Document ID: EPA-HQ-OPP-2014-0374-0067
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2016-03-10T05:00Z

Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for Potassium Salts of Hops Beta acids (K-HBAs)

                Docket ID Number: EPA-HQ-OPP-2014-0374 (NOF), 
                              Date: July 15, 2015
                                       
Section 408(c)(2)(A)(i) of FFDCA allows the U.S. Environmental Protection Agency (EPA or the Agency) to establish an exemption from the requirement for a tolerance (the legal limit for a pesticide chemical residue in or on a food) only if the EPA determines that the exemption is "safe." Section 408(c)(2)(A)(ii) of FFDCA defines "safe" to mean that "there is a reasonable certainty that no harm will result from aggregate exposure to the pesticide chemical residue, including all anticipated dietary exposures and all other exposures for which there is reliable information." This includes exposure through drinking water and in residential settings but does not include occupational exposure. Pursuant to FFDCA section 408(c)(2)(B), in establishing or maintaining in effect an exemption from the requirement of a tolerance, the EPA must take into account the factors set forth in FFDCA section 408(b)(2)(C), which require the EPA to give special consideration to exposure of infants and children to the pesticide chemical residue in establishing a tolerance exemption, and to "ensure that there is a reasonable certainty that no harm will result to infants and children from aggregate exposure to the pesticide chemical residue...." Additionally, FFDCA section 408(b)(2)(D) requires that the EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."
The EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide residues. First, the EPA determines the toxicity of pesticides. Second, the EPA examines exposure to the pesticide through food, drinking water, and through other exposures that occur as a result of pesticide use in residential settings.
I.  Summary of Petitioned-for Tolerance Exemption
In the Federal Register of September 05, 2014 (79 FR 53012) (FRL-9914-98), EPA issued a notice pursuant to FFDCA section 408(d)(3), 21 U.S.C. 346a(d)(3), announcing the filing of a pesticide tolerance petition (PP 3E8217) by Interregional Research Project Number 4, on behalf of BetaTec Hop Products, Inc.  (the Petitioner), 500 College Road East, Suite 201W, Princeton, New Jersey, 08540. The petition requested that 40 CFR part 180 be amended by establishing an exemption from the requirement of a tolerance for residues of potassium salts of hops beta acids (K-HBAs) in or on honey and honeycomb. The notice referenced a summary of the petition prepared by the Petitioner, which is available in Docket ID Number EPA-HQ-OPP-2014-0374 via http://www.regulations.gov.
II. Toxicological Profile
Consistent with section 408(b)(2)(D) of FFDCA, the EPA reviewed the available scientific data and other relevant information on K-HBAs, and considered its validity, completeness, and reliability, as well as the relationship of this information to human risk. The EPA also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children.
A.  Overview of Potassium Salts of Hops Beta Acids	

K-HBAs are derived from the resin components of the cones of female hop plants, Humulus lupulus.  The three major components of hop beta acids (HBA) are lupulone (30-55% with an isopropyl side chain), colupulone (20-55% with an isobutyl side chain); and adlupulone (5-10% with a secbutyl side chain). K-HBAs are classified as a biochemical pesticide because they are naturally occurring, have a non-toxic mode of action against the target pest(s), and are structurally similar, and/or functionally identical to a naturally-occurring active ingredient. K-HBAs are intended to be applied inside bee hives to the brood supers via plastic strips. The strips will be treated with a 16% solution of K-HBAs (two strips per hive; 1.92 g/strip).  
		
There is a long history of safe use of HBA. Humans are exposed to HBA from its use as a preservative on meats (estimated range 4.4 mg/kg of cooked meat - 5.5 mg/kg of frankfurter) (Ref 10,) and the presence of HBAs in the beer brewing process (Ref 14).  HBAs are considered to be Generally Recognized As Safe (GRAS) by FDA (Ref 10). In addition, HBAs have been shown to inhibit inflammations in mice and exhibit anticarcinogenic activity without any known side effects (Ref 2).

EPA granted a Section 18 Emergency Exemption in 2011 for K-HBA in over 20 States to control Varroa mites (Varroa destructor) in honeybee hives. The Section 18 was renewed in 2012 and 2013 in up to 37 States to control Varroa mites (Ref 3).

Based on the data submitted in support of this petition (summarized in Unit II. B., below) and the comprehensive risk assessment conducted by the Agency (References 1-3, below), EPA concludes that there is a reasonable certainty of no harm from aggregate exposures to potassium salts of hops beta acids, including the consumption of food treated with this active ingredient in accordance with label directions and good agricultural practices. EPA has made this determination because available toxicology data indicate that the active ingredient is of low acute toxicity and is not a developmental toxicant, a mutagen, or toxic via repeat oral exposure.

