1 **UNITED STATES ENVIRONMENTAL PROTECTION AGENCY**

WASHINGTON, D.C. 20460

MEMORANDUM

SUBJECT: Review of Product Chemistry, Manufacturing Process, and A cute 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 t he registrant provides official documentation

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

man de s 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

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

BACKGROUND:

Spexit ® ( EPA File Symbol 69553-U ) is a n 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 10 12 viral

occlusion bodies/L. The end use product contains a minimum of 3.8 x 10 12 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 10 12 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 sam e 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 m ammals.

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

RECOMMENDATION : ACCEPTABLE on condition that t he 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

strai n 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 enzyme s 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.

Deficienc i es : 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 me t

the specification of a minimum of 3.75 x 10 12 viral occlusion bodies /L

. Deficienc i es : None.

ENFORCEMENT ANALYTICAL METHOD : (MRID # 49409502)

Deficiencies : None.5 PHYSICAL AND CHEM ICAL 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 1 2 months was 5 °C within a variance of ±

2. 5 °C. Short temperature increases after open i ng the door of the climatic chamber for

sampling were disregarded.

Sta bility of the Original Test Item Co n tainer

No change to the container shape or size was observed over the entire test period of 1 2

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 1 2 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/ 201 3

Month 3 0 3 /01/2014

Month 6 0 6 /04/2014

Month 9 09/07/2014

Month 1 2

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 # 495 78802 )

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

( Spodoptera exigua MNPV ), w as administered to mice once by intraperitoneal (IP) injec tion. 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 10 6 Viral

Occlusi on Bodies (OB) per animal. The control gro up was sham dosed with an equivalent volume

of sterile saline. Observations for mortality and signs of p harmacologic 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 termin ation. 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 non

toxic to mice when administered by intrap eritoneal injection at a dose of l 0 6 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 Day 7 NOA

21

- F Day 7 NOA

2 4

- F Day 7 NOA

Group 2

- Spexit ( Spodoptera exigua MNPV ) Batch# 003

3

- F Day 7 NOA

1 2

- F Day 7 NOA

13

- F Day 7 NOA

26

- F Day 7 NOA

30

- F Day 7 NOA

Grou p 3

- Spexit ( Spodoptera exigua MNPV ) Batch# 005

2

- F Day 7 NOA

8

- F Day 7 NOA

1 8

- F Day 7 NOA

20

- F Day 7 NOA

2 2

- 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

2 7

- F Day 7 NOA

2 9

- F Day 7 NOA

Group 5

- Spexit ( Spodoptera exigua MNPV ) B atch# 007

4

- F Day 7 NOA

5

- F Day 7 NOA

10

- F Day 7 NOA

28

- F Day 7 NOA

F

- Fe m a l e ; N OA

- N o Ob se rv a bl e A bnorm a l i ti es8 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 M NPV was the first commercial baculovirus insecticide and is one of the

most ext ensively tested entomo pathogenic 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, 19 75)

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 addi tional toxicology data which support the negative response of viral replication in

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

reques ting a cell culture study."

6. Although baculovirus uptake has been obser ved, 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 n o evidence for baculovirus induced cytogenic, carcinogenic, mutagenic or teratogenic

effects ha s ever been found ( OECD, 2002).

CONCLUSION: ACCEPTABLE



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