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ApPENDIX I The Determinative Method for Assaying Residues in Tissue and Plasma

G. v. Downing

A. Summary B. Reagents C. Solutions D. Apparatus E. Chromatographic Apparatus F. Standards for the Determinative Assay G. Procedure for the Determinative Assay of Tissues H. Suitable Stopping Places for Tissue Assays I. Analytical Procedure for Asay in Plasma

A. Summary

The tissue sample is homogenized with -water, and dihydro B1a is extracted from the tissue with isooctane. The isooctane is evaporated, and solvent-solvent distributions into acetonitrile out of hexane and into hexane out of acetonitrile-water are performed. Solvent is removed and fluorescence produced by heating at 95° C with the -derivatizing reagent. Mter adding , the reaction mixture is washed through a silica gel SEP-PAK, the solvent is removed, and HPLC analysis (fluorescence detection) on Zorbax CI8 is performed on the residue dissolved in . A flow diagram of the assay is shown in Figure AI. I. Proteins are precipitated from plasma by addition of acetone and then water. The ivermectin dihydro B la is extracted with ethyl acetate from the acetone-water-plasma mixture. The solvent is removed and fluo• rescence produced by heating at 95° C with the derivatizing reagent. Mter adding chloroform, the reaction mixture is washed through a silica gel SEP-PAK, the solvent is removed, and the HPLC analysis (fluorescence detection) on Zorbax CI8 is performed on the residue dissolved in methanol. Homogenize 5g ti_ Extract Into evaporate Dissolve In methanol centrtfuge Dissolve In with -a-Iweter l-- laooctane end cool -20"C evaporate hexane ~ , - A

Exnctlnto Welhwlth concentrate Extract Into eveporate Dissolve In • KetonItrtte r---- hexane add weter hexane 1.0 mI methanol

-- -- t B

0.5 mI ellquot AdcI .. egent end Dilute with chlorofonn eveporate Dissolve In Inject Inlo LC end pass through eveponte heet85-100·C r---- methanol ~ with IIU0f'8scenc:e for 1 hour Sep·Pe~ cletec:llon t C

FIGURE ALl. Flow diagram of determinative assay. A, Band C are possible stopping points during the assay. 326 G.V. Downing B. Reagents

1. Acetic anhydride-Fisher or Baker, reagent grade. 2. Acetone-Mallinckrodt, Nanograde. 3. Acetonitrile-Fisher, HPLC grade; Mallinckrodt, Nanograde; or Burdick & Jackson, distilled in glass. 3. Chloroform-Burdick & Jackson, distilled in glass (1% preservative). 5. Dimethyl Formamide (DMF)-Baker or Fisher, reagent grade; or Mallinckrodt, AR. 6. Ethyl Acetate-Burdick & Jackson, distilled in glass; or Fisher, pesticide grade. 7. Hexane-Burdick & Jackson, distilled in glass; or Mallinckrodt, Nanograde. 8. Methanol-Burdick & Jackson, distilled in glass; or Mallinckrodt, Nanograde. 9. Methylene Chloride-any grade available. 10. 1-Methylimidazole-99%, Aldrich Chemical Company. 11. -the equivalent of Matheson extra-dry compressed gas. 12. Sodium Chloride-Baker or Fisher, reagent grade. 13. Sylon-CT-Supelco, Inc. 14. 2,2,4-Trimethylpentane-isooctane, Burdick & Jackson, distilled in glass; or Mallinckrodt, Nanograde. 15. Water, Filtered Millipore-Distilled water is treated with a Milli-Q system and then filtered through a Millipore Type HA 0.451Lm disc.

C. Solutions

1. Derivatizing Reagent-Mix 0.9 ml of DMF with 0.3 ml of acetic anhydride and 0.2 ml 1-methylimidazole. Make up just before use. 2. Acetone/Water-50% (v/v). 3. 95% Methanol/Water-Make 100 ml of filtered Millipore water to 2 liters with methanol. Mix. With the aid of vacuum, filter mixture through Millipore FH 0.51Lm filters. Deaerate by slowly bubbling nitrogen through for 10 minutes.

D. Apparatus

1. Balance-analytical, capable of weighing 1 mg accurately. 2. Balance-capable of weighing 5 g accurately into an approximately 50-g tube. 3. Bath, water-variable temperature 40° to 80° C. 4. Bath, oil-95° to 100° C. 5. Centrifuge, IEC Model HN-S-II, with 6-place rotor IEC #958 and 15- Appendix I. Assaying Ivermectin Residues in Tissue and Plasma 327

and 50-ml cups. The centrifuge is run at 2000 to 2500 rpm. The centrifuge used gives ca. 700 to 750 x g maximum centrifugal force (RCF). 6. Centrifuge tubes-glass, 15 to 50 ml with polyethylene stoppers to fit. 7. Centrifuge tubes-50-ml polyproplyene, Corning#25331 (used only for storing standard solutions). 8. Centrifuge tubes, 15 ml, silylated approximately once every 2 months (used only for the derivatization reaction). Tube stoppers must fit tightly. Fill each tube to the top with Sylon-CT. Let stand 20 minutes. Immediately and quickly rinse, first thoroughly with and then with methanol. Fill with methanol. Let stand 20 minutes, rinse thoroughly with acetone, and dry. All glassware used should be completely free of all acidic and alkaline residues. (These tubes should be cleaned by hand by first soaking in methylene chloride immediately after use and then in detergent for at least several hours, followed by thorough rinsing with hot water, distilled water, and acetone before thorough drying. Variations in the washing regimen are not recommended, since some analysts have had poor results when the standard washing method was not followed.) 9. Dispensing pipettors-lO, 15, and 20 ml. 10. Filtration unit, complete with Millipore FHLP 04700 filter. 11. Freezer-capable of reaching temperatures of -200 C. 12. Gloves-disposable PVC from Fisher Chemical. 13. Graduate cylinder-25 ml, l00ml. 14. Graduate cylinder-2000 ml with ground-glass stopper. 15. Homogenizer, Polytron-Brinkmann Instruments, #27-11-200-5 with PTA lOS generator #27-11-330-3. 16. Pipets-disposable, Pasteur. 17. Pipets-graduated 1.0 ml and 2.0 ml. 18. Pipets-volumetric 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml. 19. Repipet-5 to 10 ml volumes. 20. SEP-PAK-silica cartridge, Part No. 51900-Waters Associates. 21. Spatula-stainless steel. 22. Syringe-50ILI, 250ILI, and 5 ml and 10 ml. 23. Tape-#13 2956, ~ inch from Ace Scientific. 24. Ultrasonic bath-Sonogen Automatic Cleaner-Branson Model 520 or equivalent. 25. Vortex mixer.

E. Chromatographic Apparatus

A Beckman-Altex Model IIOA liquid chromatographic pump complete with Waters Wisp Autosampler and Kratos-Schoeffel Instruments Model FS950 fluorescence detector with a I-millivolt recorder was used. A 328 G. V. Downing

51Lm, 4.6-mm ID, CI8 standard Brownlee Labs guard column (Spheri-5 RP-18 OD-GU, obtained from Rainin Instrument Co., Inc.) was used before the analytical column. This guard column is replaced monthly unless the pressure reaches 2000 psi, in which case it is replaced immediately. The ivermectin H2Bla derivative has a retention time of about 12 to 13 minutes when the indicated conditions are used.

Conditions: 15 cm length x 4.6 mm ID Zorbax ODS-CI8 column. Mobile phase-5% water in methanol (v/v). Column temperature-30° C. Flow-1.8 mil minute (usual pressure 1000 psi, 500 to 2000 psi acceptable). Detector Settings: Excitation lamp-FSA 110, standard 365 nm. Standard Kratos Flowcell-FSA 210. Excitation filter-FSA 403, 365 nm band filters. Emission filter-418 nm. Sensitivity range-0.2 A. Time constant-about 6.

F. Standards for the Determinative Assay

For tissue assays using the most sensitive scale, accurate aliquots of 50, 100, 150,200, and 250 ILl of ca. 500 ng/ml standard solution of dihydro Bla in methanol (working standard solution) were added to silylated 15-ml centrifuge tubes, and the samples were evaporated to dryness at 60° C with nitrogen purge. For plasma assays, accurate aliquots of 20, 40, 60, 80, and 100 ILl of working standard solution were added to silylated tubes and evaporated to dryness. Mter reaction, SEP-PAK treatment, etc., these derivatized samples are redissolved in I ml of methanol to give 25, 50, 75, 100, and 125 ng/ml standard solution equivalents for tissue assays and 10, 20, 30, 40, and 50 ng/ml standard solution for plasma assays. Starting with solutions of about 0.045% (w/w) dihydro Bla in propylene glycol, stock solutions of about 10 micrograms/ml can be prepared by weighing accurately about 1 gram of the solution into a 50-ml volumetric flask. Methanol is used to dilute to the mark, and the solution is thoroughly mixed. Storage of this stock solution and all dilutions of same should be in polypropylene tubes at -20° C. Analysts attempting the ivermectin residue analysis for the first time are advised to run a standard curve first before attempting the complete method. Appendix I. Assaying Ivermectin Residues in Tissue and Plasma 329 Procedure for the Determinative Assay of Tissues

Homogenization

All Tissues 1. Weigh accurately 5.0 grams of sample into a 50-ml centrifuge tube.

Liver, Muscle, Kidney, and Dose Site 2. Add 15 ml of 50% acetone/water to the tube from a dispensing pipettor. 3. Homogenize for 15 to 20 seconds or shorter time with a Polytron setting of 7. Homogenize until a homogeneous-looking mixture re• sults. 4. Put a clean 50-ml tube in place containing 15 ml of isooctane from a dispensing pipettor. 5. Homogenize 5 to 10 seconds and pour the isooctane into the 50-ml tube with the homogenate. Clean the generator probe between samples with isooctane, distilled water, and acetone.

Fat Only 2. Add 15 ml of the acetone/water and 10 ml ofisooctane to the tube and homogenize until a homogeneous mixture results. 3. Rinse probe with 10 ml isooctane in a clean 50-ml tube. 4. Pour isooctance into the 50-ml tube with the homogenate. 5. Add 1 gram of solid sodium chloride to the tube containing the homogenate.

Extractions

All Tissues 6. Stopper the tube, shake well for 1 minute, and centrifuge for 10 minutes. 7. Transfer the upper or isooctane layer to a second 50-ml centrifuge tube with a disposable pipet. Completely avoid the lower layer. 8. Break up the plug by using a vortex mixer and/or shaking; add 15 ml of clean isooctane, repeating the extraction and combining isooctane layers. 9. Evaporate the combined isooctane layers to a small volume (or ca. 5 ml for fat) using an 80° C bath and nitrogen purge. 10. Repeat the extractions with 2 more passes of 15 ml each of isooctane, adding the isooctane in each case to the same tube which had the previous 2 isooctane layers. 330 G.V. Downing

11. Again evaporate as far as possible using the 80° C bath and purge. 12. Add 6 ml of methanol to all samples except fat and dissolve or resuspend completely with an ultrasonic bath and/or vortex mixer. 13. Place all samples except fat in a refrigerator until thoroughly cooled (at this point the sample is best left overnight in the refrigerator or freezer).

