1

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

 WASHINGTON, D.C. 20460 

 

MEMORANDUM

SUBJECT: Review of Product Chemistry, Manufacturing Process, and Acute Toxicity 

Studies of the EP Spexit containing the TGAI Spodoptera exigua 

nucleopolyhedrovirus strain BV-0004 for section 3 product registration. 

FROM: Ibrahim S. Barsoum, Ph.D., Microbiologist 

Microbial Pesticides Branch, Biopesticides and

Pollution Prevention Division (7511C) 

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

Microbial Pesticides Branch, Biopesticides and

Pollution Prevention Division (7511C) 

TO: Michael Glikes, Regulatory Action Leader

Microbial Pesticides Branch, Biopesticides and

Pollution Prevention Division (7511C) 

ACTION REQUESTED: 

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

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

toxicity studies for section 3 registration of the EP Spexit containing the TGAI Spodoptera exigua 

nucleopolyhedrovirus strain BV-0004 (EPA Reg. No. 69553-U) 

CONCLUSION: ACCEPTABLE on condition that the registrant provides official documentation 

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

man des QA testing for microbial contamination when the product is 

manufactured in a different country. 
2

CONTAINS FIFRA CONFIDENTIAL BUSINESS INFORMATION

DATA REVIEW RECORD

Active Ingredient: Spodoptera exigua nucleopolyhedrovirus 

Product Name: Spexit 

Company Name: Andermatt Biocontrol AG 

Reg. No: 69553-U 

Chemical Number: 129345 

Submission Number: 942667 

DP Barcode: 418390 

 MRIDs No: 492387-01 - 06; 494095-01, -02; 495788-01 - 03

BACKGROUND:

Spexit® (EPA File Symbol 69553-U) is an end use product to be used in greenhouses and open 

fields to control the beet armyworm (Spodoptera exigua). The active ingredient is 0.6% w/w 

Spodoptera exigua nucleopolyhedrovirus strain BV-0004, containing at least 6.9 x 1012 viral 

occlusion bodies/L. The end use product contains a minimum of 3.8 x 1012 viral occlusion bodies/L. 

Spodoptera exigua nucleopolyhedrovirus strain BV-0004, a member of the family Baculoviridae, is 

a naturally-occurring, non-genetically modified strain originally isolated from S. exigua larvae in 

China. It was deposited at the German Collection for Microorganisms and Cell Cultures (DSMZ) in 

2006 under the accession number BV-0004. Spodoptera exigua multi nucleopolyhedrovirus strain 

BV-0004 acts very specifically against larva of S. exigua. After the larva ingest it, the virus rapidly 

multiplies within the host insect, resulting in cell destruction and death, typically within three to 

seven days. 

DISCUSSION:

The end use product contains a minimum of 3.8 x 1012 viral occlusion bodies/L. The CSF

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

 as an impurity. 

The inert ingredient is Spodoptera exigua multi nucleopolyhedrovirus is 

exempt from the requirement of a tolerance under 40 CFR 180.1118. is exempt under 

180.950. Baculovirus species are extremely host specific, with a host range limited to one or a few 

species of the same genus. They occur only in arthropods, predominantly in the orders Lepidoptera, 

Diptera, and Hymenoptera. Baculoviruses are not infective to mammals, and no pathogenic, 

genotoxic, mutagenic, or carcinogenic effects of baculoviruses have been observed in mammals. 

Since baculoviruses have no independent metabolism, they do not produce metabolites. 

RECOMMENDATION: ACCEPTABLE on condition that the registrant provides official 

documentation that 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.
3

SUMMARY OF DATA SUBMITTED:

Product Identity and Composition: (MRID No: 49238701) 

Spodoptera exigua nucleopolyhedrovirus strain BV-0004, a member of the family Baculoviridae, is 

a naturally-occurring, non-genetically modified strain originally isolated from S. exigua larvae in 

China. It was deposited at the German Collection for Microorganisms and Cell Cultures (DSMZ) in 

2006 under the accession number BV-0004. Spodoptera exigua nucleopolyhedrovirus (SeMNPV) 

has been isolated in different locations throughout the world. Based on DNA restriction 

endonuclease analysis of the viral genomes, all the wild-type SeMNPV isolates are closely related 

to SeMNPV-US1, an isolate that originated in California (Caballero et al., 1992; Gelernter and 

Federici, 1986; Hara et al., 1995; Kondo et al., 1994). This isolate has also been proposed as the 

type strain of SeMNPV (Caballero et al., 1992). Another isolate of SeMNPV originated in Florida, 

and was designated SeMNPV-US2 (Hamm and Chandler, 1996; Kolodny-Hirsh et al., 1993). This 

strain has been marketed under the trade name Spod-X® (EPA Reg. No. 70051-46). DNA restriction 

endonuclease analysis was used to compare the DNA enzyme profile of Spexit® to the reference 

product Spod-X®. The viral DNAs were digested with the restriction enzymes BamHI, PstI, XbaI, 

BglII, ScaI, and NdeI. Results showed that Spexit® is identical to the pattern of SeMNPV-US1, 

while Spod-X® is identical to SeMNPV-US2. The genomes are estimated to be more than 93% 

identical. 