B.  Biochemical Pesticide Toxicology Data Requirements

All applicable mammalian toxicology data requirements supporting the request for an exemption from the requirement of a tolerance for residues of K-HBAs in or on honey and honeycomb have been fulfilled with data submitted by the petitioner. The following is a summary of EPA's review of the toxicity profile of this biochemical:
  
Acute Toxicity: The applicant did not submit any Guideline studies in support of the Tier I toxicity data requirements for the TGAI/EP. In lieu of Guideline studies, the applicant submitted rationales submitted by data and information from the technical literature. 

   1.   In lieu of an acute oral toxicity study, the registrant submitted a rationale supported by toxicity and exposure information obtained from the technical literature in fulfillment of the acute oral toxicity data requirement for the TGAI/EP. A single intragastric (oral gavage) administration of hops beta acids (as lupulon) to mice and rats resulted in LD50s of 1500 mg/kg and 1800 mg/kg, respectively. These data classify HBA in Toxicity Category III for acute oral toxicity (Ref 2).

   2.   In lieu of an acute dermal toxicity study, the applicant submitted a rationale supported by toxicity and exposure information (Ref 2) obtained from the technical literature in fulfillment of the acute dermal toxicity data requirement for the TGAI. Much of the information was not useable in that it did not specifically address the acute dermal toxicity route of exposure. The submitted information (Ref 2 and Ref 8) did not specifically address the acute dermal toxicity route of exposure. In addition, humans were used as the test subjects and the study was not submitted to the Human Studies Review Board (HSRB) for a review of its suitability for use in supporting a pesticide registration.  In the absence of this information, the Biochemical Pesticides Branch (BPB) reviewer notes that acute dermal exposure to Hops Beta Acids is highly unlikely and would only occur to the beekeeper/applicator, as the end use product is for Commercial applicators only.  There are no residential uses and therefore, based on the method of application (manually applied via plastic strips inside beehives), non-occupational exposure to Hops Beta Acids will be minimal. Commercial/Occupational exposure is mitigated by protective clothing normally worn by beekeepers, including gloves, protective suits, and a hat with a face mask or netting. Precautionary statements on the label must require the use of gloves and eye protection for beekeepers/applicators.  

   3.   In lieu of an acute inhalation study, the registrant did not generate or submit Guideline studies for either the TGAI or the EP but addressed the data requirement with information from the open literature.  Inhalation exposure to the active ingredient is highly unlikely based on the non-volatile nature of the active ingredient and method of application (manual application of treated plastic strips into beehives.  Inhalation exposure to the active ingredient will be minimal. The applicant also provided the following exposure rationale: "HopGuard (the end-use product containing 16% Hops Beta Acids), is non-volatile, viscous, is not sprayed, and there is no dust associated with the strips so inhalation exposure is highly unlikely."  Based on these data, K-HBAs are classified as Toxicity Category IV for acute inhalation toxicity.

   4.   A guideline primary eye irritation study was conducted using potassium salts of Hops Beta Acids (the TGAI) on rabbits.  The normal pH of the test substance is pH 11 and was adjusted to pH 9 prior to use.  Each of three test rabbits was administered a 0.1 mL of the test substance to the conjunctival sac of the right eye; the left eyes were used as the controls.  Test and control eyes were observed for signs of irritation for 21 days post treatment.  Corneal opacity, iritis, and conjunctivitis were observed a 1-hour post treatment.  By Day 21 post treatment, none of the ocular responses had completely resolved in any of the test eyes. Based on these data, K-HBAs are classified as Corrosive, Toxicity Category I, for primary eye irritation.  

   5.  In lieu of a primary dermal irritation study, the applicant submitted the following rationale supported by toxicity and exposure information (Ref 2) obtained from the technical literature in fulfillment of the primary dermal irritation data requirement for the TGAI/EP: "Contact with growing hop plants is known to cause an allergic contact dermatitis and is attributed to the myrcene oil in fresh hop oil. Growing hop plants can also cause a mechanical dermatosis attributed to the rough climbing hairs on the stem and secretions of the yellow glandular hairs on hops.  It is hypothesized that the hop maturing process may turn certain hop proteins into allergens, in rare cases leading to a diagnosis of immunological contact urticaria.  Hops Beta Acids were not implicated in this process (Ref 2). Based on the rationale (Ref 2) submitted above, and the mitigation of exposure with appropriate PPE, it is recommended that Toxicity Category I PPE precautionary language for acute dermal toxicity be placed on the label. More importantly, oils, rough climbing hairs and secretions from the yellow glandular hairs of fresh hops are not present in the final, extracted hops beta acids active ingredient.  
   