Fat Only 12. Either add 2 ml of hexane and S ml of acetonitrile to the still hot and molten fat or freeze overnight at -20° C. For samples frozen overnight, remelt in a 80° C bath and add the hexane and acetonitrile the next day. 13. Shake the still warm mixture thoroughly for ca. 1 minute and immediately centrifuge. 14. Cool in ice water until the lower or fat layer congeals and the upper or acetonitrile layer can be decanted completely into a fresh IS-ml centrifuge tube. IS. Repeat the melting, extraction with S ml of acetonitrile, and transfer into the same IS-ml tube. 16. Add 2 ml of hexane to the IS-ml tube, shake thoroughly, centrifuge, and remove the upper, hexane layer to waste by disposable pipet or by aspiration. Go to Step 22.

Liver, Muscle, Kidney, and Dose Site 14. Centrifuge the cold SO-ml tube for S minutes, and decant off the clear supernatant to a clean IS-ml centrifuge tube. IS. Wash the residue in the SO-ml tube with 1 ml of methanol using a vortex mixer, centrifuge, and decant off into same IS-ml tube. (If solids are decanted in either step, recentrifuge the IS-ml tube and decant into a new IS-ml tube to get a clear solution to continue the assay with.) ISa.For muscles and dose sites only, do a 2-ml hexane extraction of the residue solids and add to the combined methanol. 16. Using a 60° C bath and nitrogen purge, evaporate off all the methanol. 17. By repipet add 3 ml of hexane to the tube previously containing the methanol and use an ultrasonic bath to remove all material from the walls of the centrifuge tube. 18. Add by repipet 4 ml of acetonitrile and repeat the ultrasonic treatment briefly. 19. Shake thoroughly, centrifuge S minutes, and move the lower or acetonitrile layer to a clean IS-ml tube. 20. Reextract the hexane layer with a second 4 ml of acetonitrile and combine acetonitrile layers. 21. Extract the combined acetonitrile layers with 1 ml of hexane, centri- Appendix I. Assaying Ivermectin Residues in Tissue and Plasma 331

fuge to clear the layers, and move the upper, hexane layer to waste by disposable pipet or by aspiration.

All Tissues 22. Evaporate the acetonitrile solution to 1.0 ml using a ca. 60° C bath and nitrogen flush. 23. If the acetonitrile is less than 1 ml, make up to 1 ml with fresh acetonitrile. 24. Use an ultrasonic bath to get a homogeneous mix if necessary. 25. Add 4 ml of distilled water (2 ml for kidney samples) and 5 ml of hexane; shake ca. 1 minute and centrifuge for 5 minutes. 26. Move the upper, hexane layer to a clean 15-ml centrifuge tube using a disposable pipet. Avoid the lower layer. 27. Repeat with 5 ml and, then, 4 ml hexane extractions combining all 3 hexane extracts. 28. Evaporate to dryness (or as near dryness as possible) using a 40° C bath and nitrogen purge. (To get the sample more completely dry, more heat may need to be applied at the end of the evaporation. The bath may go up to ca. 80° C at this point.) 29. Redissolve the residue in exactly 1.0 ml of methanol using a vortex mixer and ultrasonic bath. 30. Mix thoroughly and centrifuge for 5 minutes.

Derivatization 31. Pipet exactly 0.5 ml of the solution from Step 30 into the bottom of a silylated 15-ml centrifuge tube. Use a O.5-ml volumetric pipet. 32. Completely evaporate the methanol carefully, using at 60° C bath and nitrogen purge. Avoid spattering. 33. With a 1.0-ml graduated pipet, add 0.1 ml of freshly made acetic anhydride-methylimidazole-DMF mix to each tube and a series of standard tubes (see standard section F preceding). 34. Stopper, vortex briefly, and centrifuge for a few seconds. 35. Tape the stoppers in place and put all samples and standards in a well-stirred 95° C oil bath for 1 hour. The bottoms of the tubes should be about 1 inch below the oil surface. 36. Remove the tubes, wash the oil off the outside of the tubes with acetone in a wash bottle, and cool briefly. The residue should be black. 37. Add about 1 ml of chloroform to each tube and vortex to mix. 38. Wash a SEP-PAK cartridge with 3 to 4 ml of chloroforM, using a syringe to force the liquid through. 39. Add the sample by disposable pipet to the syringe and force it through the cartridge. 332 G.V. Downing

All Tissues 40. Wash the centrifuge tube 3 times with 1 ml each of chloroform into the syringe and through the cartridge. 41. Elute the column with a further 8 to 9 ml of chloroform (with aliquots of 3, 3, 2, or 3 ml). 42. Collect all the chloroform eluate from the SEP-PAK in a 15-ml centrifuge tube. 43. Evaporate the chloroform off with a 60° C bath and nitrogen purge. Get the residue completely dry.

HPLC Analysis 44. Pipet 0.5 ml of methanol (or other suitable quantity) into the tube and use a vortex mixer and ultrasonic bath to completely dissolve the residue. 45. Centrifuge briefly and load WISP inserts if autosampler is used. 46. Inject 50 ILl of the supernatant of each sample and standard into the HPLC. 47. Measure the peak heights at the retention time of the derivative of dihydro-B 1a as indicated by the standards. 48. Generate a standard curve by performing a linear regression analysis of response (peak height) versus concentration (ng/ml) for standards. The curve should be linear with a regression coefficient of 0.97 or better. Use the regression equation to determine the concentration of unknowns with an observed peak height. Calculate the sample concentration as follows: VI VI ppb = (ng/ml)s x V2 x DIG = V2 x (ng/ml)s x DI5

Where: (ng/ml)s for the unknown is determined from the standard curve. VI = ml sample dissolved in at end of assay (Step 44). V2= ml of sample taken to make derivative (Step 31). D= dilution of sample at end of assay or 1 if no dilution is made. G= grams of sample taken = 5.

H. Suitable Stopping Places for Tissue Assays

The following stopping places allow storage for a maximum of 4 days: 1. In a refrigerator at 4° C. a. Only in methanol after Step 12. 2. In a freezer at -20° C. a. After Step 11 for fat only. b. After Step 30, before derivatization. Appendix I. Assaying Ivermectin Residues in Tissue and Plasma 333

c. After Step 36, the derivatization. d. After Step 44, dissolution of the residue in methanol. If stopping places other than the above are needed, assay stability at said stopping place should be demonstrated first.

I. Analytical Procedure for Assay in Plasma

1. Pipet 5 ml of plasma into a 50-ml centrifuge tube, pipet in 7.5 ml of acetone, shake briefly, and let stand 15 minutes. 2. Pipet 7.5 ml of distilled water and 15 ml of ethyl acetate into the tube and shake by hand moderately for 1 minute. Shaking should be thorough enough to allow extraction but not violent enough to cause emulsions. 3. Centrifuge thoroughly and move the upper, ethyl acetate layer to a second 50-ml tube as completely as possible, using a disposable pipet. Move absolutely no lower phase. 4. Thoroughly break up the plug in the bottom of the first tube by shaking or with the wide end of a disposable pipet and repeat the ethyl acetate extraction a second time. Combine the ethyl acetate layers. 5. Evaporate the ethyl acetate completely with a nitrogen purge in a 75° C water bath. 6. Pipet exactly 1 ml of methanol into the dry tube. 7. Dissolve the residue as much as possible by using an ultrasonic bath with intermittent shaking. (The methanol must contact all the tube.) 8. Centrifuge and pour the supernatant solution into a 15-ml centrifuge tube and pipet exactly 0.5 ml of this solution into the bottom of a silylated 15-ml centrifuge tube. 9. Evaporate completely to dryness with a nitrogen purge and 60° C bath. 10. Using a I-ml graduated pipet, add 0.1 ml of derivatizing reagent to samples and standards, stopper, and use a vortex genie to dissolve the residue. 11. Centrifuge briefly, tape the stoppers, and put unknowns and standards together in a well-stirred 95° C oil bath for 1 hour. 12. Remove tubes and rinse the oil off the outsides with acetone. The residue should be black. (If the residue is not black, the remaining sample from Step 8 can be used to repeat the derivatization with the next set of standards.) 13. Add ca. 1 ml of chloroform to the residue in each tube. 14. Use a vortex genie to thoroughly mix. 15. Wash a silica SEP-PAK cartridge with 3 to 4 ml of chloroform, using a syringe to force the liquid through and discard to waste. 16. Add the chloroform sample to the syringe with a disposable pipet and force the liquid through the cartridge. 17. Wash the centrifuge tube 3 times with ca. 1 ml each of chloroform into 1 . : i., ·-'-i-'-i- ... ' :...L!--r;'-;-t- ~'8¥!-i';c _'I~'i;-:!t '_"j_,.Jr~W-':-d'F' .i·+=m+~ .-H+=tl:··t D-irri-·ITj,+,'·Hi- '!+' i#-~"1-:"~.--i-:'~'i'+"i,' ., ,,'-, -~,',;,-'+ , ,,!_~,_L.•- -.-+. H-1':'1:: ".,-~t-J:.t;-,fffi"-++;-+t-t-'.T'T;' '-Ltt +!.J -rl-H-t-r-i-h .+~.---~-r-l:t-t-:"""'1 '-",~.-,-+- , ! 1-' ·d· i-' -t- . ." f" " ,_. t+t-;-H-j ,-tt , T T!'--,'" - -t'l-': "-,-;- .I-t+- ~-:--!-'- 4~'Trt- -t+ - 1_'--+ - _.h- -It, ,- '-i-+J-r " ~~..~ -,-t--""~-'-i''', :-l't-:- :.',]-,::1+ !E'" ' -"t.' " , I I I .' I L ' 1-:·:1"+:: ' - -r-7TT Determinative assay - Itf+ 41-, ::.rT , ~

;~~;.~...,..;r:!i '+:rt..J~ Lll'~ -~H+H-' .+H:;'-8- j .';=rl=T '- ~--j+ - +,-+-H--fW- i , IT ':--- -. Ii-' -I,i-t-ritt+ + H- .,. , , -:-r- !j- " ' - =I-+-. L:- - f-W-:- -+ H- . ...: +H+t- H++ +~- r ,: , -t+ - I , " ~ ~ , ti-L,-<-+-tl' tt '; , ; '_:':-1- LL _.j ' .. - H B - .- ,,'+ _L.,'-+ t- ' H- . q*l=i". 0. ' ~$:+FH-W + ':, '::;'-",:::_~-;:-. 2.' , L!.+. -- _ -h+ h -f + -;-i .-rj-t H- ~I~:·1 T ~r Ll-,' :i1 1--i-i-ttR-l±t-!-;-, -+H+..l- ~--- .) ,++-1+++' -4=1: ' -H+ -llit 'h"';'" -,~-++- '-i-j - ' j - - _. ffi~~~-' .J.-i-!±-H+ , - +-++ +'-1-+--o.::L1" - - j - --'-4- • - L ... _ to+- r -1- -+-t. --. - +-t ,--I--t-r-- !ll ~I-j+ "+ "1--'+l:I±P+b.·-ri-~--1 ~+,-*', , =t-clttlfflu+ r 4--:- -1- ~+tt+ =+ =.n -l±r± Ettr rH...;-ti..., ' ' r-++--l~-l-.'i-! ~--:H~ -r+: "r;" . ;, - _. -j". ., ' , "

