UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D. C. 20460 

MEMORANDUM 

SUB.J ECT: 

FROM: 

T HROUGH: 

TO: 

Review of Product Chemistry, Manufacturing Process. and Acute Toxicity 

Studies of the EP llelicovex containing I Jelicoverpa annigera 

Nucleopolyhedrovirus (Hear NPV BV003) TGAI for section 3 product 

registration. 

Ibrahim S. Barsoum, Ph.D .. Microbiologist 

Microbial Pesticides Branch, Biopesticides and 

Pollution Prevention Division (75 11 C) 

John L. Kough, Ph.D., Senior Scientist 

Microbial Pesticides Branch. Biopesticid 

Pollution Prevention Division (751 1 C) 

Michael Glikes, Regulatory Action Leader 

Microbial Pesticides Branch. Biopesticides and 

Pollution Prevention Division (751 I C) 

ACTION REQUEST ED: 

To review product chemistry, manufacturing process, formation of unintentional ingredients. 

analysis of samples, and certification of limits, storage stability, and vvaiver request for acute 

toxicity studies for section 3 registration of the EP Helicovex containing Helicoverpa armigera 

ucleopolyhedrovirus (! lear PV BV003 . EPA Reg. No. 69553-E) 

CONCLUSION: ACCEPTABLE on condition that the registrant provides official documentation 

that Shigella and Vibrio are not pathogens of concern in Switzerland where the product is presently 

manufactured, and provides QA testing for microbial contamination when the product is 

manufactured in a different country. 

CONTAINS FIFRA CONFIDENTIAL BUSINESS INFORMATION 

1 
OAT A REVIEW RECORD 

Active Ingredient: 

Product Name: 

Company Name: 

Reg. o: 

Chemical N umber: 

Submission Number: 

DP Barcode: 

MRlDs No: 

BACKGROUND: 

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 virus family 

Baculoviridae, genus Alphabaculovirus. HearNPV is a non-genetically modified, naturallyoccurring strain originally isolated by the Institute of Zoology, Chinese Academy of Sciences, from 

several 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 

Collection for Microorganisms and Cell Cultures (DSMZ) under the accession number B V -0003. 

HearNPV has a similar, if not identicaL host range and virulence with Heliothis zea single NPV 

(HzS IPV) (Hamm, 1982: Hughes et al.. 1983), which was first detected in infected H. zea larvae in 

South Africa in 1891. Since they were originally isolated from different hosts, they were previously 

named as different species. Analysis of the genome using DNA restriction analysis showed that the 

genomes of both viruses are very similar (Genig and McCarthy. 1982). This was corroborated by 

genome sequencing. The sequence identity of HzSNPV and HearNPV showed a 97% nucleotide 

identity and a 99% amino acid identity (Chen ct al., 2002). On the basis of common host range and 

virulence, the common physical properties, and the common sequence, Chen et al. (2002) concluded 

that HearN PV and HzSN PV are ·'variants of the san1c virus species." 

DISCUSSION: 

Helicovex is an end usc product to be used in greenhouses and open fields to control the corn 

earworm (Helicoverpa zea), tobacco budworm (Helicove1pa virescens), and African cotton 

bollworm (Helicoverpa armigera). The active ingredient is 0.6% w/w Helicoverpa armigera NPV 

strain BV-0003, containing at least 1.7 x 10 13 viral occ lusion bodies/L. The end usc product 

contains a minimum of 7.5 x I 0 12 viral occlusion bodies/L. The CSF and product label are in 

agreement concerning the content of active ingredient in the product. 

llelicm·e,pa armigera NPV is exempt from the requirement of a tolerance per 40 CFR 

180.1027. Five acceptable certifi cates of analysis arc provided. The certified limits for the active 

and inert ingredients arc within their respective OCSPP-recommended ranges. The enforcement 

analytical method is a bioassay using the target organism, with results compared to results for a 

standaJd batch. An acceptable description of the physical/chemical characteristics is provided. 

