UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D
. C. 20460 

### ME MOR AND U M SUB .J E C T:

FR O M :

T HRO UG H :
TO: Review of Product C hemi s tr y, Manufacturin g Process . and Acute To xicity Studies of th e EP ll e lico vex containing I J elicoverpa annigera 

### Nuc leo po lyhed rov iru s ( Hear NPV BV003 ) TGA I for section 3 product re gis trat io n. Ibrahim S. Barsoum, Ph.D
. . Mic rob iologi s t Microbial P estic id es Branch, Biop es ticides and Pollution Pr eve nti o n Di vis io n (75 11 C) John L. Kou g h , Ph .D., Senior Scientist Microbia l P es ticid es Branch
. Bi opest icid Pollu ti on P revention Di visio n (751 1 C) 

### Michae l Glikes, Regu lato r y Act io n Leader M i crobial P es ticides Branch. Bi opes ticid es a nd Polluti on Prevention Divi s io n (751 I C) 

### ACT IO N REQUES T E D : To rev iew product chemistry , manufacturing p r ocess, formation of unintenti o nal in g redi en ts . analys is of samples, and certifi catio n of limi ts, storage s tabilit y, and vvaiver request for acu t e toxicity s tudie s fo r sect ion 3 re g istrati on of the EP He lic ovex co nt a ining He licoverpa armi ge ra 

### ucleopolyhedrov iru s (! lear PV BV 003
. EPA Reg. No
. 69553

- E) CONCLUS IO N: ACCEPTABLE on c o ndition that the regi s trant provides offic ial documentation that Shigella 

### and V ibr io 

### are not pathogens of concern in Swit zerland where th e product is presently manufactured, and provides QA testing f o r microbial co nt aminat ion when the product is manufactured in a different country.
CON T A I NS F I F RA C O NFI D ENT I A L B US I NESS I NF O RMA TI ON

1

### OAT A REVIEW RECORD Active Ingredient: Product Name: Co mpany Name: Reg. o: Chemica l N umb e r: Sub mis s ion Number: DP Barcode: MRlDs No: 

### BAC KGRO UN D: Helicoverpa armigera nucleopolyhedrovirus Helicovex Andermatt Biocontrol AG 69553-E 965729 426616 492393-01 , -02 , -03,-04,
- 05; 494097-01, -02 ; 495915-01 ,-02
Helicoverpa armigera NPV (HearNPV , previously HaSNPV) is a member of the v irus family Baculoviridae , genus Alphabaculovirus. HearNPV is a non-genetically modifi ed, naturallyoccurring st rain o riginall y isolated by the Institute of Zoo log y, Chinese Academy of Sciences, from severa l H. armigera larval cadavers infected with nuclear polyhedrosis disease in a cotton field in a suburb of Beijing , China. 

### It was deposited by the registrant on April 7. 2006, with the German Collect ion for Microorganisms and Cell Cultures (DSMZ) under the accession number B V-0003. HearNPV has a simi lar , if not identicaL host range and v irulence with Heliothis zea single NPV (HzS I PV ) (Hamm , 1982: Hugh es et al.. 1983), which was fir st detected in infected 

### H. zea larvae in South Africa in 1891. S in ce they were orig in ally isolated from differe nt ho s t s, they were previously named as different species. Analysis of the genome u s ing DNA restriction analysis s howed that the genomes of bo th v iruse s are ve ry simi lar ( Geni g and McCarthy. 198 2). Thi s was corroborated by genome sequencing. The sequence ide ntity of HzSNPV and HearNPV showed a 97% nucleotide identity and a 99% amino acid identit y (C hen ct al., 2002). On the basis of common host ran ge and virulence, the common physical propert ies, and the common se quence , Chen et al. (2002) concluded that HearN PV and HzSN PV are ·'variants of the san1c virus species." 

