Arch. Toxicol. 34, 81 --88 (t975) 9 by Springer-Verlag 1975

Original Investigations The Excretion in Urine of Four Insecticidal Carbamates and Their Phenolic Metabolites after Oral Administration to Rats F. v a n H o o f and A. Heyndrickx Laboratory of Toxicology, State University of Ghent, Ghent/Belgium Received February 3, t975

Abstract. 4 insecticidal carbamates: 3-methyl, 5-isopropylphenyl N-methyl-carbamate (Promecarb), 4-methylthio, 3,5 dimethylphenyl N-methylearbamate (Mesurol), 4-dimethylamino, 3,5 dimethyIphenyl N-methylcarbamate (Zectran), and 4-benzothienyl N-methylearbamate (Mobam) were orally administered to male Wistar rats. The excretion of the unchanged products was followed in 24 hrs urine collections. The corresponding phenols of the different carbamates could be identified as metabolites by gaschromatography-mass spectrometry. These metabolites were quantified afterward by gas-liquid chromatography. In all cases only minor quantities of unchanged products and metabolites were excreted in the urine over the 48 hrs after administration. Key words: Insecticidal Carbamates -- Excretion in Urine after 0ral Administration -Wistar Rats -- Identification of Metabolites -- Quantitative Determination. Zusamrnenfassung. 4 insecticide Carbamate: 3-Methyl, 5-isopropylphenyl N-methylcarbamate (Promecarb), 4-Methylthio, 3,5 dimethylphenyl N-methylcarbamate (Mesurol), 4-Dimethylamino, 3,5 dJmethylphenyl N-methylcarbamate und 4-Benzothienyl N-methylcarbamate wurden einmalig peroral an miinnliche Wistar-Ratten verabreicht. Geringe Mengen der unveri~nderten Produkte wurden im Ham gefunden bis 48 Std nach der Verabreichung. Verschiedene phenolische Metaboliten wurden durch GaschromatographieMassenspektrometrie identifiziert und danach quantitativ bestimmt. In alien Experimenten wurden nur geringe Mengen der unver~nderten Insecticide und ihrer phenolischen Metaboliten ausgeschieden. L%Idiisselw6rter: Insecticide Carbamate -- Ausscheidung im Ham nach oraler Verabrei. chung Identifikation der Metaboliten -- Quantitative Bestimmung. -

-

Carbamate insecticides are frequently used as pest controls. I n spite of their widespread use, limited knowledge is available on their fate in m a m m a l s (Baron and Doherty, t967; Baron and Locke, t970; Dorough, t970; Fishbein and Zielinski, t969; Fukuto, 1972; K n a a k et al., t965; K n a a k eta/., 1966; K n a a k and Sullivan, 1968; K n a a k et al., 1968; K n a a k et aZ., t970; Knowles, t970; Krishna and Casida, 1966; Leeling and Casida, t966; Meikle, t973; Miyamoto et aZ., 1969; Oonnithan and Casida, 1968; Robbins et al., t969; Robbins et al., t970; Slade and Casida, 1970; Skrinjaric-Spoljar and Reiner, 1968; Strother, t970) and still fewer methods have been developed to quantify them in biological specimens (Burnett and Lanham, t970; Gajduskova, t972; Stanley, t972). The present paper deals with their isolation from urine samples with subse. quent qualification b y gas chromatography.

82

F. v a n Hoof a n d A. Heyndrickx

The main aim of this work was to find out whether monitoring unchanged insecticides and some simple metabolites might be valuable indicators in case of intoxication with these products.

Experimental A. Chemicals The 4 insecticides were obtained as reference products from producing firms. Before use they were recristallyzed from benzene or a mixture benzene -- n-hexane (50:50). Reference products of metabolites were obtained from the same source, they were recristallyzed from water before use. All reagents used were of analytical-reagent-grade quality.

B. Animal Treatment All products were administered to rats as a single oral dose dissolved im maize oil. During experiments animals were kept in a metabolism cage a n d 24-hrs urine samples were collected.