   6.  In response to the hypothesis described above, EPA believes that any potential proteins that may hypothetically turn into allergens by the hops plant maturing process would be removed during the hops beta acids extraction process.  Furthermore, hops beta acids do not contain protein and even in the unlikely event that proteins were present in the hops beta acids as a result of the extraction process, the proteins would likely be denatured and therefore would not pose the threat of developing into a potential allergen.   

   7.  In lieu of a dermal sensitization study, the applicant submitted a rationale supported by toxicity and exposure information (Ref 2) obtained from the technical literature in fulfillment of the dermal sensitization data requirement for the TGAI/EP but addressed the data requirement with information from the open literature. Much of the information was not useable in that it did not specifically address primary dermal sensitization.  However, the applicant did provide the following exposure rationale: "Contact with growing hop plants is known to cause an allergic contact dermatitis and is attributed to the myrcene oil in fresh hop oil. Growing hop plants can also cause a mechanical dermatosis attributed to the rough climbing hairs on the stem and secretions of the yellow glandular hairs on hops.  It is hypothesized that the hop maturing process may turn certain hop proteins into allergens, in rare cases leading to a diagnosis of immunological contact urticaria.  Hops Beta Acids were not implicated in this process (Ref 2). Based on the rationale (Ref 2) and the mitigation of exposure with appropriate PPE.  It is recommended that Toxicity Category I PPE precautionary language for acute dermal toxicity be placed on the label, based on the results of the Primary Dermal Irritation study. Commercial/Occupational exposure is mitigated by protective clothing normally worn by beekeepers, including gloves, protective suits, and a hat with a face mask or netting.  Precautionary statements on the label must require the use of gloves and eye protection for beekeepers/applicators.  More importantly, oils, rough climbing hairs and secretions from the yellow glandular hairs of fresh hops are not present in the final, extracted hops beta acids active ingredient.  In response to the hypothesis described above, EPA believes that any potential proteins that may hypothetically turn into allergens by the hops plant maturing process would be removed during the hops beta acids extraction process.  Furthermore, hops beta acids do not contain protein and even in the unlikely event that proteins were present in the hops beta acids as a result of the extraction process, the proteins would likely be denatured and therefore would not pose the threat of developing into a potential allergen.    
  
Mutagenicity:  Mutagenicity studies and information were not submitted by the applicant.  However, the Agency does not consider mutagenicity studies or information to be required. HBAs are not structurally-related to known mutagens, nor are they in a chemical class known to contain a known mutagen. Significant exposure (occupational and non-occupational) to female humans is highly unlikely, based on the method of product application (manual insertion of HBA-treated strips into beehives), the requirement for appropriate PPE (gloves and eye protection worn by workers/applicators), 
low application rate (two strips per hive; 1.92 g/strip), and appropriate PPE (gloves and eye protection) worn by workers/applicators.  Moreover, residues of HBA have been shown not to exceed 0.44 ppm in honey (observed in only one sample in three field studies between 2010 and 2012). When fed to rats in drinking water at up to 50 ppm daily for 7 months, HBAs have been shown to have potent anticarcinogenic activity against experimentally-induced colon cancers with no observed toxic effects (Ref 13). This subchronic exposure in drinking water (50 ppm) is approximately 114X greater than the residues detected in one sample of honey (0.44 ppm).  Mutagenicity resulting from exposure, if any, to HBAs is considered by the Agency to be highly unlikely. 

Subchronic Toxicity: In fulfilling the 90-day oral data requirement, the applicant did not submit Guideline studies for the TGAI. Repeated, subchronic exposure to the active ingredient is highly unlikely based on the method of application (manual application of treated plastic strips into beehives).  Dietary exposure also, is highly unlikely, based on the lack of residues in edible honey (see residue studies above).  In three different studies conducted over three years, only one honey sample from one honey super was shown to have residues of hops beta acids (0.44 ppm), and the residue level was only slightly above the analytical limits of detection (0.41 ppm) and well below the Limit of Quantitation (1.2 ppm).  Additional information provided by the registrant (Ref 2; discussed in paragraphs 1-4 below), supports the rationale that HBAs have minimal subchronic toxicity:

   1. When fed to rats in drinking water at up to 50 ppm daily for 7 months, HBAs have been shown to have potent anticarcinogenic activity against experimentally-induced colon cancers with no observed toxic effects (Ref 13). This subchronic exposure in drinking water (50 ppm) is approximately 114X greater than the residues detected in one sample of honey (0.44 ppm).

