UNITED STATES DEPARTMENT OF AGRICULTURE
ANIMAL AND PLANT HEALTH INSPECTION SERVICE
VETERINARY SERVICES

THE CEN TER FOR VETERINARY B IOLOGICS



# Environmental Assessment

For Field Testing of a
DNA Immunostimulant

June 201 3

Prepared by: United States Department of Agriculture Animal and Plant Health Inspection Service Veterinary Services The Center for Veterinary Biologics

_________________________________________________________________________________________________

__________________ Environmental Assessment Field Trial of an Experimental DNA ImmunostimulantEnvironmental Assessment

For Field Testing
DNA Immunostimulant

I

. Proposed Action

APHIS is considering granting authorization to ship an unlicensed DNA
Immunostimulant for field testing in chickens and cattle

. Bayer HealthCare LLC, Animal
Health Division , Shawnee Mission

, KS , has requested authorization to conduct a field safety trial in the United States (US) under production conditions that are typ ical of the two species of interest. The product is proposed for use in 18

- day old embryonated eggs as an aid in the reduction of mortality associated with Escherichia coli in embryonated eggs and newborn chicks. It is also proposed for intramuscular use in cattle 4 months of age or older as an aid in the reduction of lung lesions associated with Bovine Respiratory
disease due to Mannheimia haemolytica. The safety of the product will be evaluated in animals participating in the studies.

Under the prov isions of the Virus

- Serum

- Toxin Act of 1913, as amended in 1985, the USDA must ensure that veterinary biologics are pure, safe, potent, and efficacious and not worthless, contaminated, dangerous, or h armful. Accordingly, APHIS has conducted

an assessment of the safety risks to animals, public health, and the environment and
concluded that the risks are low. A copy of the firm’s risk analysis with confidential
business information re dact ed is available online and by request, as indicated in the Federal Register Notice announcing the proposed field testing

. The APHIS evaluation of

r isk include s th e firm’s document

, review of other studies and tests conducted by the

firm , the confirmat ory testing completed by APHIS personnel on the Master Seed, re sults of literary and regulatory review, and comparison with similar p reviously tested or licensed products.

II. Background

Immunostimulants are endogenous or synthetic substances that activate the immune system by inducing or increasing activity of any of its components. Immunostimulants can be either specific or non

- specific, i.e. acting regardless of antigenic specificity to improve the immune response to various antigens. In some circumstances it can be
be nefi cial to use an immunostimulant to incre ase protection against disease agents and to treat immunosuppressive conditions (Kahn, 2008)

.

The experimental immunostimulant to be used in the proposed field tests is a non

- coding plasmid complex which is r eplication

- incompetent in eukaryotic cells

. The proposed field safety test will be conducted in three different geographical locations for each species and the product will be used according to instructions on the product circular. The potential for escape and dispe rsal of the experimental product from the proposedrelease sites is low. The personnel to conduct the study are experienced in poultry and
bovine health management.

Plasmid DNA ha s been used previously to express foreign genes for vaccine development. Examples of USDA

- licensed plasmid DNA vaccine s include West Nile
Virus Vaccine licensed in 2005, and Canine M elanoma Vaccine licensed in 2007

. No safety issues have arisen from the use of these plasmid DNA vaccines

.

III. Need for the Proposed Action

A replication

- incompetent DNA Immu nostimulant can potentially be both safe and efficacious, inducing a broad immune res ponse in the host animal

. The purity and identity of the Master Seed were confirmed by the APHIS Cent er for Veterinary Biologics

. The plasmid DNA is produced and extract ed; all viable bacteria are removed during the extraction process. It is expected that the data from these monitored field trials
will confirm the safety of th e DNA immunostimulant for use in chickens and cattle
within the environment of the United States

.

IV. Areas of Concerns

The three areas of concern to APHIS are: 1) animal safety, 2) public health, and 3) environmental safety. APHIS has assess ed potential risks associated with the proposal to field test this experimental immunostimulant . The safety characteristics of this product
have been careful ly evaluated. The conclusions derived from the risk a s s essment for
each of the areas of concern are summarized below.

A. Animal Safety

The risk to animals is low.

 The bacterial host producing the plasmid DNA Immunostimulant is highly
attenuated and can be handled safely under N ational I nstitutes of H ealth /C enter
for D isease C ontrol and Preventions Biosafety Level (BSL) 1 conditions (the least
restrictive BSL )

. The bacterium will not be release d outside of the production plant.