C. Isolation and Quantitative Identification o/Unchanged Compounds 10-ml urine samples were extracted b y shaking over a 30-rain period with 100 ml of diethyl ether. The urine was discarded and a known a m o u n t of internal standard was added to the ether phase. The ether extract was washed twice with 10 m110 -8 N N a O H a n d dried with I g of anhydrous sodium sulfate. I t was then evaporated to dryness on a water b a t h a t 37 ~ C, u n d e r a mild stream of nitrogen. Each extract was dissolved in a mixture of acetic anhydride a n d methane sulfonic acid (40/1) a n d heated a t 100 ~ C over a 30-min period. After cooling, 4 ml of ice-cold water were added and the solution was extracted twice with 10 ml of benzene. The benzene layer was dried over 0.2 g of anhydrous sodium sulfate a n d evaporated a t 70 ~ C. The residue was dissolved in 20 [zl of ethanol a n d 1 ~l was injected in a Perkin-Elmer F20 F E gas chromatograph using the following conditions: column: 2.5 % OV-17 on Chromosorb W AW-DCMS 80 to 100 mesh, 2 m long, 2 ml inner diameter. column temperature: 170 to 195 ~ C detector: F I D a t 205 ~ C injectionport temperature: 212 ~ C gas flows: hydrogen 25 ml/min nitrogen 25 ml/min air 250 ml/min electrometer settings: R = 1 A = 64 recorder: Hitachi 159 1 m V F o r each compound quantitatively assayed, a standard curve was drawn from mixtures containing different amounts of the internal standard a n d assayed product b y plotting the relationship of peak heights versus weight ratio. The exact a m o u n t of unchanged compound in each sample was derived from the standard curve.

A n example of a gas chromatogram is shown in Fig. 1.

D. Isolation and Identification o/ Metabolites Conjugated metabolites were set free b y adding 5 ml of acetic acid-sodium acetate buffer (pH = 5.2) a n d 0.5 ml enzyme solution (800000 I.U. glucuronidase + 100000 I.U. sulphatase/ ml) to a 5 ml urine sample collected during the first 24 hrs after administration, which h a d previously been extracted with dicthayl ether. The solution ws kept a t 37 ~ C for 24 hrs. Afterwards it was extracted with 100 ml of diethylether. The ether phase was dried with I g of anhydrous sodium sulfate and finally evaporated a t 37 ~ C under a stream of nitrogen.

The Excretion in Urine of F o u r Insecticidal Carbamates

83

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Fig. 4_, Gas chromatogram showing urine extract containing 3-methyl, 5-isopropylphenyl N-methyloarbamate (2); internal standard is 2-isopropoxyphenyl l~-methylearbamate (1)

The residue was t a k e n u p in i 0 i~l ethanol a n d J ILl was injected in a gas chromatograph, containing a 2-m long 2 To SE-30 on Chromosorb W AW-DCMS 100--120 mesh column, coupled to a l~innigan 3000 quadrupole mass spectrometer. The column t e m p e r a t u r e was in the range of 170 to 190 ~ C. Mass spectra were obtained u n d e r the following conditions: b e a m current: 0.04 m A ion energy: 8.9 V electron energy: 70.7 V collector: 6.7 V emission: 0.61 emission H v : 0.61 kV extractor: 18.3 V lens: 143.5 V

E. Quantitative Assay o/Metabolites 5 ml of distilled water a n d I ml concentrated hydrochloric acid were added to 5 ml urine samples which h a d previously been extracted with diethyl ether. The solution was kept a t t_00~ C for l h r while rcfluxing. After cooling a n d neutralization with 12 N N a O H extraction was done twice with 100 ml of n-pentane.

84

F. van Hoof and A. Heyndrickx

The pentane layers were put together, an appropriate amount of internal standard was added, and they were dried with l g of anhydrous sodium sulfate. Evaporation of n-pentane was performed under a mild stream of nitrogen at 35~ C. The residue was taken up in l0 ~tl ethanol and 1 ~tl was injected into a gas chromatograph equipped with a 2-m long 5 % XE-60 column at a column temperature of between 150 and 170~ C.