   2. Hops beta acids are approved by the USDA/FSIS for use on ready to eat meat products at up to 5.5 mg/kg (Ref 1, Ref 2 and Ref 3) to control pathogens, and has been approved for use on control Listeria moncytogenes on frankfurters at up to 2.5 mg/pound on cooked meat and poultry products (Ref 1, Ref 2. and Ref 3).

   3. In a review of information used to support FDA GRAS status, it was stated that there is a long history of dietary exposure to HBAs [comprised primarily of lupulone (C26H38O4), adlupulone (C25H37O4), and colupulone (C28H38O4)], via beer consumption (FDA, 2001). The same notice provided estimates of dietary intake, via the use of hops beta acids as antimicrobial agents in cooked ready-to-eat meats and on frankfurters, to be up to 0.27 mg per person per day. A review of the literature in support of the FDA GRAS status demonstrated that no estrogenic activity was observed for polarity extracts of hops beta acids (FDA, 2001).  

   4. The active ingredient occurs naturally in the lupulin glands of the flowers (cones) of hops (Humulus lupulus and contain up to 10% hops beta acids (Ref 14). Hops are used extensively in the brewing of beer and have been used to inhibit acute localized inflammations in mice at concentrations of up to 0.4 ppm (intravenous injection) with no side effects (Ref 6). 

In lieu of a Guideline study, the applicant submitted a rationale supported by toxicity and exposure information obtained from the technical literature (Ref 2) in fulfillment of the 90-day dermal toxicity data requirement for the TGAI/EP: "Contact with growing hop plants is known to cause an allergic contact dermatitis and is attributed to the myrcene oil in fresh hop oil. Growing hop plants can also cause a mechanical dermatosis attributed to the rough climbing hairs on the stem and secretions of the yellow glandular hairs on hops.  It is hypothesized that the hop maturing process may turn certain hop proteins into allergens, in rare cases leading to a diagnosis of immunological contact urticaria.  Hops Beta Acids were not implicated in this process (Ref 2). HBAs is highly unlikely based on the method of application (manually inserted into beehives), low application rate, lack of appreciable residues in honey, and appropriate PPE (gloves and eye protection) worn by beekeepers/applicators.  

The applicant did not submit Guideline studies for the 90-day inhalation data requirement for either the TGAI. However, the Agency does not consider 90-day inhalation studies or information to be required.  Repeated, subchronic inhalation exposure to the active ingredient is highly unlikely based on the non-volatile nature of the active ingredient and method of application (manual application of treated plastic strips into beehives. Non-occupational exposure to the active ingredient does not occur.  The applicant also provided the following exposure rationale: "HopGuard (the end-use product containing 16% Hops Beta Acids), is non-volatile, viscous, is not sprayed, and there is no dust associated with the strips so inhalation exposure is highly unlikely."  

Developmental Toxicity: The applicant did not submit Guideline studies for the TGAI. Based on 40 CFR part 158.2050, if the use of the product under widespread and commonly recognized practice will not result in significant exposure to female humans or susceptible populations for the following reasons (discussed in detail under mutagenicity): Occupational and non-occupational exposure is expected to be minimal due to the method of application and the lack of appreciable residues in honey. Hops Beta Acids are applied inside the hive to the brood supers, on plastic strips treated with a 16% solution of Hops Beta Acids (two strips per hive; 1.92 g/strip). Residues have been shown not to exceed 0.44 ppm in honey (see residues studies above), well below any known acute (1500 mg/kg; Ref 5) or subchronic (50 ppm in drinking water (; Ref 13) endpoints for Hops Beta Acids.  No additional data are required at this time.  Developmental toxicity resulting from exposure, if any, to Hops Beta Acids is considered by the Agency to be highly unlikely.

III.  Aggregate Exposure

In examining aggregate exposure, FFDCA section 408 directs the EPA to consider available information concerning exposures from the pesticide residue in food and all other non-occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses).