2 

107310
RECOMMEN DATION: ACCEPTABLE on condition that the registrant provides official 

documentation that Shigella and Vibrio are not pathogens of concern in Switzerland where the 

product is manufactured. and will test for microbial contaminants when the product is 

manufactured in another country. 

SUMMARY OF DATA SUBMITTED: 

Product Identity and Composition: (MRID No: 492393-01) 

Helicovex (EPA File Symbol 69553-E) is an end use product to be used in greenhouses and open 

fields to control the corn earworm (J-Ielicoverpa zea), tobacco budworm (Helicoverpa virescens). 

and African cotton bolhvorm (Helicoverpa armigera). The active ingredient is 0.6% w/w 

fle!icoverpa armigera NPV strain BV-0003, containing at least 1.7 x 10 13 viral occlusion bodies/L. 

The end use product contains a minimum of7.5 x 10 12 viral occlusion bodies/L. The CSF and 

product label are in agreement concerning the content of active ingredient in the product. 

Helicoverpa armigera NPV is exempt from the 

requirement of a tolerance under 40 CFR 180.1027. .Helicoverpa 

armigera PV (Hear PV. previously HaSNPY) is a member of the virus family Baculoviridae, 

genus Alphabaculovirus. HearNPY is a non-genetically modified, naturall y-occurring strain 

originally isolated by the Institute of Zoology, Chinese Academy of Sciences. from several fl. 

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 Collection for 

Microorganisms and Cell Cultures (DSMZ) under the accession number BV-0003. 

Manufacturing Process: (MRID o: 492393-01) 

3 
Production of Helicoverpa a rmiger a NPV (MRIO No: 495915-02) 

Deficiencies: None 

Discussion of Forma tion of Unintentiona l Ingredients: (M RID No: 492393-0 I) 

4 
The registrant states in MRID 4940970 I that Helicovex® is produced in Switzerland, and that 

Shigella and Vibrio arc not pathogens of concern to human health there. Therefore, according to 

OECD guidelines, there is no need to provide data on the possible contamination of products with 

these two pathogens. 

Deficiencies: The registrant 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 the product is manufactured in another country. 

ANALYSIS OF SAMPLES: 

Certificates of analysis for five batches of I lelicovex00 are provided in MRID 49239302. Each batch 

met the specification of a minimum of 7.5 x 1012 viral occlusion bodies/L. 

Deficiencies: None. 

CERTIFICATION OF LIMITS: 

Table 2 lists the nominal concentration and certified limits for Helicovex!J\1 as given on the CSF. The 

certified limits for the active and inert ingredients are within their respective OCSPP-recommended 

ranges. A lower certified limit is given for the impurity, but is not required 

TABLE 2. Nominal concentration and certified limits for Helicovexlll"a 

Ingredients (CAS number) PC Code Purpose 

- •contams a m1ntmum ol 7.) x I 012 vtral occluston bodtes/L 

Deficiencies: None. 

5 

Concen tration (0

/., by weight) 

Nominal Lower Upper 
PHYSICAL AND CHEMICAL CHARACTERISTICS: 

TABLE 3. Physical and Chemical Properties for Helicovex'

1911 

Gu ideline Reference 'o./Propcrty Description of Result Methods 

830.6302 Color Grey-brO\\ n Visual inspection 

830.6303 Physical State Liquid Visual inspection 

830.6304 Odor Typical organic odor Olfactory inspection 

830.63 13 Stability Not applicable. the product will not be stored 

in metal packaging. and will be stored at 5°C 

&30.6314 Ox idation/Reduction: Not applicable. the product docs not contain 

Chemical Incompatibility oxidizing/reducing agents 

830.6315 Flammability Not applicable. the product does not contain 

combustible liquids 

830.6316 Explodability Not applicable. the product docs not contain 

potentially explosive ingredients 

830.6317 Storage Stability Stable at 5°C lor 36 months CIPAC MT75.3. MT 160. 

MT 184. MTI85. tv1T I48. 