:t~~+ - r1~t~ r:i+l~~t-i:+++- J-1+=·:H++-lql++lq.::' l1+~ ·nti= +FI:++ffiFf-t-:'::ffi+-rlf+H:p:++~*~;·++::q· i- n:f" · ~1ftittrt-i+ ", tm' +-- -H H-t++i-H ' "t.E.; ,'r-;-' tl+,-i-L fl' t- -: .. cf ~," -H'::;+I-HI-~tt± -:-T', ,!.j.4-t-t++-f-jt+Jt+!+f-t+I~ttt~ .. !+It~tH±r,1:11 '-W-I- ' j_L '_~c'" , ""L.1...:-r-+-oo- -L-i...J..- U+"r:·5i+··,-'-j-t-l-t-I-h-t.. B1-+'-l-f+IJ::J:+i1++H:a.B,o. @- 1 . ~-+ H 8 --;;,:.... - 14J::ttt ~~,_q:ttti-t:::tt - t-t"H-F-~T, , . i '- - tt - -HL + -: +t- LLH-'- -H---- li-ri- H- .+- :-h-H-I-· l rt l fJ-l _', " .. ' '_ 2.. • b ,.LLLL .' , L I ' ~u....u. -' ... nTT TT"1 ,,+,. ~ ai'-, ...:..J..~LLLLL._LJ...L . _l__ .-I-l-I-_I.J._.I...LLLt_L.~iLLLl.!~++ 'I" : :+~ '" .. C.w.._ ' l~.'-L ;.-+..l}.....l...!_~.~~--:-, t-r- ..:..:::...:--r:-T- --!"T,- -,;LL++ I,. ~~~I __ ~ +-+.... ±. _.'..:- ..··.·+-i '-:-J.~.-I-1-+-=1f-t-~+.;.--...... -.-+·-l~--·"";-~-·-.··;""-'-t· -,-,-+ .. !- •• -.-. . .•• , ..... -- ••.•.• • ~:-:," __ .. _ t±ttt~~J.. ,+H·-i-'-+t· '- ' ' .L-.. -h·1-t--r-ri-t+-!i++-H' . _f.llI +, ,\A .-i--j-H-' r- rr 1-+- '-r'- .. -H'T-+- i .. ;':'~. ' - '~- 'l-~.~~;' . -t-++ J.~-t' - ,- ,+~p+,. J i+ --t:H=t+t··! -I+H-H+ ,-H-t-rt-: ...;t-' H- -r-- ""i-r+I:t~+I+I-.." '--=t··'··H·'·! h '.. L.LL , I- InJections.,-!.!._ -,-rL:... --11"1+,' .. '1-1--1'+ ~i,"rh" 1l_:...-V I . t--I'll I .' •••• , ••.• ',.,.. , • I I -"'-r-Thi"--',I-H++-, . + 1,, I I

FIGURE AL2. Chromatographic tracings of (A) standard and (B) plasma sample containing ivermectin. Appendix I. Assaying Ivermectin Residues in Tissue and Plasma 335

the syringe and through the cartridge, using the same disposable pipet. 18. Elute the cartridge with a further 8 to 9 ml of chloroform (aliquots of 3, 3, 2, or 3 mI). 19. Collect all the chloroform, leaving the SEP-PAK from Steps 16, 17, and 18 (eluent) in a IS-ml centrifuge tube. 20. Evaporate the chloroform completely off with a 60° C bath and nitrogen purge. The residue must be completely dry. 21. Pipet exactly 0.5 ml of methanol (or other larger volumes for high-assay plasmas) into the tube and use a vortex mixer and ultrasonic bath to completely dissolve the residue. 22. Centrifuge and inject SO ILl of sample from each sample and each standard in turn into the HPLC unit using the WISP Autosampler. 23. Measure the peak heights of the derivative at the retention time indicated by the standards. 24. Calculate results as described in the tissue method starting at Step 48, substituting ng/ml for ppb and S ml for G in the equation. ApPENDIX II Confirmatory Assay for Assaying Ivermectin Residues in Liver

G. V. Downing

A. Summary of the Confirmatory Residue Method B. Reagents C. Solutions D. Apparatus E. Chromatographic Apparatus F. Standards for the Confirmatory Assay G. Procedure for the Confirmatory Method

A. Summary of the Confirmatory Residue Method

The tissue is homogenized with acetone-water and the dihydro B1a is extracted with isooctane with the tissue present. Following removal of the isooctane, solvent-solvent distributions into acetonitrile out of hexane and into hexane out of acetonitrile-water are performed. Solvent is removed from aliquots of the resulting solution, and monosaccharide and aglycone derivatives are formed. Extractions into methylene chloride/ hexane/isobutyl out of methanol/water are followed by solvent removal and fluorescence production by heating at 95° C in imidazole reagent. Extractions are performed out of methanol/water into hexane/ isobutyl alcohol. Solvent is removed and HPLC analysis performed on Zorbax C18. Confirmation results when peaks are observed at appropriate levels for H2B1a itself and the monosaccharide and aglycone derivatives.

B. Reagents

1. Acetic anhydride-Baker or Fisher, reagent grade. 2. Acetone-Mallinckrodt, Nanograde. 3. Acetonitrile-Fisher, HPLC grade; or Mallinckrodt, Nanograde. 4. Dimethyl Formamide (DMF)-Baker or Fisher, reagent grade. 5. Hexane-Burdick & Jackson, distilled in glass; or Mallinckrodt, Nanograde. Extract Into Add IUIfurIc DIsecIM In G.1mI DIeeoMtIn 0.5 mt aliquot Lelstenelst { Methylene chloride Enporste KId rugenta Hexe.,. rugent and hNt 2.0 mI meIhenoI RTovemlght oHlOhent ".ponIte Iodrytubn IDA IS-100°C 1 hour and teke thInI.. aliquot f I

Add 1 mlMeoH InJectInto DIaoIw In and extract Into Enpcnte 0.5 mI LCwlth {Henne oHIOIvMt MeoHforLC FIuoreecence__ 1kIn IDA

~ -~ -

FIGURE All. I. Flow diagram of confirmatory assay. B is a possible stopping point during the assay. 338 G. V. Downing

6. Isobutyl alcohol-Burdick & Jackson, distilled in glass. 7. Isopropanol-Burdick & Jackson, distilled in glass. 8. Methanol-Burdick & Jackson, distilled in glass; or Mallinckrodt, Nanograde. 9. Methylene Chloride-any grade available. 10. Methylene chloride-Burdick & Jackson, distilled in glass. 11. I-Methylimidazole-99%, Aldrich Chemical Company. 12. Nitrogen-the equivalent of Matheson extra-dry compressed gas. 13. Sylon-CT-Supelco, Inc. 14. 2,2,4-Trimethylpentane-isooctane, Burdick & Jackson, distilled in glass. 15. Water, doubled distilled-Distilled, deionized water is redistilled in an all-glass apparatus.

C. Solutions

1. Derivatizing Reagent-Mix 0.2 ml of I-methylimidazole with 0.3 ml of acetic anhydride and 0.9 ml of DMF . Make up just before use. 2. Acetone/Water-50% (v/v). 3. Methanol/Water Mobile Phase (90/I0)-Make 200 ml of doubly dis• tilled and filtered water to 2 liters with methanol. Deaerate by slowly bubbling nitrogen through for 10 minutes. Make other water content mobile phases correspondingly. 4. 40% Methylene Chloride-Hexane-Isobutyl Alcohol-Fill a 500-ml stoppered graduate to the 200 mark with methylene chloride. Add hexane to the 500-ml mark and, then, 20 ml of isobutyl alcohol. Mix. 5. 1% Isobutyl Alcohol in Hexane-Make 5 ml of isobutyl alcohol to 500 ml in a graduated cylinder. Mix. 6. 1% Sulfuric Acid in Isopropanol-Pipet 0.5 ml of sulfuric acid carefully into about 40 ml of isopropanol in a 50-ml volumetric flask. Mix. Dilute to the mark with isopropanol and thoroughly mix. Make fresh just before use. 7. 1% Sulfuric Acid in Methanol-Pipet 0.5 ml of sulfuric acid carefully into about 40 ml of methanol in a 50-ml volumetric flask. Mix. Dilute to the mark with methanol and thoroughly mix. Make fresh just be• fore use.

D. Apparatus

1. Balance-analytical, capable of weighing 1 mg accurately. 2. Balance-capable of weighing 5 g accurately into an approximately 60-g tube. Appendix II. Assaying Ivermectin Residues in Liver 339

3. Bath, water-variable temperature 40° to 80° C. 4. Bath, oil-95° to 100° C. 5. Centrifuge, IEC Model HN-S-II, with 6-place rotor IEC 958 and 15- and 50-ml cups. The centrifuge is run at 2000 to 2500 rpm. The centrifuge used gives ca. 700 to 750 X g maximum centrifugal force (RCF). 6. Centrifuge tubes, glass, 15- and 50-ml with polyethylene stoppers to fit. 7. Centrifuge tubes-50-ml polypropylene, Corning 25331 (used only for storing standard solutions). 8. Centrifuge tubes, 15 ml, silylated approximately once every 2 months (used only for the derivatization reaction). Tube stoppers must fit tightly. Fill each tube to the top with Sylon-CT. Let stand 20 minutes. Immediately and quickly rinse, first thoroughly with toluene and then with methanol. Fill with methanol. Let stand 20 minutes, rinse thoroughly with acetone, and dry. All glassware used should be completely free of all acidic and alkaline residues. (These tubes should be cleaned by hand by first soaking in methylene chloride immediately after use and then in detergent for at least sev• eral hours each, followed by thorough rinsing with hot water, dis• tilled water, and acetone before thorough drying. Variations in the washing regimen are not recommended, since some analysts have had poor results when the standard washing method was not followed.) 9. Dispensing pipettors-lO, 15, and 20 ml. 10. Gloves-disposable PVC from Fisher Chemical. 11. Freezer-capable of reaching temperatures of -20° C. 12. Graduate cylinder-25 ml. 13. Graduate cylinder-500 ml. 14. Homogenizer, Polytron-Brinkmann Instruments, 27-11-200-5 with PTA lOS generator 27-22-330-3. 15. Parafilm-American Can Co. 16. Pipets-disposable. 17. Pipets-graduated 1,2,5, and 10 ml. 18. Pipets-volumetric 0.5 ml, 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml. 19. Reciprocating shaker, variable speed-Eberbach, J. T. Baker Catalog 8287-E30 or equivalent. 20. SEP-PAK, silica catridge, Part No. 51900-Waters Associates. 21. Spatula-stainless steel. 22. Syringe-50 ILl and 250 ILL 23. Tape-13 2956, 112 inch from Ace Scientific. 24. Ultrasonic bath-Sonogen Automatic Cleaner-Branson Model 520 or equivalent. 25. Vortex mixer. 340 G.V. Downing E. Chromatographic Apparatus