### DI SCUSS ION : Helicovex is an end usc product to be u se d in g reenhouses and open fields to control the corn earworm (Helicoverpa zea), tobacco budworm (Helicove1pa virescens), and Af ri can cot ton bollworm (Helicove rpa armigera). The active ingredient is 0.6% w/ w Helicoverpa armigera N PV st r ain BV-0003 , conta inin g at least 1.7 x 10 1 3 viral occ lu s ion bodies/ L. The end usc product contains a minimum of 7.5 x I 0 12 viral occlusion bodi es / L. The CSF and product label are in agreement concerning the content of active in g redient in the product. llelicm·e,pa armigera N PV is exempt from the requirement of a to leranc e per 40 CF R 180
. 1027. Five acceptable cert ifi cate s of ana l ysis arc prov id ed. The certified limit s for the ac tive and inert in g redients arc within their re s pective OCSPP-recommended ranges. The enforcement analytical method is a bioassay u s ing the targ e t organism, wi th result s compared to results for a standaJd batch. An acceptab le description of the physical /chem ical c h aracter istics i s provid e d. 2

### RECOMM EN DATIO N : ACCEPTABLE on condition that the registrant p rovides official documentation that Shigella 

### and Vib rio 

### are not pat hoge ns of conce rn in Sw itzer land where the product is manufactured
. and will test for microbial contaminants when the product is manufactured in another cou ntr y. SUMMA RY OF D ATA SUBM ITT ED: Pro du c t Id e ntity and Compo s ition: ( MRID No: 49 2393-0 1 ) Helicovex (EPA File Symbol 69553-E) is an end use product to be used in greenhouses and open fields to contro l the co rn earworm (J-Ielicove rp a zea), 

### tobacco budworm ( Hel i coverpa virescens). 

### and African cotton bolhvorm (Helicoverpa armigera). 

### The active ingredient is 0.6% w /w fl e !icoverpa armigera 

### NPV stra in BV-0003, contain ing at leas t 1 .7 x 10 13 

### vi ral occlusion bodies/ L. The end use product co ntain s a minimum of7.5 x 10 12 

### v iral occ lu s ion bodies/ L. The CSF and product label are in ag re ement concerning the co nten t of active in g r edient in th e product. Helicoverpa armigera 

### N PV is exempt from the requirem e nt of a tol e r ance under 40 CFR 180
. 1 027. .Helicoverpa armigera 

### PV (Hear PV. previously HaSNPY) is a member of the virus family Ba culoviridae, genus Alphabacu lovirus. 

### Hea rNPY is a non-g e neticall y modified , natura ll y- occu rrin g s train originally iso lated by the Institute of Zoo logy , Chinese Acade m y of Sc iences. from severa l fl. armigera 

### larval cadave rs inf ected with nuc l ear po lyhed rosis d isease in a cotton field in a s uburb of Beijing. China. It was deposited by the registrant on Apri l 7 , 2006. with the German Collection for Microorganisms and Cell Cult ure s ( DSMZ) und e r the accession numb er BV-0003. Ma nuf ac turing Process : 

### (M RID o: 492393-0 1)

3P r o du c ti o n o f H e li cove rp a a rmi ge r a N P V (M R I O No : 495915-02)

D e fi c i e nci es : None Di s cu ss ion o f F orm a ti o n o f U ninte nti o n a l Ingredi e nt s : (M RID No: 492393-0 I )

4

### The registrant states in MRID 4940970 I that Helicovex ® is produced in Switzerland, and that Shigella 

### a nd Vibrio 

### arc not pathogens of conce rn to human health th ere. T herefore , according to OECD guidelines, th e re is no need to provide data on the po ss ible contamination of products with these two pathogens. De ficienci es: The r eg istrant should provide official documentation that Shigella 

### and Vibrio 

### are not pathogens of concern in Switzerland where the product is manufactured and provides testing for microbial contaminants when th e produc t is manufactured in ano th er country. ANALYSIS OF SAM PL ES : Ce rtificate s of analysis for five batches o f I lelicovex 00 