Manufacturing Process: (MRID No: 49238701, 49578801) 

Discussion of Formation of Unintentional Ingredients: (MRID # 49409502) 
4

The registrant states in MRID 49409502 that Spexit® is produced in Switzerland, and that 

and are 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 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 and Certification of Limits: (MRID # 49238702) 

Certificates of analysis for five batches of Spexit® are provided in MRID 49238702. Each batch met 

the specification of a minimum of 3.75 x 1012 viral occlusion bodies/L. 

Deficiencies: None. 

ENFORCEMENT ANALYTICAL METHOD: (MRID # 49409502) 

Deficiencies: None. 
5

PHYSICAL AND CHEMICAL CHARACTERISTICS: (MRID # 49238705)

TABLE 2. Physical and Chemical Properties for Spexit ®a

Guideline Reference No./Property Description of Result Methods

830.6302 Color Grey-brown Visual inspection 

830.6303 Physical State Viscous liquid Visual inspection 

830.6304 Odor Aromatic Olfactory inspection 

830.6313 Stability Not applicable, the product will not be stored 

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

830.6314 Oxidation/Reduction: 

Chemical Incompatibility

Not applicable, the product does not contain 

oxidizing/reducing agents

830.6315 Flammability Not applicable, the product does not contain 

combustible liquids

830.6316 Explodability Not applicable, the product does not contain 

potentially explosive ingredients

830.6317 Storage Stability Stable at 5° for one-year study 

830.6319 Miscibility Not applicable, the product is not an 

emulsifiable liquid to be diluted with 

petroleum solvents

830.6320 Corrosion Characteristics Non-corrosive to container for one-year 

830.6321 Dielectric Breakdown Voltage Not applicable, the product is not for use 

around electrical equipment

830.7000 pH 6.35 – 6.84 (1% aqueous solution) CIPAC MT 75.3

POS BT 158

830.7100 Viscosity Non-Newtonian fluid. 

3120.4 mPa/s at 2.5 rpm at 20°C

610.4 mPa/s at 50 rpm at 20°C

3027.2 mPa/s at 2.5 rpm at 40°C

410.3 mPa/s at 50 rpm at 40°C

OECD 1114

POS BT 164

830.7200 Melting Range Not required for EP

830.7220 Boiling Range Not required for EP

830.7300 Density/Relative Density/Bulk 

Density

Relative density = 1.160 g/mL Pycnometer

OECD 109

EC Reg. 440/2008 method 

A.3

POS BT 286

830.7370 Dissociation Constant in Water Not required for EP

830.7550 Partition Coefficient Not required for EP

830.7840 Water Solubility Not required for EP

830.7950 Vapor Pressure Not required for EP

Deficiencies: None.
6

Storage Stability and Corrosion Characteristics (MRID # 49578803)

Storage Procedure

The temperature during storage over 12 months was 5°C within a variance of ±

2.5°C. Short temperature increases after opening the door of the climatic chamber for

sampling were disregarded.

Stability of the Original Test Item Container

No change to the container shape or size was observed over the entire test period of 12

months at a temperature of 5

o

C.

Physical state, Color, and Odor of the Test Item

The typical characteristics of the test item did not change during the test period (Table 3).

Color and odor of the test item were stable throughout the entire test period of 12 months

at a temperature of 5°C. The product remained a f lowable single-phase liquid. Clumps and

other particles outside specification of the product were not detected. The physical state of 

the test product can therefore be considered as stable at a temperature of 5°C for one year.

Table 3. Results for physical state, color and odor of the test item at each sampling date

09/07/2013

Month 0

12/07/2013

Month 3

03/01/2014

Month 6

06/04/2014

Month 9

09/07/2014

Month 12

State viscous

liquid, single

viscous liquid,

single phase,

viscous liquid,

single phase,

viscous liquid,

single phase,

viscous liquid,

single phase,

Color: grey-brown grey-brown grey-brown grey-brown grey-brown

Odor: characteristic

of aromatic 

characteristic

of aromatic

characteristic of 

aromatic

characteristic

of aromatic

characteristic of 

aromatic

ACUTE TOXICITY STUDIES

Spexit Intraperitoneal Acute Mouse Toxicity/Infectivity (MRID # 49578802)

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

(Spodoptera exigua MNPV), 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 IP 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 mortality and signs of pharmacologic and/or toxicological effects 

were 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. 
7

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 

abnormalities. The test substance, Spexit (Spodoptera exigua MNPV), was determined to be nontoxic to mice when administered by intraperitoneal injection at a dose of l06 OB/mouse.

