, 0.80)” Other extracted Unextracted Total Cyano-labeled deltamethrin Deltamethrin Other extracted Unextracted Total

Fraction

Time after dosing (min)

0.08 0.25 1.17 1.50

0.46 1.12

0.54 0.04

0.08

180

TABLE 2 Levels (nmollg) of Deltamethrin and Acid-. Alcohol-. und Cyano-Labeled Metabolites in Various Fructions of Ethyl Acetate Extracts of Blood Samples Taken after iv Administration of Deltamethrin at 1.75 mglkg

0.04 0.09 1.40 1.53

240

2

F

m 2 E 2: 5

0”

210

GRAY

AND

not extracted (Table 1) were proportionately too great to be accounted for by poor extraction of deltamethrin. This finding was confirmed when the blood of rats administered alcohol-labeled compound was extracted with ethyl acetate and the extracts analyzed for their metabolite content (Table 2). Less radiolabel remained in the residue fraction and after 2 min the majority of the radioactivity extracted, but not measured as deltamethrin, was present as a single metabolite (Table 2) that cochromatographed with PBacid. The total radiolabel and the amounts of deltamethrin detected by extraction of blood with ethyl acetate (Table 2) or pentane (Table 1) were similar. The amounts of cyano- or acidlabeled deltamethrin detected in blood after ethyl acetate extraction (Table 2) were similar to that measured with alcohol-labeled compound (Tables 1 and 2). When acidlabeled deltamethrin was administered, the majority of the non-deltamethrin, ethyl acetate-soluble radiolabel was also present as a single metabolite (Table 2). This metabolite was presumably the dibromovinyl chrysanthemic acid as it had a different Rf value (0.80) to the alcohol-labeled metabolite (0.74) and was found between the PBacid and PBald on the TLC plates developed twice in the BFE solvent system. The blood concentration of this acid moiety metabolite reached a maximum at IO-15 min and then declined with a half-life of approximately 30 min (Table 2). The amounts of radiolabel remaining after extraction of blood samples taken 30 and 60 min after iv administration of acid-labeled deltamethrin were lower than those measured after dosing with alcohol-labeled compound (Table 2) and also resulted in a lower total level of blood radioactivity (Tables 1 and 2). In contrast to the results found with the other two deltamethrin preparations, no metabolites of deltamethrin were observed after TLC of the ethyl acetate extracts of blood samples taken from rats administered cyano-labeled deltamethrin (Table 2). The extracted label not measured as deltameth-

RICKARD

rin was probably entirely due to decomposition of parent compound during analysis. However, the amounts of radiolabel not extracted from 10 min onward were similar to those determined with alcohol-labeled deltamethrin (Table 2). Brain and spinal cord. The distribution of radiolabel in the various extraction fractions of the three regions of the CNS were similar with all three labeled preparations of deltamethrin (see Fig. 1). The results obtained were, therefore, combined to give the mean -+ SE values shown in Table 3. Lower values were obtained with the spinal cord due to entrapment of radiolabel in a white material (probably myelin) that was extracted by the pentane. This material precipitated when the pentane was evaporated and contained approximately 20% of the extracted radiolabel and was, therefore, not analyzed by TLC. The initial levels of radiolabel in the cerebellum were slightly higher than those of the cerebrum or spinal cord (Table 3, Fig. 1). The greatest total label and parent compound levels were achieved within 1 min of dosing and decreased rapidly to around 0.5 and 0.2 nmol/g, respectively, by 15 min, and then more gradually over the next 4 hr (Table 3, “0,

TOXICOKINETICS

OF

DELTAMETHRIN

TABLE 3 Levels (nmollg) of Deltamethrin and Other Pentane-Extractable Spinal Cord after iv Administration of ‘T-Alcohol-, -Acid-,