A Beckman-Altex Model 110A liquid chromatographic pump complete with Valco sample valve with syringe-loading sample loop or Waters Wisp Autosampler and Kratos-Schoeffel Instruments Model FS950 fluo• rescence detector with a I-millivolt recorder is used. A 51Lm, 4.6-mm 10, C18 standard Brownlee Labs guard column (Spheri-5 RP-18 OD-G, obtained from Rainin Instrument Co., Inc.) is used before the analytical column. This guard column is replaced monthly unless the pressure reaches 2000 psi, in which case it is replaced immediately. Conditions: 15 cm lengthx4.6 mm 10 Zorbax ODS-CI8 column. Mobile phase-IO% water in methanol (v/v). Column temperature-43° C. Flow-1.8 mIl minute (usual pressure 1000 psi, 500 to 2000 psi acceptable). For FS950: Excitation Lamp-FSA 110, standard 365 nm. Standard Kratos Flowcell-FSA 210. Excitation Filter-FSA 403, 365 nm band filters. Emission Filter-418 nm. Sensitivity Range-0.2 A or higher. Retention Time-14 minutes (or greater with Modifications for I, the parent compound) Time Constant-about 6. F. Standards for the Confirmatory Assay

The objective is to have standards for the parent (I), aglycone (A), and monosaccharide (M) at approximately the concentration observed in the determinative assay or at the RM. To achieve this, dilute an aliquot of standard ivermectin solution in methanol with methanol. The final concentration of the solution should be 10 times the level to be detected (±10%). This results from the following equation: C=5xL where: C= the weight of ivermectin in 2 ml of the diluted standard. 5= number of grams of liver sample used. L= level of analyst wishes to determine in ppb or ng/g.

Because the unknowns are dissolved in 2 ml before aliquoting into I, M, and A, the standard is figured on the basis of 2 ml. Two 0.5 ml aliquots of the diluted standard solution should be run through each part of the assay (M, A, and I). The M and A standards should start in the procedure at the Step 4 evaporation. The I standard should start at Step 15. Appendix II. Assaying Ivermectin Residues in Liver 341 G. Procedure for the Confirmatory Method

1. Follow the method described in the determinative assay procedure up through Step 28. 2. Take the residue up in exactly 2 ml of methanol, using vortex mixer and ultrasonic bath. 3. Mix thoroughly and centrifuge for 5 minutes. 4. Pipet exactly 0.5 ml of the solution from Step 3 into each of 2 fresh 15-ml tubes and blow completely to dryness with nitrogen in a 70° C bath. The dried samples generally look like a small drop of oil. All solvent must be completely removed. Store the remaining 1 ml from Step 3 in a freezer until later. 5. Add 0.1 ml of 1% sulfuric acid in methanol to 1 ofthe 2 samples (the A sample) and 0.1 ml of 1% sulfuric acid in isopropanol to the second (the M sample). 6. Vortex mix the sample for 10 seconds, thoroughly ultrasound it, and repeat the vortex mixing. 7. Let stand at room temperature for 16-18 hours (overnight in the dark). 8. To all (A) samples and (A) standards at one time, add 0.9 ml of methanol, using a 5-ml graduated pipet. 9. Add the 40% methylene chloride-hexane-isobutyl alcohol solvent mixture to the 7-ml mark and mix. 10. Add 4 ml of distilled water, using a lO-ml graduated pipet, shake for 1 minute, and centrifuge for 5 minutes. 11. Move the mixed solvent upper phase by disposable pipet to a fresh silylated 15-ml tube. Move as much upper phase as possible but absolutely no lower phase. 12. Repeate the extraction of the lower phase with a second 6 ml of the mixed solvent and combine the extracts in the silylated tube. 13. Do the same extractions with the (M) samples, except that the shaking must be only moderate and the centrifuging should be for 10 or more minutes (otherwise emulsions may result). 14. Into a third 15-ml silylated tube, pipet another 0.5 ml of the sample from the freezer stored in Step 4 (the I sample). 15. Blow the (M) and (A) extracts and the (I) sample completely to dryness. Again, all solvent must be removed and no moisture picked up. A bath temperature of 40° C is suitable for the (M) and (A) samples and temperatures up to 70° C for the (I) sample. 16. Add 0.1 ml of derivatizing reagent to all (M), (A), and (I) dry tubes and all standards, vortex mix 10 seconds, tape the stoppers in place, centrifuge briefly, and place in a 95-100° C oil bath for 1 hour, all at the same time. 17. Cool to room temperature, add 0.9 ml of methanol using a 5-ml graduated pipet, and mix.

Appendix II. Assaying Ivermectin Residues in Liver 343

18. Add 1% isobutyl alcohol in hexane to the 7-ml mark and mix. 19. Add 4 ml of water from a graduated pipet, shake 1 minute, and centrifuge for 5 minutes. 20. Move the upper phase as completely as possible to a fresh 15-ml tube. Move no lower phase. 21. Repeate the extraction with a second 6 ml and combine extracts. 22. Blow completely to dryness with a nitrogen flush and a 40° C bath. At the end of this evaporation, the temperature can be allowed to rise to 60-70° C. 23. Take up residue in exactly 0.5 ml of methanol, using vortex mixing and ultrasound. In all above (17-23), the (M) samples should be handled first and placed on the LC before handling the (A) or (I) samples. After extraction, protect the (M) samples from light as much as possible. (If the (A) and (I) samples are to be delayed signifcantly, they should be stored in freezer at - 20° C before adding the methanol.) 24. Centrifuge and inject 50 ILl of the clear phase (M samples) onto the HPLC with the modified mobile phase doing the first standard of a type, the unknowns, and finally the second standard, in that order. 25. Do the same with the (A) and (I) samples. 26. Examine the unknown sample chromatograms for the presence of the (A), (M), and (I) peaks at the same elution time as the standard peak. (That is, compare the unknown (A) to standard (A), etc.) 27. Average the 2 peak heights for each standard (A, M, and I) and calculate each unknown as a precent of that standard average. 28. A value of 60% or more of all 3 (A, M, and I) is proof that that particular level of ivermectin is present in the tissue (liver). ApPENDIX III List of Registrations

J. Di Netta

Ivermectin was released for registration by Merck & Co., Inc., in 1981 and first registered in France as IVOMEC Injection for Cattle that same year. Since that time ivermectin has been approved for use in over 60 countries. It is currently registered for use in cattle, sheep, horses, goats, swine, dogs, camels, reindeer, and bison. The following lists the products and the countries in which ivermectin is registered.

IVERMECTIN APPROVALS IVOMEC' byection for Cattle ALGERIA GUATEMALA PHILIPPINES ARGENTINA HOLLAND POLAND AUSTRALIN HONDURAS PORTUGAL AUSTRIA HUNGARY ROMANIA BELGIUM INDIA S. AFRICA BRAZIL IRELAND SPAIN BULGARIA ISRAEL SUDAN CANADA ITALY SWEDEN CHILE JAMAICA SWITZERLAND COLOMBIA JAPAN THAILAND COSTA RICA JORDAN TRINIDAD C.S.S.R KENYA TUNISIA DENMARK LUXEMBOURG TURKEY ECUADOR MALAYSIA U.K. EGYPT MEXICO URUGUAY ELSALVADOR MOROCCO U.S.A. FINLAND NEW ZEALAND U.S.S.R. FRANCE NORWAY VENEZUELA G.D.R. PANAMA YUGOSLAVIA GERMANY PARAGUAY ZIMBABWE GREECE PERU IVOMEC' Oral Solution for Cattle FRANCE NEW ZEALAND U.K. HOLLAND SOUTHAFRICA Appendix III. List of Registrations 345

IVOMEC' Paste for Cattle CANADA U.S.A. IVOMEC' Pour-On for Cattle ARGENTINA HOLLAND NEW ZEALAND BRAZIL MEXICO U.K. CANADA IVOMEC-F' Injection for Cattle BRAZIL IRELAND SPAIN FRANCE MEXICO U.K. HOLLAND PORTUGAL IVOMEC' or ORAMEC' Liquid for Goats BRAZIL NEW ZEALAND U.K. HOLLAND SOUTH AFRICA IVOMEC' or ORAMEC' Liquid for Sheep ARGENTINA HOLLAND MOROCCO AUSTRALIA INDIAN NEW ZEALAND BELGIUM IRELAND SOUTH AFRICA BRAZIL JORDAN SUDAN CANADA KENYA U.K. FRANCE LUXEMBOURG U.S.A. URUGUAY ZIMBABWE IVOMEC' Injection for Sheep ALGERIA DENMARK POLAND ARGENTINA FRANCE PORTUGAL BELGIUM HOLLAND ROMANIA BRAZIL JORDAN SOUTH AFRICA BULGARIA LUXEMBOURG SPAIN C.S.S.R. MEXICO SUDAN CANADA MOROCCO TUNISIA CHINA PARAGUAY URUGUAY YUGOSLAVIA IVOMEC' Injection for Swine AGRENTINA GERMANY PORTUGAL AUSTRIA HOLLAND ROMANIA BELGIUM HONG KONG SINGAPORE BRAZIL IRELAND SOUTH AFRICA BULGARIA ITALY SPAIN CANADA JAPAN SWEDEN CHILE KOREA SWITZERLAND CHINA LUXEMBOURG TAIWAN COLOMBIA MALAYSIA THAILAND C.S.S.R. MEXICO U.K. DENMARK NEW ZEALAND U.S.A. ECUADOR NORWAY URUGUAY FINLAND PARAGUAY VENEZUELA FRANCE PHILIPPINES YUGOSLAVIA G.D.R. POLAND ZIMBABWE IVOMEC' Injection for Young Pigs HOLLAND U.S.A. 346 J. Di Netta

HEARTGARD-30' or CARDOMEC' or CARDOTEK-30' Tablets for Dogs AUSTRALIA HOLLAND SPAIN CANADA JAPAN U.S.A. FRANCE MEXICO EQV ALAN' Paste for Horses ALGERIA HOLLAND PARAGUAY ARGENTINA HONDURAS PERU BELGIUM HONG KONG PHILIPPINES BRAZIL IRELAND POLAND CANADA ITALY PORTUGAL CHILE JAMAICA SINGAPORE COLOMBIA JAPAN SOUTH AFRICA COSTA RICA JORDAN SPAIN DENMARK KENYA SUDAN ECUADOR KOREA SWEDEN EGYPT LEBANON SWITZERLAND ELSALVADOR LUXEMBOURG THAILAND FINLAND MEXICO TRINIDAD FRANCE MOROCCO U.K. G.D.R. NEW ZEALAND U.S.A. GERMANY NORWAY U.S.S.R. GUATEMALA PANAMA URUGUAY VENEZUELA EQV ALAN' Liquid for Horses CANADA MEXICO U.S.A HOLLAND IVOMEC' Injection for Camels ALGERIA MOROCCO SUDAN JORDAN

