UNITED STATES DEPARTMENT OF AGRICULTURE

ANIMAL AND PLANT HEALTH INSPECTION SERVICE

VETERINARY SERVICES

THE CENTER FOR VETERINARY BIOLOGICS

Environmental Assessment

For Field Testing of a

DNA Immunostimulant

June 2013

Prepared by:

United States Department of Agriculture

Animal and Plant Health Inspection Service

Veterinary Services

The Center for Veterinary Biologics

_________________________________________________________________________________________________

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Environmental Assessment

Field Trial of an Experimental DNA Immunostimulant

Environmental 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 typical 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 provisions 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 harmful. 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 redacted is available online and by request, as indicated in the

Federal Register Notice announcing the proposed field testing. The APHIS evaluation of 

risk includes the firm’s document, review of other studies and tests conducted by the 

firm, the confirmatory testing completed by APHIS personnel on the Master Seed, results 

of literary and regulatory review, and comparison with similar previously 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 

beneficial to use an immunostimulant to increase 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 replication-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 dispersal of the experimental product from the proposed 
release sites is low. The personnel to conduct the study are experienced in poultry and 

bovine health management.

Plasmid DNA has been used previously to express foreign genes for vaccine 

development. Examples of USDA-licensed plasmid DNA vaccines include West Nile 

Virus Vaccine licensed in 2005, and Canine Melanoma 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 Immunostimulant can potentially be both safe and 

efficacious, inducing a broad immune response in the host animal. The purity and 

identity of the Master Seed were confirmed by the APHIS Center for Veterinary 

Biologics. The plasmid DNA is produced and extracted; 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 the 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 assessed potential risks associated with the proposal to 

field test this experimental immunostimulant. The safety characteristics of this product 

have been carefully evaluated. The conclusions derived from the risk assessment 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 National Institutes of Health/Center 

for Disease Control and Preventions Biosafety Level (BSL) 1 conditions (the least 

restrictive BSL). The bacterium will not be released 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 significant adverse 

events detected.

 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 nontarget 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 Health

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 inoculation 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 

effects. 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 (Grosenbaugh, 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 immunostimulant is 

based on highly purified and well-characterized plasmid DNA that does not 

replicate in inoculated animals.

 The plasmid cannot readily transfer to other bacteria, since it lacks critical transfer 

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 other denaturing conditions will result in degradation 

of the DNA.

The information above supports the claims made 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. The only alternative considered other than the 

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 public 
interest and responsibilities and any potential environmental impact. Based upon the 

results of our risk analysis 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 

implementation 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 Merck 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 efficacy 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. Plasmid 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.