MT 4.7. bioassay with 

target organism 

830.6319 Miscibi lity Not applicable. the product is not an 

emu lsi fiablc liquid to be diluted with 

petroleum solvents 

830.6320 Corrosion Characteristics Non-corrosive tO PET container when stored Visual observation. 

at 5° for 36 months container weight 

830.6321 Dielectric Breakdown Voltage Not applicable. the product is not for usc 

around electrical equipment 

830.7000 pH 6.43 (undiluted at 23.4°C) CIPAC MT 75.3 

6.92 ( 1% w/v aqueous solution at 24.1 °C) POSBT 

830.7 100 Viscosity Non-Newtonian fluid. OECD 11 14 

2863.7 mPa/s at 2.5 rpm at 20°C 1

1 0S BT 164 

690. I mPals at 50 rpm at 20°C 

1490.3 mPals at 2.5 rpm at 40°C 

405.00 mPa/s at 50 rpm at 40°C 

lD0.7200 Melting Range Not required for EP 

830.7220 13oi ling Range Not required tor EP 

830.7300 Density/Relative Density/Bulk Rdative density = 1.163 g/mL at 20°C OECD 109 

Density EC Reg. 440/2008 J\.3 

POS BT 286 

830.7370 Dissociation Constant in Water Not required for EP 

830.7550 Partition Cocl'lkicnt Not required for EP 

830.7840 Water Solubilit) Not required for El' 

830.7950 Vapor Pressure Not requin.:d lor EP 

Defic•cnc•es: None 

6 
ACUTE TOXICITY STUDIES 

H clicovex Intra peritoneal Acute Mouse Toxicitvllnfectivitv (MRID # 4959 1502) 

This study was conducted to assess the toxicity/infectivity of 5 batches of the test substance. The 

animals were treated \·Vith the test substance on 08 Jan 15, and the in-life portion of the study was 

terminated on 15 Jan 15. Helicovex (He/icoverpa armigera NPV), was administered to mice once 

by intraperitoneal (IP) injection. Thirty female mice were randomly divided into six groups of five 

mice per group. Five groups were each administered one batch of the test substance by lP injection 

at a dose of approximately 106 Viral Occlusion Bodies (OB) per animal. The control group was 

sham dosed with an equivalent volume of sterile saline. Observations for mortali ty and signs of 

pharmacologic and/or toxicological effects we re made twice on Day 0 and once daily through Day 

7. Body weights were recorded on Day 0 and at study termination. All mice were sacrificed on Day 

7 and gross necropsies were performed. 

Results 

There was no mortality during the study and no observed abnormalities during the study for the 

general health observations. There were no significant differences between body weights or body 

weight gain. The gross necropsy conducted at termination of the study revealed no observable 

abnorma lities. The test substance, Helicovex (He!icoverpa armigera NPV), was determined to be 

non-toxic to mice when administered by intraperitoneal injection at a dose of 106 OB/mouse. 

Tctble 1 -l ntJ·apedtoncal Acute Mo use Toxicity/Infectivity 

G •·oup Day of S aCI'ificc Necropsy 

Gt·oup 1 - Helicovex (Helicol'erpa armigera NPV) Batch# 200 

3-F Dav 7 N01\ 

7-F J)ay 7 NOA 

8-F Day 7 NOA 

I 9-F Day 7 ~OA 

~ 1-F Day 7 OA 

Group 2- Hclicovex (He/h·ot·erpa armigera NPV) Batch# 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 armigera 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·oup .t- Hcliconx (Helicoverpa armigera NPV) Batch# 203 

1-F DH\' 7 NO/\ 

7 
11 -F 

14-r: 

17-F 

20-F 

Da\' 7 

Dav 7 

Dav 7 

Day 7 

1'\0 A 

NOA 

NOA 

NOA 

GroupS- Hclicovcx (llelicol'erpa armigera NPY) 

Batch# 204 

4-F Day 7 NOA 

12-F Dav 7 NO/\ 

16-F Dav 7 'OA 

28-r: Da) 7 'OA 

F - F~mak : 1\!0A- No Obsc.:n ·ablc Abnormaliti es 

Deficiencies: None 

Rationale for waiver regucst for acute toxicity studies of Hclicovcx (MRID # 49409700) 

l ."Baculoviruscs are naturally occW'ring pathogens of arthropods. Their host range is exclusively 

restricted to arthropods. No member of this virus family is infective to plants or vertebrates. 