1 AVOMEC 'Trademark of MERCK & CO., Inc., Rahway, New Jersey, U.S.A. Index

A AGRI-MEK, 288 Abamectin Airblast treatment, 202, 203 antibacterial activity, 29 (Table 2.3) See also Worker exposure. antifungal activity, 30 (Table 2.4) Alaska, 271 biological activity, 20 (Table 1.8) Algae, toxicity, 168 clinical trials, cattle, 216-222, Algeria, 268, 344, 346 230-233 Alkylating agents, use in mutational compared to ivermectin, 102-103 procedures, 35-36 toxicity, 103 (Table 6.5), 110 Alkylations, 15 crop protection, 288-305 Almonds, 305 environmental effects, 184-198 Alpacas, clinical trials, 271 photoinstability, 292 AI Salvador, 344 structure of, 90 (Fig. 6.1), 184 (Fig. Amblyomma americanum, 265-266 13.1) Amidine,219 toxicity studies, 102-107 Amitraz, 254 worker exposure study, 201-213 Analytical techniques, tissue residues, See also Specific topics. 137-138, 144-145 Acarines, 288 Ancylostoma caninum, 251-252 Acetylcholine release, ivermectin, 79 Ancylostoma spp., 255, 269, 320 Acid-sensitivity, , 8 Angiostrongylus cantonensis, 265 Aculops lycopersici, 302 Anguillicola crassus, 279 Acute toxicity Anorexia, 96, 108 abamectin, 193-194 (Table 13.4) Antelope, clinical trials, 273-274 ivermectin, 92-95, 169 Anthelmintic market, 24 Aelurostrongylus abstrusus, 255 Anthelmycin, 24 AFFIRM, 289-290 Anthelvencin, 24 nonagricultural land, 289 Antifungal/antibacterial activity Africa, 275 abamectin, 29-30 (Tables 2.3, 2.4) Agitators, and fermentation, 47-48, Streptomyces auermitilis, 28-31 49-50 (Figs. 3.7,3.8,3.9,3.10) Aphids, 298, 300 Aglycones Aphodius, 178 , 10-12, II (Fig. 1.5), 16 Aquatic life. See Environmental ivermectin, 12 effects of abamectin 348 Index

Argentina, 344, 345, 346 I H N MR data, 5 (Table I. I) Armyworm, 299, 302 isolation of, 26 Ascaris Ilimbricoide.l', 74 macrocyclic lactones, I, 10 Ascaris suum, 76 mass fragmentation, 4 (Fig. 1.2) Asia, 275 mode of action Aspiculomycin, 24 acetylcholine release, 79 A.I'piculliris tetraptera, 262 A VM binding sites, 76-78, 80-81 Aspis cracciuora, 300 A VM effect on Aspis gossypii, 300 binding sites, 82-84 Assay method A VM effect on [3Hl GABA apparatus, 326-327, 338-339 binding, 81-82 chromatographic apparatus, A VM stimulated neurotransmitter 327-328, 340 release, 80 determinative assay of tissues, chitin synthesis inhibition, 79-80 329-332,341-343 chloride uptake, 78-79, 84-85 determinative assay standards, 328, difficulties related to, 73-74 340 electrophysiological studies, fat, 329, 330 74-76 liver/muscle/kidney tissues, 329, GABA binding sites, 77, 81 330 glycine binding sites, 85 plasma, 333-335 in invertebrates, 74-80 reagents, 326, 336, 338 protein kinase C activity, 85 solutions, 326, 328 retinol binding proteins, stopping places, 332-333 interaction, 79 Ataxia, 93, 94, 96, 108, 150, 151, 152, in vertebrates, 80-85 153, 155, 157, 158, 159 most important, Bla, 3 Australia, 175, 177,345,346 photoisomerization, 9 (Fig. 1.4) Austria, 344, 345 structure of, 55, 56 (Fig. 4.1) Auxotropic strains, 36 naturally occurring avermectins, A vermectins 1-3 antifungal activity, 31 A vermectin aglycone chemical properties, 7-8 glycosyltransferase, studies with, acid-sensitivity, 8 66,70 stability studies, 7 A vermectin B I a. See Abamectin chemistry Avermectin B O-methyltransferase, alkylations, 15 studies with, 66, 67-68 (Figs. 4.5, glycoside syntheses, 15 4.9) hydroxy group characteristics, Avermectin 5-ketoreductase, studies 9-10, 15 with, 66, 69-70 (Figs. 4.6, 4.7) interconversions, 15 AVID, 288, 294, 295 monosaccharides and aglycones, A VOMEC, cattle, 230-233 10-\2 Axenomycins, 24 oxidations, 12-13 radiolabeled derivatives, 13, 15 reductions including ivermectin, B 13 Bancroftian filariasis, 320 l3C NMR data, 6-7 (Table 1.2) Beans, 298 discovery of, 24 Bees, toxicity, 195 dosage, 10 Beet armyworm, 302 Index 349

Beetles, dung, effect of ivermectin, [methyl-14C] methionine, 176-178 60 (Table 4.4) Belgium, 344, 345, 346 results of studies, 62 Benomyl,203 valine, isoleucine, isobutyrate, Benzimidazoles, 222 57, 60 (Table 4.2) Benzodiazepine, binding sites, proposed pathway, 71 (Scheme I), ivermectin, 82-84 72 Benzyl alcohol, 118, 122 specific inhibitors. sinefungin, 70, , 78, 80, 81 72 Biexponential decay Birds, clinical trials cattle, 114, 115 (Fig. 7.1), 116 Ascaridia. 276-277 (Fig. 7.2) Capillaria spp .• 277 sheep, 118-119 (Fig. 7.5) mites, 277-278 Bighorn sheep, clinical trials, 273 Oxyspirura sp .• 277 Bioavailability analyses Pelecitus sp .• 277 cattle, 114-115 (Fig. 7.1),117 toxicity, 195-196 (Fig. 7.3), 118 (Fig. 7.4) Bison, clinical trials, 274 dogs, 122-123 (Fig. 7.6) Blackeye peas, 301 horses, 125-126 (Fig. 7.8) Blood, 135, 136 (Table 8.3) humans, 127-128 Bollworm, 300 sheep, 121 Boophilus decoloratus. 219 swine, 124-125 (Fig. 7.7) Boophilus microplus. 219,220,221, Biosynthesis 231,232 bioconversion of intermediates, Bordetella hronchiseptica. 28 62-65 Bots, 223 desfurano avermectins, feeding Brain, 135 of, 63, 65 (Table 4.8) Brazil, 344, 345, 346 radioactivity distribution after Brine shrimp, 80 feeding, 64 (Table 4.8) Broad mite, 291 results of studies, 65 Brugia malayi. 276 studies, 66-70 Brugia pahangi. 264, 267, 276 avermectin aglycone Budgies, 278 glycosyltransferase, 66, 70 Buffalo, clinical trials, 274 avermectin B Buffalo flies, 174 O-methyltransferase, 66, Bulgaria, 344, 345 67-68 (Figs. 4.5, 4.9) Bush fly, 174, 175 avermectin 5-ketoreductase, 66, 69-70 (Figs. 4.6,4.7) C incorporation of labeled precursors, C-076,25 56-62 Caenorhahditis elegans. 74, 76-77 [1_ 18°2, I-Bc] acetate and California, 213 propionate, 57, 61 (Table Caligus elongatus. 279 4.6),62 Camels, clinical trials [14C] methionine, 60 (Table 4.3) ectoparasites, 270-271 [ 13C] precursors, 57, 58-59 (Table endoparasites, 270 4.1) Cameroon, 315 labeled glucose, 62 (Table 4.7) Canada, 344, 345, 346 [methyl-13C] methionine, Canaries, 278 61 (Table 4.5) Cape hunting dog, clinical trials, 269 350 Index

Capillaria spp., 253, 256 Cattle feedlot environmental fate Captan, 203 study, 171 , 202 Celery, abamectin, 303 Carbaryl, 203 Cell mass on-line, prediction of, Carnation foliage, 288, 295, 296, 297 45-47 Catabolite repressible, avermectin Cephalopina titillator. 271 production, 43 Cerelose, in seed media development, Cats, clinical trials 38 Aelurostrongylus abstrusus. 255 Chabertia ovina. 231 Ancylostoma spp., 255 Chelonians, 279 Capillaria spp., 256 Cheyletiella blakei. 269 Ctenocephalides cati. 256 Chickens, 277 Nodoedres cati. 256 Chile, 344, 345, 346 Otodectes cynotis. 256 Chimpanzees. clinical studies, 318 Toxocara cati. 255 China, 345 Cattle Chitin synthesis inhibition, clinical trials, 216-222, 230-233 ivermectin, 79-80 dosage/administration, 230 Chiarella pyrenoidosa. 168 ectoparasite activity, 232-233 Chloride endoparasite activity, 216, 219, ions, 74, 76, 81, 82. 84, 86 220-221,231-232 uptake, ivermectin, 78-79, 84-85 grubs, 217, 219-220, 221 Chlorobenzilate, 203 lice, 218, 220. 221 Chorioptes bovis. 218, 221 mites, 218, 219 Chromatographic methods, 144-145 oral formulation, 219 Chrysanthemum foliage, 203, screwworms, 219 208-209 (Table 14.4), 210-213, sustained-release bolus, 221-222 294-295, 296, 301 ticks, 219 Chrysomya bezziana. 219 topical formulation, 220 Citrus crops, abamectin, 290-294 fat metabolism studies, 139-141 and beneficial organisms, 293-294 liver metabolism studies, 135-139 broad mite, 291 pharmacokinetics of ivermectin, citrus red mite, 291-292 114-119 citrus rust mite, 290-291 biexponential decay, 114, 115 citrus thrips, 293 (Fig. 7.1),116 (Fig. 7.2) decomposition of abamectin, 293 bioavailability analyses, 114-115 Cleft palate, 99, 100, 103, 109 (Fig. 7.1), 117 Clubbed forepaws, 100, 109 (Fig. 7.3); 118 (Fig. 7.4) Cnemidocoptes pilae. 278 dosage formulations and results, Cochliomyia hominivorax. 219 117-119 Cockroach, 76, 78-79 high-pressure liquid Coleoptera, 178, 179 chromatographic (HPLC) Collies, 246 method, 114 Colombia, 344, 345, 346 metabolic transformation, 114 Coma, 99 plasma concentration, 116 Convulsions, 91, 99, 105 (Fig. 7.2), 117, 118, Cooperia oncophora. 216 119 (Table 7.1) Cooperia punctata. 216 safety of ivermectin, 151-153 Copepods, 278, 279 tissue residues, 132-134 Cornea, microfilariae, 313 Index 351