Results and Discussion The unchanged products could be satisfactorily isolated from urine b y the extraction procedure described under C. The method was tested for several blank urine samples and gave no interfering peaks. The recoveries were tested for each compound b y extracting 5 i0-ml samples fortified with i00 ~g of the product under investigation. The results are shown in Table t. The higher recovery rate obtained for 4-benzothienyl N-methylcarbamate is due to a second extraction with diethyl ether, thus proving t h a t nearly quantitative recovery can be achieved through this procedure. The lower recovery of 4-dimethylamino, 3,5 xylyl N-methylcarbamate is caused b y a further purification step based on the presence of a tertiary amino function in the molecule to extract it from the ether phase with 0.1 N HC1. After neutralization the insecticide was extracted back in the etherphase. For each compound at least 2 rats were given a single oral dose and excretion of the unchanged product in urine was monitored. Results are compiled in Table 2. All results shown in Table 2 indicate t h a t excretion of unchanged compounds in rat urine occurs mainly within 48 hrs after administration, the amounts excreted being v e r y small: in most cases less than 2 % of the dose administered. These results are in good agreement with studies on other C14-1abeled insecticidal carbamates which were rapidly but almost entirely metabolized, excreted into rat urine (Baron and Doherty, i967; Baron and Locke, i970; Dorough, i970; Fukuto, i972; K n a a k et aL, i965; K n a a k et al., i966; K n a a k and Sullivan, 1968; Knowles, 1970; Krishna and Casida, 1966; Leeling and Casida, i966; Meikle, i973 ; Miyamoto et al., t969; Oonithan and Casida, 1968; Robbins et al., i969; Skrinjaric-Spoljar and Reiner, i968; Strother, i970). Those studies clearly indicated t h a t a large fraction of the radioactivity was excreted as conjugates (glucuronates and sulphates). I t therefore seemed of interest to look more closely at those products. Table I. Recoveries of unchanged products from urine by ether extraction Product investigated

Internal standard

Mean % recovery

% Standard deviation

3-methyl, 5-isopropylphenyl N-methylcarbamate 4-methylthio, 3,5 xylyl N-methylcarbamate 4-dimcthylamino, 3,5 xylyl N-methylcarbamate 4-benzothienyl N-methylcarbamate

2-isopropoxyphenyl N-methylcarbamate 1-naphthyl N-methylcarbamate 2-chloro, 4,5 xylyl N-methylcarbamate 1-naphthyl N-methylcarbamate

77.6

6.5

76.0

5.7

54.0

3.3

93.5

3.1

The Excretion in Urine of Four Insecticidal Carbamates

85

Table 2. Excretion of unchanged carbamate in urine after oral administration to rats Product investigated

Amount Amount excreted (in mg) Total amount exadministered 0--24 24--48 48--72 72--96 96--120 creted (as % of (in rag) dose administered)

A 3-methyl, 5-isopropyl phenyl N-methylcarbamate I. 5.0 2 . 5.0 3. 5.0 4 . t0.0 B 4-methylthio, 3,5 xylylN-methylcarbamate I. 5.0 2. 5.8 3. 5.8 C 4-benzothienyl N-methylcarbamate t. 6.8 2. 5.8 D 4-dimethylamino 3,5 xylyl N-methylcarbamate I. 10.0 2. 10.0 3. 10.0 4. t0.0

20 2.5 12.5 56

8 12 2.5 89

75 34 44

13 . 5

97 63

47 36

75 21 66 t3

t6 22 47 38

5 10 -14

.

--

--

6

--

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--

9

5

--

--

--

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2.3 0.58 0.84

-32

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44 46 23

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--

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.

0.66 0.67 0.30 1.7

Table 3. Major fragments in mass spectra of met~abolites Metabolite

Major fragments

I. Promecarb Mesurol I I I . Mobam IV. Zectran

150 168 t50 165

H.