### are provided in MRID 49239302. Each batch met the spec ification of a minimum of 7.5 x 10 12 

### vira l occlusion bodies/ L. D efic ien cies: None. 

### CERTI F I CA TIO N OF LIMIT S: Tab le 2 li st s the nominal concentration and certified limits for Helicovex !J\1 as given on the CSF
. The certified limit s for the active and inert in gred ient s a r e within their respective OCSPP-recommended ranges. A lower certified limit is given for the impurity, but is not required T A BL E 2. No minal co n ce n tration a nd ce rtifi ed limit s for H elicovex lll "a
In g redi e nt s (CAS number) P C Code Purp ose



## - •co ntam s a m1ntmum ol 7.) x I 0 12 vtral occluston bodtes / L 

### Deficiencie s : No ne. 5 Concen t ratio n ( 0 /., by weig ht ) No minal Lower Up p e rPHYSI C AL AN D CHEMICAL CHARACTE RI ST ICS : TABLE 3. Phy s ical and Chemical Properti es for Helicov ex ' 19 11 Gu id e line R e feren ce ' o ./Propcrty Des cripti o n of Resu lt Method s 830 .6302 Co l or Grey-brO\\ n Visual inspection 830 .6303 Ph ysical State Liquid Visual inspection 830 .6304 Odor Ty pical organic odor Olfactory inspecti o n 830 .63 13 Stability No t applicable. the product will not be stored in metal packag ing. and will be st ored at 5 °C &30
. 6314 Ox i dation/ Redu ction : Not applicab l e. the product docs not conta in Chemical Inc ompatibility oxidizing/redu cing agents 830.6 3 15 Flammability Not app l icable. th e product does not contain combustib l e liquid s 830
. 6316 Explodabili t y Not applicable. the product docs not contain potentiall y explosive ingredient s 830.6317 Storage Stability Stable at 5°C l or 36 mon th s C IP AC MT75.3. MT 160. MT 184. MTI85. tv1T I 48.

MT 4.7. bioassay with target o r ganism 830.6319 Mi sc ibi lity No t applicable. the product is not an emu lsi fiablc liquid to be diluted with petroleum solve nts 830
. 6320 Co rros i on C haracter i s tic s Non-co rro sive tO PET co n tai n er when s tored Visual obse rvati on. at 5 ° for 36 months container wei ght 830
. 632 1 Dielectric Bre akdown Voltage No t applicable. the product i s not for u sc around electrical equipmen t 830 .7000 pH 6.43 (undiluted at 23.4 ° C) C IPA C MT 75
. 3 6.92 ( 1% w / v aqueous so lution at 24
. 1 °C) POSBT 830 .7 100 Vi scos ity Non-Newtonian fluid
. OECD 11 1 4 2863
. 7 mPa/s at 2
. 5 rpm at 20°C 1 1 0S BT 164 690. I mP als at 50 rpm at 20° C 1490
. 3 mP als at 2.5 rpm at 40° C 405
. 00 mP a/s at 50 rpm at 40 °C lD0
. 7200 Meltin g Range Not required for EP 830.7220 13 oi lin g Ran ge Not r equired tor EP 830
. 7300 Den si t y/Relati ve Den s it y

/ Bulk Rdati ve density = 1
. 163 g/ mL at 20 °C OEC D 109 Density EC Re g. 440 /2008 J\.3 POS BT 286 830 .7370 Dis so ciation Co nstant in Water No t required for EP 830 .755 0 Partition Cocl'lkicn t No t required for EP 830
. 7840 Water Solubilit) Not required for El' 830
. 7950 Vap or Pre ss ure No t requin.:d lor EP D e fic• c n c •es: 

### None 6A CU T E TOX I C ITY STU DIES H c li co v e x In t r a p e riton ea l Acute Mo u se T ox i c i tvllnf ec ti v it v (MR I D # 495 9 1 5 02 ) This study was co ndu c ted t o assess the t ox ic ity / infe c ti v it y of 