 The firm conducted numerous efficacy and overdose studies in chickens and
cattle and a pre

- slaughter cattle withdrawal study with no s ignificant adverse

events detect ed

.

 Non

- target animal studies were performed in swine, rabbits, and non

- human primates, indicating a satisfactory safety profile.

 The extracted plasmid DNA immunostimulant is not a risk for target and non

target species since it cannot replicate in eukaryotic cells, is not infectious, and is highly purified. Should ingestion occur, DNA is readily degraded in the stomach by gastric acid and in the duodenum by pancreatic and bile secretions, without negative effect to the host (Maturin & Curtiss, 1977)

. It has been estimated that the probability of a DNA molecule to integrate into the chromosome of muscle cells after intramuscular injection is at least 3000x less than the natural mutation rate (Martin, et al.1999; Ledwith, et al. 2000).

B. Public Heal th

The risk to public health is likewise low.

 The plasmid has no virulence

- associated genes, and no known mobility or integration motifs which would allow integration into the human genome.

 Only qualified personnel, knowledgeable in the art of inoculati on and prevention of accidental exposure, will be administering the DNA immunostimulant.

 Although the safety of this specific product has not been evaluated in humans and is therefore unknown, accidental human injection is not expected to cause adverse
eff ects. A related product is being evaluated for possible use in humans. In clinical trials thus far, it has been found to be safe.

 Many doses of plasmid DNA vaccines have been administered to animals, including dogs, mice, guinea pigs, and horses (Grosen baugh, et al. 2011; Martin, et al.1999; Ledwith, et al. 2000; Manam, et al. 2000; Davis, et al. 2001 ). No significant adverse effects have been reported in the animals or the handlers

.

 Numerous DNA vaccines have entered human clinical trials (Liu & Ulmer, 2005). There has been no evidence of autoimmunity, immunological tolerance, or integration in these various trials.

C. Environmental Safety

The risks to the environment are low.

 The risks to environmental safety are low, given that the immunostimul ant is based on highly purified and well

- characterized plasmid DNA that does not replicate in inocula ted animals.

 The plasmid cannot readily transfer to other bacteria, since it lacks critical t ransfer
factors . It cannot infect bacteria without prior lab

- based chemical or physical treatment of the bacteria to generate cells competent to take up plasmids.

 The plasmid is unstable in the environment and exposure to nucleases, pH variations, oxidation, and/or ot her denaturing conditions will result in degradation
of the DNA.

The information above support s the claims m ade regarding the safety of this DNA
Immunostimulant to animals, public health, and the environment. There are no apparent
substantial issues with adverse environmental impacts concerning use of this product

.

V. Alternatives

Two alternatives were considered. T he only alternative considered other than t he preferred action alternative is not to approve the proposed field tests, the “no action” alternative. We have considered the applicants’ goals in light of the agency’s publicinterest and responsibilities and any potential environmental impact. Based upon the
results of our risk an alysis and the potential applications for this product in disease control, APHIS adopts the alternative that the proposed field tests be approved.

VI. Conclusion

Based upon the risk analysis documented in this EA, APHIS has determined that implementatio n of the proposal would not significantly affect the quality of the human environment and that the preparation of an Environmental Impact Statement is not required (Finding of No Significant Impact).

References

1. Nonspecific Immunotherapy . In: The Mer ck Veterinary Manual, Ninth Edition, 2008. C.M. Kahn (Ed), Merck & Co., Inc., Whitehouse Station, N.J. Available online: http://www/merckvetmanual.com

2. Maturin, L. & Curtiss, R. 1977. Degradation of DNA by nucleases in intestinal tract of rats. Science 196(4286):216

- 8.
3. Martin, T., Parker, S.E., Hedstrom, R., et al. 1999. Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Therapy
10:759

- 768.
4. Ledwith, B.J ., Manam, S., Troilo, P.J., et al. 2000. Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection
in mice. Intervirol 43:258

- 272.
5. Grosenbaugh, D.A., Leard, A.T., Bergman, P.J., et al. 2011. Safety and effi cacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor.

Am J Vet Res 72:1631

- 8.

6. Manam, S., Ledwith, B.J., Barnum, A.B., et al . 2000. Plasmi d DNA vaccines: tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA. Intervirol 43:273

- 81.

7. Davis, B.S., Chang, G.J., Cropp, B., et al . 2001. West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme

- linked immunosorbent assays. J Vir 75:4040

- 4047.
8. Liu, M.A. & Ulmer, J.B. 2005. Human clinical trials of plasmid DNA vaccines. Adv
Genetics 55: 25

- 40.