IN

211

RATS

Radiolabel in Cerebrum, or -Cyano-Labeled Compound

Cerebellum, and at 1.75 mglkg

Time after dosing (min) Fraction Deltamethrin Other pentane extractable

Deltamethrin Other pentane extractable Deltamethrin Other pentane extractable

1

5

15

30

60

120

240

0.44 2 0.03 0.07 2 0.02

0.33 dz 0.02 0.07 +
0.20 +-co.01 0.07 ~
Cerebrum” 0.22 r<0.01 0.06 t co.01

0.22 k
0.10 ?
0.06 *
0.58 5 0.12 0.11 k 0.03

0.44 k 0.01 0.08 2 0.01

0.22 c 0.02 0.09 + 0.02

CerebelIum* 0.29 ‘: 0.05 0.07 k 0.01

0.23 k< 0.01 0.09 k 0.01

0.10 k< 0.01 0.03 ?
0.05 r< 0.01 0.03 ?
0.49 f 0.07 0.13 2 0.03

0.27 2 0.02 0.09 ‘- 0.01

0.17 ?
Spinal cord’ 0.19 2 0.05 0.07 f 0.02

0.17 2 0.01 0.06 -c
0.08 ?
0.05 ?
Note. Minimum level of detection was calculated as 0.01 nmol/g brain or spinal cord. ’ Mean ? SE of duplicate values taken from each of three rats administered alcohol-, acid-, or cyano-labeled deltamethrin. * Mean t SE of one sample taken from each of three rats administered alcohol-. acid-, or cyano-labeled deltamethrin. ’ Mean _t SE of one sample taken from each of two rats administered acid- or cyano-labeled deltamethrin except at 60 min when the level in a spinal cord taken from an animal dosed with alcohol-labeled compound was also included.

Fig. 1). The levels of non-deltamethrin extracted label were probably the result of losses during TLC analysis as their relative amount was similar to that measured after extraction of compound added to brain prior to sonication. Only deltamethrin was observed on the TLC plates when exposed to X-ray film when pentane extracts were analyzed. However, in two experiments in which ethyl acetate extractions were made of CNS tissue samples taken 30 min after alcohol-labeled deltamethrin administration, two metabolites were isolated on TLC with mean concentrations of 0.100 and 0.008 nmohg, tentatively identified as PBacid and PBalc, respectively. A further 0.066 nmol/g was not extracted with similar levels of deltamethrin and total radiolabeled compound to the pentane extracts (Table 3, Fig. 1). The radiolabel not extracted with

pentane was of greatest interest as it correlated with the severity of the signs of toxicity. The radiolabel content of this fraction increased to a maximum of about 0.2 nmoYg at 15-30 min even though the total label content of brain was decreasing (Fig. 1). Liver. The levels of parent compound in the liver were very similar with all three labeled preparations. The mean * SE values were calculated at each time from the mean values obtained from individual animals, given each labeled deltamethrin preparation (Fig. 2). Peak deltamethrin levels were found between 1 and 5 min after dosing, and then rapidly declined over the next 10 min. The radioactivity levels in the other fractions were not similar with the three labeled deltamethrin preparations. The amount of cyano label in the nondeltamethrin, acetonitrile-extractable frac-

212

GRAY

Time (mid

After

Dosing

FIG. 2. Levels of deltamethrin times after intravenous administration rin. Each determirled obtained alcohol-. not

sho\lw

the

SE.

point is the mean from triplicute from three or cyano-labeled wherr

the

AND

itr liver

-C SE of the or quadruplicnte

ruts

administered pyethroid.

,sFmbol

used

exceeds

ut various of deltamethmean

values samples

either ucid-. Error bars nre the

limit

of

tion was greater than that of acid or alcohol label especially at time points after 15 min (Table 4). However, the identity of this additional radiolabel was not determined. In the unextracted fraction, the pattern was less consistent, but, in general, the liver of animals dosed with acid- or alcohol-labeled deltamethrin contained more nonextracted radiolabel than animals administered cyano-labeled compound (Table 4). This fraction also accounted for more than 50% of the total label content of the liver from 15 min onward with acid- and alcohol-labeled preparations, but often only about 30% when cyano-labeled deltamethrin had been administered. In Vitro Metabolism Blood. Deltamethrin was metabolized by blood to at least two metabolites. The first metabolite cochromatographed with PBacid with an Rf of 0.74 when developed twice in solvent system BFE and increased at a linear rate of 0.07 nmol/g/min for 20 min after which its rate of formation decreased.

RICKARD

TOXICOKINETICS

OF

DELTAMETHRIN

IN RATS

213

The minor second metabolite cochromatographed with PBalc in this solvent system (I$ 0.62) and increased at a linear rate of 0.02 nmol/g/min for 20 min and then its rate of formation decreased with time. The concentration of deltamethrin decreased at a rate of 0.10 nmol/g/min for the first 20 min. There appeared to be no consistent change in the amount of labeled compound which was not extracted by ethyl acetate nor in that extracted but not measured as deltamethrin or one of the two metabolites. Electron impact mass spectra were obtained of the PBacid and PBalc standards. A spectrum was given by the PBacid extract with the molecular ion at m/z 214, identical to the standard PBacid, but was very weak as difficulty was experienced in purifying sufficient material by chromatography. The considerably smaller amounts of the second metabolite produced during in vitro metabolism precluded attempts at GC-MS identification. Bruin. At neither concentration of deltamethrin (equivalent to 0.5 or 3.73 nmol/g brain) was there any evidence of metabolism. Deltamethrin recovery was 96.25 t 0.51% (n = 15) of the incubated radiolabel. At the higher concentration, approximately 0.015 nmoVg of brain remained unextracted and at the lower level (0.5 nmol/g), less than 0.005 nmol/g was found in the residue. These levels were unaltered by length of incubation. DISCUSSION