(40, 1K+), 125 (100), 117 (i0), i07 (12.5), 91 (25), 77 (i0) (100, M+), 153 (68.6), 109 (41), 91 (8), 77 ( 5 . 7 ) (100, M+), 122 (35), 121 (33), 96 (6.9), 78 (8), 77 (6.9), 75 (t0.4) (100, M+), 164 (70), 150 (71), 149 (17), 148 (12), 135 (12), 134 (22)

T h i s w a s d o n e as d e s c r i b e d u n d e r D . F o r e a c h p r o d u c t i n v e s t i g a t e d a p e a k ( w i t h t h e s a m e r e t e n t i o n t i m e as t h e d e c o m p o s i t i o n p r o d u c t o c c u r r i n g d u r i n g gas c h r o m a t o g r a p h y o f t h e c a r b a m a t e s w i t h o u t a c e t y l a t i o n , F i s h b e i n a n d Z i e l i n s k i , 1969) a p p e a r e d i n t h e g a s c h r o m a t o grams. Mass spectra were taken. The most intense m/e fragments with their r e l a t i v e i n t e n s i t i e s a r e g i v e n i n T a b l e 3.

86

F. van Hoof and A. Heyndrickx Table 4. Recoveries of metabolites added to urine

Compound

Internal standard

% Recovery

% Standard deviation

3-methyl, 5-isopropylphenol

3,5 dimethylphenol

94

8

4-methylthio, 3,5 dimethylphenol

4-dimethylamino, 3,5 dimethylphenol

92

1.7

4-dimethylamino, 3,5 dimethylphenol

3-methyl 5-isopropylphenol

94

9

4-benzothienol

4-methylthio, 3,5 dimcthylphenol

95

2.2

Table 5. Quantitative data on the excretion of metabolites Product investigated

Promecarb

Dosereceived (in rag) I. 10.0 2.

Mesurol Mobam Zectran

Excreted metabolite (in g) 0--24 24--48 48--72

5.0

3. 5.0 i. 5.8 t. 6.8 2. 5.8 1. 10.0 2. 10.0

3t

Amount excreted as % dose

--

--

0.3

--

--

--

0

-134 46 108 133 76

--19 20 30 34

--39 9 4 14

0 3.3 2.1 3.2 2.2 t.6

:

T h e m a s s s p e c t r a o b t a i n e d were i d e n t i c a l w i t h 3-methyl, 5-isopropylphenot (I), 4 - m e t h y l t h i o , 3,5 d i m e t h y l p h e n o l (II), 4-benzothienol ( I I I ) a n d 4 - N , N d i m e t h y l amino, 3,5 d i m e t h y l p h e n o l (IV). Most i m p o r t a n t f r a g m e n t s were d u e t o loss of m e t h y l g r o u p (3-methyl, 5 - i s o p r o p y l p h e n o l , 4 - m e t h y l t h i o , 3,5 d i m e t h y l p h e n o l ) , loss of CO a n d CHO (4-benzothienol) on cleavage o f t h e h y d r o x y l g r o u p (4-dimethylamino, 3,5 dim e t h y l p h e n o l ) . T h e m a s s s p e c t r a of t h e reference p r o d u c t s were o b t a i n e d u n d e r t h e s a m e conditions a n d were i d e n t i c a l w i t h t h o s e o b t a i n e d from t h e m e t a b o l i t e s . B y t h i s p r o c e d u r e no o t h e r m e t a b o l i t e s could be definitely identified. T h e m e t a b o l i t e s were e x t r a c t e d a n d quantified as described u n d e r E. T h e p r o c e d u r e was t e s t e d on b l a n k urine s a m p l e s a n d gave no interferences. Recoveries were a s s a y e d b y e x t r a c t i n g urine s a m p l e s fortified w i t h i m g m e t a b o l i t e p e r t 0 0 m l urine. R e c o v e r i e s a n d s t a n d a r d d e v i a t i o n s are s u m m a r i z e d in T a b l e 4. U r i n e s a m p l e s of rats, which h a d r e c e i v e d a single oral dose o f different c a r b a m a t e s , were a n a l y z e d . T h e results of t h e s e a n a l y s e s can be f o u n d in T a b l e 5. As w i t h t h e u n c h a n g e d p r o d u c t s , e x c r e t i o n of t h e m e t a b o l i t e s occurs m o s t l y w i t h i n 48 hrs a f t e r a d m i n i s t r a t i o n . The m e t a b o l i t e s m o n i t o r e d r e p r e s e n t o n l y a m i n o r f r a c t i o n of t h e dose given. E x c r e t i o n o f t h e Z e c t r a n m e t a b o l i t e occurs