Corn earworm, 299 Dictyocaulus viviparus. 216,230,231, Costa Rica, 344, 346 232,272 Cote d'ivore, 313 Diethylcarbamazine (DEC), 159 Cotton, abamectin, 297-300 compared to ivermectin, 311-316, aphids, 298, 300 319 lepidoptera, 299-300 Diglycerides, 52 spider mites, 297-299 22,23, dihydroavermectin Bl, 16,20 Coumaphos, 178 22,23, dihydroavermectin Bla, 3 Crenosoma striatum, 268 , 79 Crop protection Dipetalonema reconditum. 250 citrus crops, 290-294 Dipetalonema viteae. 264 cotton, 297-301 Diptera, 175, 178, 179 nut trees, 305 Dirofilaria immitis. 169, 246-250, 264, ornamental plants, 294-297 267 pears, 303-304 See also Heartworm disease, dogs. products used, 288-289 Dislodgeable foliar residues, worker red imported fire ant, 289-290 exposure, 202, 209-213 strawberries, 305 DNA synthesis, unscheduled, 92 vegetables, 301-303 Dogs Cross-feeding, mutational procedures, adverse reactions, ivermectin, 246 36-37 clinical trials C.S.S.R., 344, 345 Ancylostoma caninum. 251-252 Ctenocephalides cati. 254-255, 256 Capillaria spp., 253 Cuterebra fontinella. mice, 265 Ctenocephalides cati. 254-255 Cyclophosphorothionate, 83-84 Demodex canis. 254 Cytodites nudus. 278 Dipetalonema reconditum. 250 Dirofilaria immitis. 246-250 D dosage/formulation, 246 Dahlia foliage, 296 Filaroides osleri. 253 Daisy foliage, 295 Otodectes cynotis. 254 Damalinia bovis. 218, 221 Sarcoptes scabiei. 253-254 Daphnia magna. 169, 170 (Table Strongyloides stercoralis. 253 11.6),171 Toxascaris leonina. 251 DDT, 219 Toxocara canis. 250-251 Deer, clinical trials, 271-272 Trichuris vulpis. 253 ectoparasites, 272-273 Uncinaria stenocephala. 252-253 endoparasites, 272 pharmacokinetics of ivermectin, Demodex canis. 254 122-124 Denmark, 344, 345, 346 bioavailability analyses, Dermal penetration study, abamectin, 122-123 (Fig. 7.6) 107, 108 (Table 6.8) plasma concentration, 120 Dermanyssus gallinae. 277 (Fig. 7.5), 122 Dermatobia hominis. 217, 221 tablet fQrmulation, 122-124 Destomycins, 24 safety of ivermectin, 157-159 Developmental toxicity, 98-102, 109, toxicity, 157-158 104 (Table 6.6) abamectin, 104 Dichlorvos, 178 ivermectin, 93-94, 97, 108 Dicofol, 297 See also Heartworm disease. Dictyocaulus arnfieldi. 239 Donkeys, 239, 241 352 Index

Dopamine, 80 fate of avermectin in terrestrial Dosage formulations and results environment, 190-191 minimum toxic dose (MTD), 94, 108 mammals, 196 no-effect level (NEL), 110 microorganisms in soil, 195 See also Specific species. plant sensitivity, 194 Downstream extraction, fermentation plant uptake, 186-187 development, 45, 52-53 risk assessment, 198-199 Draschia spp., 240 thin film degradation, 185 water photolysis studies, 184-185, 197 E Environmental effects of ivermectin Earthworms, 168, 174 cattle feedlot environmental fate Ecinothrips americanus, 2% study, 171 Ecuador, 344, 345, 346 dung fauna, 173-180 Edema, 150 cattle dung breeding diptera, Edesonfilaria malayensis, 276 174-176 Edible tissues, 147 native insects, 178 See also Tissue residues, edible Onthophagus binodis, 177-178 tissues. Onthophagus gazella, Eels, 279 176-177 (Table 12.2) Egypt, 344, 346 role in ecosystem, 173-174 Eisenia foetida, 168 treated/untreated aged manure, Elaphostrongylus cervi, 272 178-179 Electrophysiological studies, environmental burden, ivermectin, 74-76 163-165 (Table 11.2) Elephants, clinical trials, 275 environmental fate, ivermectin, El Salvador, 346 166 (Table 11.3) Emesis, 91, 94, 108 freshwater organisms, acute Environmental considerations, toxicity, 169 (Table 11.5) fermentation development, 51 safety assessment, 169-170 Environmental effects of abamectin soil aquatic life photodegradation of ivermectin, acute toxicity, 192-193 (Tables 167 13.1, 13.2) soil binding, 166-167, 169 chronic toxicity, 193-194 (Table toxicity to earthworms, 168 13.4) toxicity to soil microorganisms, fate of avermectin in aquatic 167 environment, 188-189 Enzyme studies, 66-70 fish, 187-188, 193 avermectin aglycone invertebrates, 192-193 glycosyltransferase, 66, 70 risk assessment, 197-198 avermectin B O-methyltransferase, hydrolysis, 185 66, 67-68 (Figs. 4.5, 4.9) soil binding, 186 avermectin 5-ketoreductase, 66, soil metabolism, 185 69-70 (Figs. 4.6, 4.7) soil photolysis studies, 185 Epitrimerus pyri, 304 solubility, 186 Equus asinus, 241 terrestrial life EQVALAN,158 bees, 195 injectable, 235 birds, 195-196 liquid, 234 Index 353

paste, 234 survival curves, 34-35 See also Horses, clinical trials. suspensions used, 34, 35-36 Etazolate, 81 ultraviolet (UV) light, 35 Ethylmethane sulfonate (EMS), in oxygen transfer, 47-48, 49-50 mutational procedures, 35-36 (Figs. 3.7, 3.8, 3.9, 3.10), 51-52 Euseius tularensis. 293 (Fig. 3-11) Excretion production media development, ivermectin, animals. 134, 163 40-41 (Table 3.4), 42-44 residues of ivermectin, 164 (Table classical approach, 40-41 11.1) medium constituent substitutions, See also Environmental effects of 41 ivermectin. response surface methodology, Exotic mammals. See specific species 41-43 Eye shake flash fermentation, 43 corneal microfilariae, 313 (Fig. 3-1) ocular reaction index, 317 (Fig. scale-up strategy, 48, 51-52 21.3) (Fig. 3.11) seed media development, F 37-38 (Table 3.3), 39-40 Face fly, 174, 175 change in high producers, 39 Fall armyworm, 302 improved avermectin production, Fat, 135 38 (Table 3.3), 38-39 fat pad, 136 (Table 8.3) seed medium IV, use of, 39-40 metabolism of ivermectin, in vivo stirred tanks, 44-45 systems, 139-141 viscosity profiles, 48 (Fig. 3.6) Feces Ferns, 288 excretion of ivermectin, 134, 163, Ferrets, clinical trials, 267 164 (Table 11.1) Filarioidea. 79 See also Environmental effects of Filaroides osleri. 253 ivermectin. Finland, 344, 345, 346 Fenbendazole, 253 Fish, clinical trials, 278-279 Fenvalerate, 300 copepods, 279 Fermentation development nematodes, 279 agitators and, 47-48, 49-50 toxicity, 169 (Table 11.5), 170, (Figs. 3.7, 3.8, 3.9, 3.10) 187-188, 193-194 Bagner/Wildman process, 52-53 Fleas, 254-255, 256 cell mass on-line, prediction of, Flour beetle, 288 45-47 Flukes, 256 components of harvested broth, 52 , 83 downstream extraction, 45, 52-53 F1uorescence-derivatization method, environmental considerations, 51 114,121,124,137 media sterilization, 51 Fly dung, effect of ivermectin, mutational procedures, 34-37 174-176 alkylating agents, 35-36 Fly strike, 272 auxotropic strains, 36 Foxes, clinical trials cross-feeding, 36-37 ectoparasites, 269 detection of mutants, 36 endoparasites, 268-269 most avermectin produced, 37 France, 254, 344, 345, 346 mutants, 35 (Table 3.1), 36 Frankiniella occidentalis. 296 354 Index

Free residues, animal tissues, 132 Haematopinus suis. 226 Friedman's test, 237 Haematopinus tuberculatus. 274 Haemonchus contortus. 74, 222 G avermectin activity, 16 (Table 1.3), GABA 19 (Table 1.6) , 84, 89, 91 Haemonchus placei. 216,231,232 binding sites, 77, 81 Hamsters, clinical trials, 266 functions of, 91 Heart, 135, 136 (Table 8.3) Gastrophilus intestinalis. 241 HEARTGARD-30, 122, 256 Gastrophilus nasalis. 241 Heartworm disease, dogs, 122, 159, G.D.R., 344, 345, 346 246 Genotoxicity studies, 91-92 ivermectin efficacy Gerbils adult worms, 248 avermectin activity, 16, dosage, 249 (Table 18.2) 17-18 (Tables 1.4, 1.5) immature stages, 246-248 clinical trials, 266 microfilariae, 248-250 Germany, 344, 345, 346 prophylactic efficacy, 247 (Table Ghana, 312, 313 18.1) Glossina palpalis. 266, 267 See also Dogs. Glutamate, 80 Hedgehogs, clinical trials, 268 Glycene binding sites, ivermectin, 85 Heliothis virescens. 299, 300 Glycerol formal, 114, 117, 118, 119, Heliothis zea, 299, 300 122, 124 Hemiptera triatominae, 264 Glycine, 85 Hens, 277 Glycoside syntheses, avermectins, 15 Hexachlorocyclohexane, 79 Goats, clinical trials, 224-225, 273 Hickory shuckworm, 305 injectable formulation, 225 High performance liquid oral formulation, 224-225 chromatography (HPLC), 7, 10, safety of ivermectin, 155 26, 114 toxicity, 155 isolation of avermectins, 26 Goldfish, 279 Holland, 213, 344, 345, 346 Greece, 344 Homeopronematus anconai. 302 Grubs, ivermectin efficacy, cattle, Honduras, 344, 346 219-220, 221 Honey bees, 77 Guatemala, 313, 315, 344, 346 Honeydew, 304 Guinea fowl, 276-277 Hong Kong, 345, 346 Guinea pigs, clinical trials Hookworms mange mites, 266 cats, 255 ticks, 265-266 dogs, 251-253 tsetse flies, 266 human, 320-321 Gymnodia, 178 Horn fly, 174, 175, 179 Gypsophilia foliage, 295 Horses clinical trials H Dictyocaulus arnfieldi. 239 Habronema spp., 240 dosage/formulation, 234-235 Haematobia irritans. 174,221 Draschia spp., 240 Haematomyzus elephantis. 275 Gastrophilus intestinalis. 241 Haematopinus eurysternus. 218, 220, Gastrophilus nasalis, 241 221 Habronema spp., 240 Index 355