Food Exposure: Dietary exposure is highly unlikely based on the lack of residues in edible honey. In three different studies conducted over three years, only one honey sample from one honey super was shown to have residues of HBAs (0.44 ppm), and the residue level was only slightly above the analytical limits of detection (0.41 ppm) and well below the Limit of Quantitation (1.2 ppm). The acute oral toxicity LD50 = 1500 mg/kg (Chin and Anderson, 1950), a level that is approximately 3409X the level found in one sample of honey. According to the National Honey Board (NHB, 2014), annual U.S. per capita honey consumption was approximately 1.3 lbs (approximately 0.6 kg or 600 mg) in 2013. Assuming a 70 kg standard human and the lower acute oral toxicity endpoint (LD50 = 1500 mg/kg), and a honey residue level of 0.44 mg/kg, a human would need to consume [(1500 mg/kg x 70 kg)/0.44 mg/kg) = 2.39 x 10[6] kg] (approximately 5 lbs) of honey in one serving to reach the acute toxicity endpoint of 1500 mg/kg.  The 5 lb level of honey consumption is approximately 3.9X greater than the annual U.S. per capita consumption of honey in 2013. When fed to rats in drinking water at up to 50 ppm daily for 7 months, HBAs have been shown to have potent anticarcinogenic activity against experimentally-induced colon cancers with no observed toxic effects (Lamy et. al., 2007). This subchronic exposure in drinking water (50 ppm) is approximately 114X greater than the residues detected in one sample of honey (0.44 ppm).  

HBAs are approved by the USDA/FSIS for use on ready to eat meat products at up to 5.5 mg/kg to control pathogens, and have been approved for use on control Listeria moncytogenes on frankfurters at up to 2.5 mg/pound on cooked meat and poultry products (Ref 2). 

There is a long history of oral/dietary exposure of humans to K-HBAs via its use as a preservative on meats and its presence in beer making, without any reported toxic effects.  HBAs are considered to GRAS by Ref 10). HBAs have been shown to inhibit inflammations in mice and exhibit anticarcinogenic activity without any known side effects (Ref 2). 

Drinking Water Exposure:  K-HBAs are formulated into a viscous liquid and coated on fiber strips which are placed inside the beehive. The product is not sprayed or applied in any way that it would be expected to contact any source of drinking water. When fed to rats in drinking water at up to 50 ppm daily for 7 months, HBAs have been shown to have potent anticarcinogenic activity against experimentally-induced colon cancers with no observed toxic effects (Ref 2).   

Other Non-occupational Exposure: Non-occupational exposure to K-HBAs is not expected because this biochemical is formulated into a viscous liquid and coated on fiber strips which are placed inside the beehive. The product is not sprayed or applied in any way that it would be expected to contact any beekeepers directly or through any type of drift. Exposure to applicators us also limited and mitigated through personal protective equipment when handling bees. There is no anticipated exposure to other agricultural workers because the strip would be on the inside of the hive. However, minimal to no risk is expected for the general population, including infants and children, due to the low toxicity of this chemical.

IV.  Cumulative Effects from Substances with a Common Mechanism of Toxicity

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify, or revoke a tolerance, the EPA consider "available information concerning the cumulative effects of [a particular pesticide's] . . . residues and other substances that have a common mechanism of toxicity."

The EPA has not found K-HBAs to share a common mechanism of toxicity with any other substances, and K-HBAs do not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, the EPA has assumed that K-HBAs do not have a common mechanism of toxicity with other substances. Following from this, the EPA concludes that there are no cumulative effects associated with K-HBAs that need to be considered. For information regarding the EPA's efforts to determine chemicals that have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the EPA's website at http://www.epa.gov/pesticides/cumulative. 

V.  Determination of Safety for the United States Population, Infants and Children
	
FFDCA section 408(b)(2)(C) provides that, in considering the establishment of a tolerance or tolerance exemption for a pesticide chemical residue, the EPA shall assess the available information about consumption patterns among infants and children, special susceptibility of infants and children to pesticide chemical residues, and the cumulative effects on infants and children of the residues and other substances with a common mechanism of toxicity. In addition, FFDCA section 408(b)(2)(C) provides that the EPA shall apply an additional tenfold (10X) margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the database on toxicity and exposure, unless the EPA determines that a different margin of safety will be safe for infants and children. This additional margin of safety is commonly referred to as the Food Quality Protection Act Safety Factor. In applying this provision, the EPA either retains the default value of 10X, or uses a different additional or no safety factor when reliable data are available to support a different additional or no safety factor. 

Because there are no threshold effects associated with this biochemical, an additional margin of safety for infants and children is not necessary.  