2. Baculoviruses are ubiquitously present in the environment and have been used for biological 

insect control for more than I 00 years. Circumstantial evidence for the safety of baculoviruses 

emerges from the history of contact between baculoviruses and humans \ovithout any detrimental 

effect. During the 1970s, the US Environmental Protection Agency established the Guidance for 

Safety Testing of Baculoviruses, which also became a guideline of baculovirus safety tests. 

3. Helicoverpa zea SNPV was the first commercial baculovirus insecticide and is one of the most 

extensively tested entomopathogenic viruses (lgnoffo, 1975). Safety tests of more than 51 

entomopathogenic viruses including more than 30 baculoviruses resulted in a long and complete 

safety record (extensively reviewed by lgnoffo. 1973; Surges et al.l980a, 1980b; Groner, 1986). 

4. No adverse effect on human health has been observed in any of these in vestigations indicating 

that the usc of baculovirus is safe and does not cause any health hazards." 

5. EPA concluded in the lleliothis zea RED that "Due to the overall results ofTier III toxicology 

testing and the additional toxicology data which support the negative response of viral replication in 

mammals, the agency concludes there are sufficient data to satisfy this requirement without 

requesting a cell culture study." 

6. Although baculovirus uptake has been observed, there has been no evidence that viral D A 

replication, production of viral proteins or cytopathological effects have occurred. In vivo. the 

uptake of baculovirus OBs by various animals did not lead to the production of baculovirus-specific 

antibodies. 

8 
7. There is no evidence for baculovirus induced cytogenic, carcinogenic, mutagenic or teratogenic 

effects has ever been found (Groner, 1986; Ignoffo. 1975; Me William, 2007; OECD, 2002). 

CONCLUSION: ACCEPTABLE 

References 

I. Chen, X., I.J. Wu, R. Tarchine, et al. 200 I. The Sequence of the Heliocoverpa armigera 

Single Nucleocapsid NPV Genome. J. Gen. Virol 82:241-257. 

2. Chen, X., W.J. Zhang, J. Wong, et al. 2002. Comparative Analysis of the Complete Genome 

Sequences of Helicoverpa zea and Heliocoverpa armigera Single-Nucleocapsid 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 Pathology 39:255-256. 

4. Hughes, P.R., R.R. Gettig, and W.J. McCarthy. 1983. 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 !!eliot his 

zea, H. virescens, and H. cmnigera (Lep: Noctuidae) to 3 Baculoviruses. Entomophaga 28: 1-

8. 

6. Jehle, J.A., M. Lange, H. Wang, et al. 2006. Molecular Identification and Phylogenti c 

Analysis of Baculoviruses of Lepidoptera. Virology 346: I 80-196. 

7. Ogembo, J.G., B.L. Caoili , M. Shikata, et al. 2009. Comparative Genomic Sequence 

Analysis of Novel Heliocol'elpa armigera NPV (NPV) Isolated 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 Heliothine Pests Helicoverpa armigera, Heliocoverpa 

zea, and Heliothis virescens. J. Invertebr. 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 Viruses. Anna ls of Applied Biology I 04: 405-4 12. 

10. Zhang. C.S., S.C. Ma, and Z.J. Guo. 2005. Comparison of the Complete Genome Sequence 

bet\veen C l and G4 Isolates of the Helicove1pa armigera Single Nucleocapsid NPV. 

Virology 333: 190- 199. 

9