Onchocerca cervicalis, 240 Invertebrates Parascaris equorum, 235-237 aquatic toxicity, 192-193 Sarcoptes scahiei, 241 ivermectin mode of action, 74-80 Strongylus edentatus, 238-239 Ireland, 344, 345, 346 Strongylus equinus, 239 Isooctane extract, preparation of, Strongylus vulgaris, 237-238 145 pharmacokinetics of ivermectin, Israel, 344 124-125 Italy, 254, 344, 345, 346 bioavailability analyses, Ivermectin, 3 125-126 (Fig. 7.8) clinical trials dosage formulations and results, birds, 276-278 125-126 carnivores, 268-269 safety of ivermectin, 149-151 cats, 255-256 toxicity, 151-152, 153 cattle, 216-222 Humans dogs, 246-255 bancroft ian filariasis, 319, 320 elephants, 275 Loa loa, 320 ferrets, 267 nematodes, 320-321 fish, 278-279 onchocerciasis, 311-316, 320 gerbils, 266 pharmacokinetics of ivermectin, goats, 224-225 126-129 guinea pigs, 265-266 bioavailability analyses, 127-128 hamsters, 266 dosage formulations and results, hedgehogs, 268 129 horses, 234-241 plasma concentration, mice, 262-265 127-128 (Figs. 7.10, 7.11) primates (nonhuman), 275-276 See also Onchocerciasis. rabbits, 266-267 Hungary, 344 rats, 265 Hydrofoil impeller, 47 reptiles, 279-280 Hydrolysis, abamectin, 185 rodents, 268 Hydroxy group characteristics, 9-10, sheep, 222-224 15 swine, 225-227 Hygromycin B, 24 ungulates, 269-275 Hypoderma hovis, 217,219-220,221, l3C NMR data, 6-7 (Table 1.2) 274 derivatives, chemical structures, Hypoderma lineatum, 217, 220, 221 137 (Fig. 8.2) Hypoderma spp., 152 enyironmental effects, 163-179 Hypotension, 319 I H NMR data, 5 (Table 1.1) human exposure, acceptable levels, 110 pharmacokinetic properties, Identification/confirmation method, 113-129 147 reductions, 13 Impala, clinical trials, 273 registrations, listing of, 344-346 India, 319, 344 safety in, animal studies, 149-159 Inhibitors synthesis, by selective of chitin synthesis, 79-80 hydrogenation of avermectin sinefungin, 70, 72 B), 14 (Fig. I) Interconversions, avermectins, 15 toxicity studies, 91-102 356 Index

Ivermectin (cont.) Litomosoides carinii. 264 tritiated derivative, 13, 15 Liver, 135, 136 (Table 8.3), 237, 238, See also Individual topics. 263 IVOMEC, 159 metabolism of ivermectin cattle, 216-222 in vitro systems, 136 goats, 224-225 in vivo systems, 137-139 sheep, 222-224 Llamas, clinical trials, 271 swine, 225-227 Loa loa, 320 Ivory coast, 318 Lobster, 74-75 Locust, 75 J Lucilia cuprina. 288 Jamaica, 344, 346 Lung, 135, 136 (Table 8.3), 237, 238, Japan, 344, 345, 346 263 Jirds, clinical trials, 267 Lungworm, 219, 220, 222, 223, 225, Jordan, 268, 344, 345, 346 272,273 bighorn sheep, 273 K deer, 272 Keiferia lycopersicella. 302-303 Luxembourg, 344, 345, 346 Kenya, 344, 346 Lymphoma assay, 91 Kidney, 135, 136 (Table 8.3) Kidney worms, 225, 226 M Kitasato Institute, 24 MA-4680, electron micrographs of, Konigs-Knorr procedure, 15 25-26 (Figs. 2.1, 2.2) Korea, 345, 346 MA-5856, 28-30 Macaca mulatta, 93 L Macaw, 277, 278 Lactation, 101-102 Macrocyclic lactones, I, 10 concentration of ivermectin, Malaysia, 344, 345 102 (Fig. 6.3) Mali, 312, 313 toxicity studies, 97, 101 Mange mites, 269, 269-270 Leafminer, 295, 301-302, 303 guinea pigs, 266 Lebanon, 346 rabbits, 266-267 Lepeopthirus salmonis, 279 Mansonella perstans, 320 Lepidoptera, 299-300 Manure, aged, effect of ivermectin, Lepomis macrochirus, 169 178-179 Lernea spp., 279 Mass spectrometry/mass Levamisole, 177, 178,222 spectrometry (MS/MS), 10, 145, Liberia, 302, 313, 315, 318 147 Lice Maternotoxicity, 99-100, 104 (Table cattle, 218, 220, 221 6.6), 109, 206, 207 (Table 14.3), wolves, 269 210, 211, 213 (Table 14.6) Limbitis, 313 Mazzotti reaction, 313, 314 Linear regression analysis, 201 Media sterilization, fermentation Linognathus a/ricanus, 272 development, 51 Linognathus vituti, 218,220,221,230, Melanin, 36 231 Meningitis, 318 Lipophilicity, 114 Metabolism of ivermectin Liriomyza sativae, 301 fat tissue, in vivo systems, 139-141 Liriomyza trifolii. 295-297, 301 liver Index 357

in vitro systems, 136 Morantel, 222 in vivo systems, 137-139 Morocco, 268, 344, 345, 346 tissue residues, 132-136 Mosquitoes, 246 Metaseiulus occidentalis, 305 Mouse assay, 26 Methylation, avermectins, 15 Musca autumnalis, 174 Mexico, 344, 345, 346 , 77, 79 Mice, clinical trials Muscle, 135, 136 (Table 8.3) Aspiculuris tetraptera, 262 Mutational procedures, 34-37 Cuterebra fontinella, 265 alkylating agents, use of, 35-36 Hematospiroides dubius, 262 auxotropic strains, 36 microfilariae, 263-264 cross-feeding, 36-37 mites, 264 most avermectin produced, 37 Strongyloides ratti, 262 mutants Strongyloides stercora lis , 263 morphological variants, 36 Syphacia obvelata, 263 types of, 35 (Table 3.1) Toxocara canis, 263 survival curves, 34-35 triatomid bugs, 264 suspensions used, 34, 35-36 Trichinella spp., 263 ultraviolet (UV) light, use of, 35 Trichuris muris, 263 Myiasis, 271 Micellar formulation, 235 Mydriasis, 91, 94, 96, 97, 98, 104, Micrococcus luteus, 28 108, 149, 155, 158, 159 Microfilariae Myobia musculi, 264 heartworm disease, 248-250 Myxin,24 mice, 263-264 N Microgram-scale reverse isotope Necator, 320 dilution assay (RIDA) method, Necator americanus, 266 137-138 Nematodirus helvetianus, 216 Micronema deletrix, 241 Nematospiroides dubius, 24-25, 262 Microorganisms in soil, toxicity, 167, Netherlands, 213 195 Neurotransmitter release, ivermectin, Midge bites, 240 79,80 Milbemycins, 1,3, 12 New Zealand, 180, 344, 345, 346 Milk assay, 144 Nigeria, 315 Minimum toxic dose (MTD), 94, 108 Nitrification, soil, 167, 170 Mites Nitrosoguanidine (NTG), in abamectin activity, 292 (Table 20.1) mutational procedures, 35-36 birds, 277-278 Nitrosomethylurethane (NMU), in cattle, 218, 219 mutational procedures, 35 citrus crops, 290-292 Nodoedres cati, 256 foxes, 269 NOEL, for toxicological effects, mange mites, 266-267, 269-270 210-211 mice, 264 Norway, 344, 345, 346 primates (nonhuman), 275 Notoedres cati, 267 Monkeys, toxicity studies Notoedres douglassi, 268 abamectin, 107 Nuts, abamectin, 305 ivermectin, 94, 95, 97, 108-109 Monocrotophos, 297 o Monosaccharides, of avermectin, Odocoileus vorginianus, 271 10-12, II (Fig. 1.5), 16 Oedemagena tarandi, 271 358 Index

Oesophagostomum columbianum, fermentation development, 47-48, avermectin activity, 16 (Table 49-50 (Figs. 3.7, 3.8, 3.9, 3.10), 1.3), 19 (Table 1.6) 51-52 (Fig. 3-11) Oesophagostomum radiatum, 231, 232 Oestrus ovis, 222 P Oligomycin, 31, 80 Panama, 344, 346 Onchocerca cervicalis, 150, 240 Panonychus citri, 291-293 Onchocerca lienalis, 264 Panonychus ulmi, 304 Onchocerca volvulus. See Parafilaria bovicola, 175, 216 Onchocerciasis Paraguay, 344, 345, 346 Onchocerciasis, ivermectin, 311-316, Parascaris equorum, 235-237 320 Parelaphostrongylus tenuis, 271, 272 clinical reactions, 312-314, 318 Paris, 311 counterindications, 318-319 Paromomycin, 24 current use, 318-319 Partridges, 277 ocular disease, 314 Paste formulations, 125-126 Phases I through IV studies, Pears, abamectin, 303-304 311-316 mites, 304 prophylactic use, 318 psylla, 303-304 time span illustrations, 315-317 Pecan leaf scorch mite, 305 (Figs. 21.1, 21.2, 21.3) Pecan weevil, 305 transmission effects, 318 Pectinophora gossypiella, 300 Onthophagus binodis, 177-178 , 81 Onthophagus gazella, 176-177 (Table Peppers, abamectin, 301-302, 303 12.2) Periplanata americanus, 76, 77, 78 OP compounds. See Peru, 344, 346 Organophosphates Pesticide residues, measurement of, Ophinyssus sp., 280 202 Organophosphates, 202, 203, 219 Pharmacokinetic properties Ornamental plants, abamectin, cattle, 114-119 294-297 dogs, 122-124 leafminer, 295 horses, 125-126 thrips species, 296-297 humans, 126-129 two-spotted spider mite, 294-295 sheep, 119-122 Ornithodoros moubata, 267 swine, 124-125 Ornithodoros savignyi, 219 See also Specific species. Ornithonyssus sylvarium, 277 Phenothiazine, 178 OS-3153 strain, 25 Philippines, 344, 345, 346 Ostertagia, 216 Photodegradation of ivermectin, 167 Ostertagia circumcinta, 222 Photoisomerization, avermectins, avermectin activity, 16 (Table 1.3), 9 (Fig. 1.4) 19 (Table 1.6) Photolysis, abamectin, 184-185, 197 Ostertagia ostertagia, 230, 231, 232 Phototoxicity, 170 Ostrich, 278 Phyllocoptruta o/eivora, 290-291 Otodectes cynotis, 254, 256 Physostigmine, 159 Oxidations, avermectins, 12-13 , 74, 75, 78, 79, 80, 81, 83 Oxygen transfer Pigeons, 277 cell growth and oxygen uptake, Pink bollworm, 300 45 (Fig. 3.3) Pinworms, 302-303 Index 359