The total radiolabel distribution curves for the three labeled preparations in blood, liver, and brain were very similar to those previously reported (6), but the actual levels of radioactivity were often slightly less presumably due to losses during extraction and TLC analysis. The clearance of deltamethrin from blood (Tables 1 and 2), liver (Fig. 2), and brain (Table 3) was much slower than that reported for cismethrin and bioresmethrin (5) even though the dose administered was approximately half that of an equitoxic dose

214

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of cismethrin, on a molar basis. This may partly account for the longer duration of toxicity of deltamethrin when compared to cismethrin (l-6, 11). Analysis of extracts of blood at various times after administration of alcohol- or acid-labeled deltamethrin demonstrated the presence of labeled metabolites of both moieties that had different Rf values (Table 2). As no cyano-labeled metabolites were isolated, these results indicate that ester cleavage had occurred and that the cyano moiety had been lost presumably as HCN, as postulated in previous metabolic studies (12). The total radiolabel content of blood samples taken 5 min or more after administration of 14C-cyano-labeled compound were up to 50% lower than those reported earlier (6). However, as the level of parent deltamethrin was similar to that measured after administration of acid- or alcohollabeled deltamethrin (Table 2), this result implies that some volatile cyano metabolite may have been lost during analysis, probably when the ethyl acetate was evaporated. This may be due to evaporation of the volatile HCN formed after ester cleavage (12) or due to decomposition of the unstable 3-phenoxybenzaldehyde cyanohydrin that has been isolated after in vitro metabolism experiments with mouse liver homogenates (14). The alcohol-labeled metabolite in blood accumulated with time similar to 5benzyl3-furylcarboxylic acid after cismethrin administration (5) but not to such a degree as that found with bioresmethrin (5, 13). The acid-labeled metabolite had a much faster clearance presumably due to more rapid excretion in the urine (12). In vitro metabolism experiments with blood indicated that some degradation of deltamethrin could occur. The two radiolabeled metabolites formed cochromatographed with authentic PBacid and PBalc on TLC but only the identity of the major product, PBacid, could be confirmed by GC-MS. The metabolism of deltamethrin in blood may contribute to the detoxitica-

RICKARD

tion of this pyrethroid in vivo, but as the rate of metabolism was relatively slow, this is probably of only minor consequence in limiting toxicity. In contrast to the earlier studies with cismethrin (5), the onset of and recovery from toxicity, did not correlate well with the total amount of radiolabel or parent pyrethroid in the CNS. The best correlation between the severity of toxicity and levels of radiolabel in the CNS were shown by the fraction unextracted by pentane (Fig. 1). The actual amount of radiolabeled compound in this fraction peaked at 15-30 min when the signs of toxicity were most severe, and then gradually decreased over the following 90 min when recovery was almost complete. The amount of radiolabel in this fraction initially increased although the level of total label in each brain region was decreasing. It therefore seems unlikely that this was nonspecific binding. Alternatively, this radioactivity could represent a metabolite of deltamethrin that retains all three radiolabels. However, as some of this pentane-insoluble label was tentatively identified as PBacid and PBalc, in agreement with previous studies (15), the toxicological significance of the pentane-insoluble fraction requires further investigation. In contrast to the in vitro metabolism experiments with mouse brain homogenates that indicated metabolism of deltamethrin to phenoxybenzyl alcohol and its aldehyde and acid derivatives (15), our own studies failed to demonstrate any metabolism of deltamethrin by rat brain homogenates. This would suggest that either metabolism of deltamethrin in the brain requires cofactors not included in our in vitro experiments or that these metabolites are sufficiently lipid soluble to cross the bloodbrain barrier. In conclusion, this study demonstrates that deltamethrin is quickly metabolized in vivo by liver and blood enzymes and probably also in other organs (12) although not as rapidly as cismethrin and bioresmethrin (5, 13). Deltamethrin rapidly enters the CNS

TOXICOKINETICS

OF

DELTAMETHRIN

after iv injection. In agreement with previous studies (6, 7, 12), CNS levels of deltamethrin of approximately 0.5 nmoh’g are required for toxicity to occur although peak concentrations of parent compound did not correspond with peak signs of toxicity. The levels of unextracted radiolabel in the CNS may have greater relevance to the production of toxicity although this requires further investigation. ACKNOWLEDGMENTS

We thank Roussel Uclaf for the gifts of radiolabeled and nonlabeled deltamethrin, Dr. T. A. Connors for his encouragement, Dr. P. Farmer for the CC-MS analysis of blood extracts, and Dr. D. J. Cunningham for his assistance with the computer curve fitting. AJG also wishes to thank the National Research Development Corporation for financial support. REFERENCES

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