The Excretion in Urine of Four Insecticidal Carbamates

87

s o m e w h a t slower t h a n e x c r e t i o n Of t h e o t h e r m e t a b o l i t e s . E x c r e t i o n o f t h e p a r e n t c o m p o u n d showed a s i m i l a r p a t t e r n .

Conclusion B o t h u n c h a n g e d c o m p o u n d s a n d t h e i r h y d r o l y s i s p r o d u c t s were e x c r e t e d as m i n o r f r a c t i o n s o f t h e a m o u n t a d m i n i s t e r e d w i t h i n 48 hrs a f t e r a d m i n i s t r a t i o n . T h e s e results a r e in g o o d c o r r e s p o n d e n c e w i t h d i s t r i b u t i o n a n d e x c r e t i o n studies d o n e b y o t h e r a u t h o r s on similar c o m p o u n d s . Since it has b e e n s h o w n t h a t some similar p r o d u c t s are m e t a b o l i z e d in different m a m m a l s in m u c h t h e s a m e w a y (Dorough, t970 ; K_naak et al., i965 ; K n a a k et al., i 9 6 8 ; Leeling a n d Casida, t 9 6 6 ; Meikel, i 9 7 3 ; R o b b i n s et al., i 9 6 9 ; R o b b i n s et al., 1970) m o n i t o r i n g o f a n u n c h a n g e d c o m p o u n d m i g h t b e a v a l u a b l e i n d i c a t o r o f e x p o s u r e t o t o x i c dosis, as q u a n t i t a t i v e a n a l y s e s of m o r e p o l a r a n d m o r e i m p o r t a n t m e t a b o l i t e s could p r o v e t o be m u c h m o r e c o m p l i c a t e d .

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Oonnithan, E. S., Casida, J. E.: Oxidation of methyl- and dimethylcarbamate insecticide chemicals by micresomal enzymes and Anticholinesterase activity of the metabolites. J. Agr. Food Chem. 16, 28--44 (1968) Robbins, J. D., Bakke, J. E., Feil, V. J.: Metabolism of 4-benzothienyl N-methylcarbamate (mobam) in rats balance study and urinazy metabolite separation. J. Agr. Food Chem. 17, 236--242 (1969) Robbins, J. D., Bakke, J. E., Feil, V. J. : Metabolism of benzo (b)thien-4-yl methylcarbamate (mobam) in dairy goats and a lactating cow. J. Agr. Food Chem. 18, 130--t34 (1970) Skrinjaric-Spoljar, M., Reiner, E.: Enzimska razgradnja karbamata. Arh. hig. rada 19, 251--258 (1968) Slade, M., Casida, J. E.: Metabolic fate of 3,4,5- and 2,3,5-trimethylphenyl methylcarbamates, the major constituents in landrin insecticide. J. Agr. Food Chem. 18, 467--474 (1970) Stanley, C. W., Thornton, J. S.: Gas chromatographic method for residues of baygon and its major metabolite in animal tissues and milk. J. Agr. Food Chem. 20, 1269--1273 (1972) Strother, A. : Comparative metabolism of selected N-methylcarbamates by human and rat liver fractions. Biochem. Pharmacol. 19, 2525--2529 (1970) Prof. Dr. A. Heyndrickx Laboratory of Toxicology State University of Ghent Ghent/Belgium