EPA has determined that there are no foreseeable dietary risks to the U.S. population, including infants and children, from the use of K-HBAs as a pesticide (miticide) in or on honey and honeycomb when label instructions and good agricultural practices are followed. The available data and information indicate that the chemical is of minimal toxicity and not a developmental toxicant. Therefore, EPA concludes that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to the residues of K-HBAs when it is used as labeled and in accordance with good agricultural practices. Such exposure includes all anticipated dietary exposures and all other exposures for which there is reliable information. EPA has arrived at this conclusion because the data and information available on K-HBAs do not demonstrate significant toxic potential to mammals, including infants and children. 

VI.  Conclusions
EPA concludes that there is a reasonable certainty that no harm will result to the U.S. population, including infants and children, from aggregate exposure to residues of K-HBAs. Therefore, an exemption is established for residues of the biochemical pesticide potassium salts of hops beta acids when used in or on honey and honeycomb in accordance with label directions and good agricultural practices.

VII.  References

   1. EPA (2015) Science Review of a Response to Product Chemistry Deficiencies in Support of the Registration of HopGuard TGAI, HopGuard Liquid, and HopGuard. US EPA. U.S. Environmental Protection. May 21, 2015

   2. EPA (2014) Science Review in Support of the Registration of HopGuard TGAI, HopGuard Liquid, and HopGuard, Hops Beta Acids Resin, a New Active Ingredient. Review of Residue Studies, Tier I Toxicity and Tier I Non-Target Organism Studies and Rationales.  US EPA. U.S. Environmental Protection. December 04, 2014. 

   3. EPA (2014) Science Review in Support of the Registration of HopGuard TGAI, HopGuard Liquid, and HopGuard, Hops Beta Acids Resin, a New Active Ingredient.  Review of a TGAI, an MP, an EP, and a Tolerance Exemption for the New Active Ingredient. US EPA. U.S. Environmental Protection. September 02, 2014. 

   4. Breed, M. D., E. Guzman-Nova, and G. J. hunt.  2004.  Defensive behavior of honey bees:  organization, genetics, and comparisons with other bees.  Annual Review of Entomology 49:  271-296.  

   5. Chin, Y-C., and H. H. Anderson.  1950.  Toxicology and pharmacology of lupulon.  Arch. Int. Pharmacodyn. 82(1):  1-15.  

   6. Cleemput, M. V., A. Heyerick, C. Libert, K. Swerts, J. Philippe, D. D. Keukeleire, G. Haegeman, and K. D. Bosscher.  2009.  Hop bitter acids efficiently block inflammation independent of GR-alpha, PPAR-alpha, or PPAR-gamma.  Molecular Nutrition and Food Research 53:  1143-1155.  

   7. Dong, N. T. K and R. B. Ogle.  2003.  Effect of brewery waste replacement of concentrate on the performance of local and crossbred growing Muscovy ducks.  Asian-Australian Journal of Animal Science 16(10):  1510-1517.  

   8. Dumas, E. R., A. E. Mchaud, C. B. Bergeron, J. L. LaFrance, S. Mortillo, and S. Gafner.  2009.  Deodorant effects of a supercritical hops extract:  antibacterial activity against Corynebacterium xerosis and Staphylococcus epidermidis and efficacy testing of a hops/zinc ricinoleate stick in humans through the sensory evaluation of axillary deodorancy.  Journal of Cosmetic Dermatology 8:  197-204.  

   9. Estrada, J. L., F. Gorzalo, C. Cecchini, and E. Casqueta.  2002.  Contact urticaria from hops (Humulus lupus) in a patient with previous urticaria-angiodema from peanut, chestnut, and banana.  Contact Dermatitis 48:  127.  

   10. FDA, 2001.  Agency response letter GRAS Notice No. GRN 000063.  CFSAN/Office of Premarket Approval. March 15, 2001.  

   11. Huszcza, E., A. Bartmanska, M. Aniol, W. Maczka, A. Zolnierczyk, and C. Wawrzenczyk.  2008.  Degradation of hop bitter acid by fungi.  Waste management 28(8):  1406-1410.

   12. Krings, A.  2002.  Keys to the vines of Carolina wetlands.  Vulpia 1:  23-40.

   13. Lamy, V., S. Roussl, M. Chaabi, F. Gosse', N. Schall, A. Lobstein, and F. Raul.  2007.  Carcinogenesis 28:  1575-1581.

   14. Moir, M. M.  2000.  Journal of the American Society of Brewery Chemists 58(4):  131-146.