### 5 bat c hes of the tes t s ub s tanc e. The animals were tre ate d \· V

it h the te s t s ubs t ance o n 

### 08 Jan 

### 15 , a nd the in- lif e po r tion of the study was terminated on 

### 15 Jan 

### 15. Helic ovex ( H e/i cove rpa armi ge ra NPV), was admini s te r e d to mic e o nce by intraperitoneal ( IP ) injecti on. Thirty female mic e we re random ly divided into s ix g ro up s of five mice per gro up . Five gro up s we re eac h adm ini ste r ed one batch of th e t est s ub s t a nce by l P injection a t a dose of approximately 10 6 Viral Occlu s io n Bodie s (OB) pe r animal. The control g roup wa s s ham dose d with an e qu i va lent volume of s t e ril e sa lin e. Ob se r vations fo r mo r ta li ty a nd s ig ns of pharmaco logic and / or tox icological e ffect s we re made twice on Day 

### 0 a nd once daily throu gh D ay 7. Bod y we i ghts were reco rd ed on Day 0 and at s tudy termination. A ll mice we re sac rificed o n Day 7 and g ross ne c r ops ies were performed. R es ul t s There was no mor ta lity durin g th e study and no obser ved ab no rm a liti es durin g the s tudy for th e ge ne ra l health obse r vati o n s. There we re no s ig nificant differences between bod y we ig ht s or bod y weight ga in. The g ross necrop sy co nducte d a t term ination of the s tud y r evea led no obse r vab l e abnorma liti es. The t est s u bstan ce, Helicovex ( H e ! i coverpa armigera NPV) , wa s de termined to be non-tox ic to mic e when admini s t e re d by intrape riton ea l inj ec tion at a d ose of 10 6 OB/ mo us e. Tctble 1 -l ntJ ·ape dton ca l Acute Mo u s e Tox i city/ Inf ecti v ity G •·o up Day of S aCI'ifi cc Necrop s y Gt·oup 1

- H e l icovex 

### (H elicol'e rpa armig e ra NPV) Batch # 200 3-F Dav 7 N0 1 \ 7-F J)ay 7 NOA 8-F 

### Day 7 NOA I 9-F Day 7

**~OA**

**~** 1-F Day 7 OA Group 2- Hcli cove x 

### ( He/h· ot·er pa armigera N PV) Batc h # 20 I 13-F Da' 7 NOA I 5-F !Jn\' 7 NOA

**~2-F** 

### Dav 7 NOA 23-F Dav 7 NO/\ 24-1: 

### Day 7 NOA G roup 3-Hclicovex 

### ( Ilelicoverpa armig e ra NPV) Batch # 202 5-l· Dav 7 OA 

### 9-F Day 7 NOA I 0-F Day 7 NO
. .L\ 18-F Dav 7 NOA 27-F Day 7 NOA G r ·o up .t- H c li conx 

### ( H elicoverpa armige ra NPV) Batch# 203 1 -F DH\ ' 7 NO/\ 711 -F 14-r: 17-F 

### 20

- F Da \' 7 Da v 7 Dav 

### 7 Day 7 1'\0 A 

### NOA NOA 

### NO A GroupS- Hclico vcx 

### (llelicol'erpa armigera NPY) Ba tch # 204 4-F 

### Day 7 NOA



### 1 2 -F Dav 7 NO/\ 

### 16
- F Dav 7 ' O A 28-r: 

### Da) 7 'OA F
-

**F~mak :**  1\!0A- 

### No Ob sc.: n · ablc Ab norm a l iti es Defici en cie s: 

### No n e Rationale for waiver r e gu cst for acute toxicity st udi es of Hc licov cx (MRID # 49409700) 

### l
. " Baculoviruscs are natur ally occW'ring pathogens of arthropods. The ir host range is exclusively restricted to arth ropods
. No member of this v irus fami ly is infective to plants o r vertebrates. 2. Bacul ov iru ses are 