Plackett-Burman studies, 41 Radiolabeled derivatives, Plants, toxicity, 186-187, 194 avermectins, 13, 15 Plasma concentration, 136 (Table 8.3) Rats, clinical trials assay method, 333-335 Angiostrongylus cantonensis, 265 cattle, 116 (Fig. 7.2),117,118, Syphacia muris, 265 119 (Table 7.1) Trichinella spiralis, 265 dogs, 120 (Fig. 7.5), 122 Red imported fire ant, AFFIRM, humans, 127-128 (Figs. 7.10, 7.11) effectiveness, 289-290 ivermectin, 95 (Table 6.2) Red mite, 291-292, 304 sheep, 119-120 (Fig. 7.5) Reindeer, clinical trials, 271 swine, 124 Reproductive toxicity, ivermectin, Pneumonyssus, 275 98-102 Poland, 344, 345, 346 Reptiles, clinical trials, 279-280 Polynesia, 319 Response surface methodology, Polyoxyethylene sorbitan monooleate, production media development, 118, 122 41-43 Polyphagotarsonemus latus, 291 Retinol binding proteins, ivermectin, Polysorbate 80, 235 79 Polyvinylpyrrolidone, 114, 119 Rhabdochona sp., 279 Portugal, 344, 345, 346 Rhodnius prolixus, 264 Postural hypotension, 302 Rodents Pregnancy, 318 absorption/ distribution/excretion of Primates (nonhuman), clinical trials ivermectin, 134-135 mites, 275 clinical trials, 268 nematode infections, 275-276 fat metabolism studies, 139-141 Production media development, See liver metabolism studies, 135-139 Fermentation development tissue residues, 135, 136 (Table 8.3) Propargite, 297 toxicity studies Propylene glycol, 114, 117, 119, 121, abamectin, 104-107 124 ivermectin, 92-93, 94, 101, 108 Protein kinase C activity, ivermectin, Romania, 344, 345 85 Rose foliage, 295, 296 Protostrongylus, 273 Rushton radial flow impeller, 47 Protostrongylus rujescens, 223 Russet mite, 302 Psorergates ovis, 222-223, 224 Rust mite, 290-291, 304 Psoroptes cuniculi, 267 Psoroptes ovis, 218, 221, 224, 267, S 273 Salmo gairdneri, 169 Psylla pyricola, 303-304 Salmon, 278, 279 Pterolichidae, 278 Sarcoptes scabiei, 218, 221, 224, 226, Pyrethroids, 219, 304 241,253-254,269,270 Pythons, 279-280 Schistocerca gregaria, 75 Scirtothrips citri, 293 Scolothrips sexmaculatus, 305 R Scotland 179 Rabbits, clinical trials Screwworm fly, 216 mange mites, 266-267 Screwworms, 219 ticks, 267 Sculpin, 278 tsetse flies, 267 Seagulls, 277 360 Index

Seed flasks, fermentation of culture, Sphaerocerids, 178 34 Spider mites, 297-299 Seed media development, 37-40, Spinal cord parasites, 272 38 (Table 3.3) Spodoptera eridania, 299, 300, 302 See also Fermentation Spodoptera exigua, 302 development. Squalene, 52 Senegal, 311, 312 Stable fly, 174, 175 Sepsids, 178 Staphylinids, 178 Setaria equina, 241 Stephanurus dentatus, 226 Shake flash, avermectin formation, Sterilization, media, 51 43 (Fig. 3-1) Sternostoma tracheolum, 278 Sheep Stirred tanks, fermentation avermectin activity, 19 (Table 1.6) development, 44-45 clinical trials, 222-224 Stomoxys calcitrans, 175 ectoparasite activity, 222-223, Strawberries, abamectin, 305 224 Streptomyces avermitilis, 80, 89, 288 endoparasite activity, 222, 223 antifungal/ antibacterial activity, injectable formulation, 223 28-31 oral formulation, 222 compounds resulting from fat metabolism studies, 139-141 fermentation of, 1-3 liver metabolism studies, 135-139 containment of, 51 pharmacokinetics of ivermectin, cultural characteristics of, 27 (Table 119-122 2.1) biexponential decay, 118-119 electron micrographs of, (Fig. 7.5) 25-26 (Figs. 2.1, 2.2) bioavailability analyses, 121 genealogy, 37 (Table 3.2) dosage formulations and results, physiological properties of, 121 28 (Table 2.2) plasma concentration, shake flash, avermectin formation, 119-120 (Fig. 7.5) 43 (Fig. 3-1) safety of ivermectin, 153-155 source of, 24, 34 tissue residues, 132-134 taxonomy of, 25-26 toxicity, 153-154 See also Fermentation Sinefungin, as inhibitor, 70, 72 development. Singapore, 345, 346 Strongyloides fuelleborni, 275 Skin microfilariae, 314-318 Strongyloides ransomi, 226 density, 315 (Fig. 21.1) Strongyloides ratti, 262 effects, 316 (Fig. 22.2) Strongyloides stercoralis, 253, 263, Sleeping sickness, 318 320-321 Snakes, clinical trials, 279-280 Strongyloides westeri, 241 Soil. See Environmental effects of Strongylus edentatus, 238-239 abamectin; Environmental effects Strongylus equinus, 239 of ivermectin Strongylus vulgaris, 237-238 Solenopotes capillatus, 218, 221 Strychnine, 85 Solenopsis invicta, 288-290 Subchronic toxicity, 95-98 Solubility, abamectin, 186 Sudan, 268, 344, 345, 346 South Africa, 175,344,345,346 Summer sores, 240 Southern armyworm, 20, 299 Sunlight, abamectin, effects, 292 Spain, 344, 345, 346 Superphosphate, 178 Index 361

Sustained-release bolus, 221-222 guinea pigs, 265-266 Sweden, 271, 344, 345, 346 rabbits, 267 Swine Tissue residues clinical trials, 225-227 analytical techniques, 137-138, ectoparasite activity, 226-227 144-145 endoparasite activity, 226 animal studies, 132-136 injectable formulation, 225-226 distribution pattern and route of fat metabolism studies, 139-141 dose, 134 liver metabolism studies, 135-139 free residues, 132 pharmacokinetics of ivermectin, metabolism and, 133 (Table 8.l) 124-125 rodents, 135, 136 (Table 8.3) bioavailability analyses, assay method, 324-344 124-125 (Fig. 7.7) edible tissues dosage formulations and results, analytical methods, 144-145, 147 124-125 chemical assay, 144-148 plasma concentration, 124 depletion profiles, 147 (Table 9.3) safety of ivermectin, 155-157 recoveries of ivermectin, 146 tissue residues, 132-134 (Tables 9.1, 9.2) toxicity, 155-156 See also Assay method. Switzerland, 344, 345, 346 Tobacco hornworm, 299 Syphacia muris, 265 Togo, 313 Syphacia obvelata, 263 Tomato foliage, abamectin, 301-303 Tortoises, 279, 280 Toxascaris leonina, 251 T Toxicity Tablet formulation, dogs, 122-124 algae, 168 Taiwan, 345 bees, 195 Tampans, 216 birds, 195-196 Tapeworms, 241, 256 cattle, 151-152, 153 Teratology studies dogs, 157-158 abamectin, 104 (Table 6.6) earthworms, 168 ivermectin, 104 (Table 6.6) fish, 169 (Table 11.5), 170, 187-188, Tetranychus cinnabarinus, 297 193-194 Tetranychus pacijicus, 305 goats, 155 Tetranychus urticae, 20 (Table 1.7) horses, 149, 151 294-295, 297-298, 302, 304, 305 microorganisms in soil, 167, 195 Thailand, 344, 345, 346 plants, 186-187, 194 Thaimycins, 24 sheep, 153-154 Thelazia, 216 swine, 155-156 Thiabendazole, 178, 275, 320 Toxicity studies Thiacetarsamide, 269 abamectin Thin-layer chromatography (TLC), 7, chronic studies, 103 (Table 6.7) 26 dermal penetration study, 107, Thrips species 108 (Table 6.8) citrus crops, 293 dogs, 104 ornamental plants, 296-297 monkeys, 107 Ticks rodents, 104-107 abamectin efficacy, 231, 232-233 teratology studies, results, cattle, 219 104 (Table 6.6) 362 Index

Toxicity studies (cont.) Twospotted spider mite, 17, 294-295. ivermectin 302, 304 acute toxicity, 92-95 developmental/reproductive U toxicity, 98-102, 109 Ultraviolet methods, 145 dogs, 93-94, 97,108 use of, mutational procedures, 35 genotoxicity studies, 91-92 Uncinaria stenocephala. 252-253 lactation, 97, 10 1 Ungulates. See specific species minimum toxic dose (MTD), 94, United Kingdom, 80, 344, 345, 346 108 Uruguay, 344, 345, 346 monkeys, 94, 95, 97, 108-109 U.S.A., 344, 345, 346 plasma concentrations, 95 (Table U.S.S.R., 344, 346 6.2) rodents, 92-93, 94, 101, 108 V subchronic toxicity, 95-98 Vegetables, abamectin, 301-302 teratology studies, results, armyworms, 302 104 (Table 6.6), 109 and beneficial arthropods, 303 Toxocara canis, 250-251,263,269 leafminer, 301-302 Toxocara cati, 255 pinworms, 302-303 Translocation, 166 russet mite, 302 Transuterine/transmammary twospotted spider mite, 302 transmission, 250 Venezuela, 344, 345, 346 Tremors, 91, 94, 96, 99, 104, 105, 106, Vertebrates, ivermectin mode of 108 action, 80-85 Triatomid bugs, mice, 264 Volta River Basin, 315 Tribolium confusum, 288 Trichinella spiralis, 265 W Trichinella spp., 263 Warbles, 273 Trichodectes canis. 269 West Africa, 319 Trichostrongylus axei. 231, 232 Whipworm, dogs, 253 avermectin activity, 16 (Table 1.3), Wild boar, clinical trials, 274 19 (Table 1.6) Wolves, clinical trials, 269 Trichostrongylus colubriformis. 216, Worker exposure study, 201-213 222, 266 abamectin dissipation curve, avermectin activity, 16-18 (Tables 208-209, 210 (Fig. 14.1) 1.3, 1.4, 1.5), 19 (Table 1.6) during airblast treatment of citrus Trichuris muris. 263 groves, 203-208 Trichuris uulpis. 253 clothing and protection, 205, 211 Triglycerides, 52 dermal exposure, dosimeters, 204 Trinidad, 344, 346 dermal penetration, 206 Trioxabicyclooctaines, 79 dislodgeable foliar residues, 202, Trixacarus cauiae. 266 209-213 Trout, 278 exposure model used, 201-202 Tsetse flies during harvest, 208-213 guinea pigs, 266 harvester exposure data, 213 (Table rabbits, 267 14.6) Tunisia, 344, 345 individual worker exposure data, Turkey, 344 212 (Table 14.5) Index 363

margins of safety (MOS) Y calculations, 206-207 Yugoslavia, 344, 345 exposure data, 207 (Table 14.3) foliar residues, 210-211, 212-213 Z routes of exposure, 202 Zimbabwe, 344, 345 Zweig-Popendorf Factor, 202-203, Zimectrin paste, 234 208, 209, 211 Zweig-Popendorf Factor, worker Wuchereria bancrofti, 319-320 exposure, 202-203, 208, 209, 211