Table 1-Intraperitoneal Acute Mouse Toxicity/Infectivity

Group Day of Sacrifice Necropsy

Group 1 - Spexit (Spodoptera exigua MNPV) Batch# 002

6-F Day 7 NOA

14-F Day 7 NOA

17-F Day7 NOA

21-F Day 7 NOA

24-F Day 7 NOA

Group 2 - Spexit (Spodoptera exigua MNPV) Batch# 003

 3-F Day 7 NOA

12-F Day 7 NOA

13-F Day 7 NOA

26-F Day 7 NOA

30-F Day 7 NOA

Group 3 - Spexit (Spodoptera exigua MNPV) Batch# 005

 2-F Day 7 NOA 8-F Day 7 NOA

18-F Day 7 NOA

20-F Day 7 NOA

22-F Day 7 NOA

Group 4 - Spexit (Spodoptera exigua MNPV) Batch# 006

 1-F Day 7 NOA

 7-F Day 7 NOA

 9-F Day 7 NOA

27-F Day 7 NOA

29-F Day 7 NOA

 Group 5 - Spexit (Spodoptera exigua MNPV) Batch# 007

4-F Day 7 NOA 

5-F Day 7 NOA

10-F Day 7 NOA

28-F Day 7 NOA

F - Female; NOA - No Observable Abnormalities
8

Deficiencies: None.

Rationale for waiver request for acute toxicity studies of Spexit

1."Baculoviruses are naturally occurring 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 100 years. Circumstantial evidence for the safety of baculoviruses 

emerges from the history of contact between baculoviruses and humans without 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. Spodoptera exigua MNPV was the first commercial baculovirus insecticide and is one of the 

most extensively tested entomopathogenic viruses. Safety tests of more than 51 entomopathogenic 

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

(extensively reviewed by Ignoffo, 1975)

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

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

5. EPA concluded in the Heliothis zea RED that "Due to the overall results of Tier 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 DNA 

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.

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

effects has ever been found (OECD, 2002).

CONCLUSION: ACCEPTABLE

References 

Caballerro, P. D. Zuidema, C. Santgiago-Alvarez, et al. 1992. Biochemical and Biological 

Characterization of Four Isolates of Spodoptera exigua Nuclear Polyhedrosis Virus. Biocontrol Sci. 

Technol. 2:145-157.

Gelernter, W.D., and B.A. Federici. 1986. Isolation, Identification and Determination of Virulence 

of a Nuclear Polyhedrosis Virus from the Beet Armyworm Spodoptera exigua (Lepidoptera: 

Noctuidae). Environ. Entomol. 15:240-245.
9

Hamm, J.J., and L.D. Chandler. 1996. Effects of a Nuclear Polyhedrosis Virus and a Fluorescent 

Brightener on Colonies of Spodoptera exigua (Hubner). J. Entomol. Sci. 31:355-362.

Hara, K., M. Funakoshi, and T. Kawarabata. 1995. In vivo and in vitro Characterization of Several 

Isolates of Spodoptera exigua Nuclear Polyhedrosis Virus. Acta Virol. 39:215-222.

Heldens, J. G., van Strien, E. A., Feld mann, A. M., Kulcsar, P., Munoz, D., Leisy, D. J., Zuidema, D.,

Goldbach, R. W., and J. M. Vlak. 1996. Spodoptera exigua multicapsid nucleopolyhedrovirus deletion

mutants generated in cell culture lack virulence in vivo. J Gen Virol. 77: 3127 -3134.

Ignoffo, C.M., 1975. Evaluation of in vivo specificity of insect viruses. In: Baculovirus for 

Insect Pest Control: Safety Considerations (MD Summers, R. Engler, LA Falcon, PV Vail, 

Eds) ASM, Washington.

Kolodny-Hirsch, D.M., D.L. Warkentin, B. Alvarado-Rodriguez, et al. 1993. Spodoptera exigua

Nuclear Polyhedrosis Virus as a Candidate Viral Insecticide for the Beet Armyworm (Lepidoptera: 

Noctuidae). J. Econ. Entomol. 86:314-321.

Jehle, J.A., M. Lange, H. Wang, et al. 2006. Molecular Identification and Phylogenetic Analysis of 

Baculoviruses of Lepidoptera. Virology 346:180-196.

Munoz, D., Castillejo, J. I., and P. Caballero. 1998: Two naturally occurring deletion mutants are

parasitic geno-types in a Spodoptera exigua nucleopolyhedrovirus strain. Appl. Environ. Microbial. 64:

4372-4377.

Murillo, R., Munoz, D., Lipa,, J., and P. Caballero. 2001. Biochemical characterization of three

nucleopolyhedrovirus isolates of Spodoptera exigua and Mamestra brassicae. J. Appl. Entomol. 125:

267-270

OECD (2002). Consensus Document on Information used in the Assessment of Environmental

Applications involving Baculovirus (Monograph). Series on Harmonization of Regulatory Oversight in

Biotechnology, No. 20, OECD Environment, Health and Safety Publications, 90 S.