### ubiquitously present in the environment and have been used for biological insect co ntro l for more than I 00 years. Circu mstantia l evidence for the safety of baculoviruses emerges from the history of contact between baculoviruses and humans \ovithout any detrimental effect. During the 1970 s, the US Environmental Protection Agency established the Guidance for Safety Testing of Baculoviru ses, wh ich a lso became a gu ide lin e of bacu lovirus safety tests. 3. Heli cove rpa zea SNPV was the first commercia l bacu lovirus in sect icide and is one of the mos t extensively tested entomopathogenic v iru ses ( l gnoffo , 197 5). Safety tests of more than 51 entomopath oge ni c v iru ses including more than 30 baculovi ruses resulted in a lo ng and complete safety record (exte nsivel y reviewed by l gnoffo. 1973 ; Surges et al.l980a, 198 0b; Groner, 1 986). 4. No adverse effect on hum an hea lth ha s be en observed in any of th ese in vestigat ions indi cat in g that the usc of baculovirus is safe and does not cause any healt h hazards." 5
. EPA concluded i n the ll eliothis zea RED that " Due to the ove ra ll results ofTie r III toxicolog y testing and the additional toxicology data whic h support the negative response of viral replication in mammal s , the age ncy concludes there are s ufficient data to sa ti sfy this requireme nt without reque st in g a ce ll culture st ud y ." 6. Although baculovirus uptake has been ob s erved , there has been no evidence that v iral D A replicati on, production of vira l proteins or cy topatholog ic al effects have occu rred. In vivo. the uptake of baculovirus OB s by various animals did not lead to the production of baculovirus-specific antibodies.

87. There is no evidence for baculovirus induced cytogenic, carcinogenic, mutagenic or teratogenic effects has ever been found (Groner, 1986 ; Ignoffo. 1975 ; Me William, 2007; O ECD, 2002). C O NCLUS IO N: ACCEPTABLE References I. C he n , X., I.J. Wu, R. Tarchine, et al. 200 I. The Sequence of the Heliocoverpa armigera S ingle N ucleocap sid NPV Genome. J. Gen. Virol 82:241-257. 2. C he n , X., W.J. Z hang , J. Wong, et al. 2002. Comparative Analysis of the Complete Genome Seq uenc es of H elicove rp a zea and H eliocoverpa armigera S ingle- Nucleocaps id NPVes. J. Gen. Virol. 83:673-684. 3. llamm, .J.J. 1982. Relati ve Susceptibility of Several Noctuid Species to a Nuclear Polyhedrosis Virus from 1/e/iothis armigera. Joumal of Invertebrate Patho l ogy 39:255-256. 4. Hughes, P.R., R.R. Gettig, and W.J. McCarthy. 198 3. Comparison of the Time-Mortality Response of Heliothis zea to 14 Isolates of Heliothis uclear Polyhedrosis Virus. Journal of Invertebrate Pathology 41 :256-26 1
. 5. lgnoffo. C.M., A. H. Mcintosh, and C. Garcia. 1983. Susceptibility of Larvae of !!eliothis zea, H. virescens, and H. c mnige ra (Lep: Noctuidae) to 3 Baculoviru ses . Entomophaga 28: 1

- 8. 6. J e hle, J.A. , M. Lange, H. Wang, e t al. 2006. Molecular Identification and Ph y logenti c Anal ys is of Baculoviruses of Lepidoptera. Virology 346: I 80-196. 7. Ogembo, J.G., B.L. Cao ili , M. Shikata, et al. 2009. Comparative Genom ic Sequence Analysis of Novel Heliocol'elpa armigera N PV (NPV) I solated from Kenya and Three Other Previously Sequenced Helicoverpa spp. NPVs Virus Genes. Reference not provided. 8. Rowley. O.L., l

-l.J. Popham, and R.L. Harrison. 20 II. Genetic Variation and Virulence of NPVes Isolated Worldwide from the H e li o thine Pests Helicoverpa armigera, Heliocoverpa zea, and H e liothis virescens. J. Inv e rtebr. Pathol. I 07: 112-1 26. 9. Williams, C .F. , and C.C. Payne. 1984
. The Susceptibility of He/iothis armigera Larvae to Three Nuclear Polyhedrosis Viru ses. Anna ls of App li ed Biology I 04: 405-4 1 2. 10. Zhang. C.S., S.C. Ma, and Z.J
. Guo. 2005. Compa ri son of the Complete Genome Sequence bet\veen C l and G4 I so lates of th e H e licov e 1pa armigera Sing le Nucleocapsid NPV. Virology 333: 1 90- 199
.

9