                                     Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for Natamycin 

                 Docket ID Number: EPA-HQ-OPP-2015-0811 (NOF)
                              Date: June 16, 2016

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.

 Summary of Petitioned-for Tolerance Exemption

In the Federal Register of April 25, 2016 (81 FR 24044) (FRL-9944-86), 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 5F8407) by Keller and Heckman, LLP, 1001 G Street, NW, Washington, DC, 20001 on behalf of DSM Food Specialties (the Petitioner) B.V., P.O. Box 1, 2600 MA Delft, The Netherlands. The petition requested that 40 CFR part 180 be amended by establishing an exemption from the requirement of a tolerance for residues of natamycin in or on  citrus, pome and stone fruit crop groups, avocado, kiwi, mango and pomegranates when used as a fungistat and used as a dip drench, flood or spray in enclosed packing house facilities. The notice referenced a summary of the petition prepared by the Petitioner, which is available in Docket ID Number EPA-HQ-OPP-2015-0811 via http://www.regulations.gov.

     Toxicological Profile

Consistent with section 408(b)(2)(D) of FFDCA, the EPA reviewed the available scientific data and other relevant information on natamycin, 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.

 Overview of Natamycin 
    
Natamycin is a naturally occurring antifungal agent produced during fermentation by the common soil microorganisms Streptomyces natalensis, Streptomyces lydicus, and Streptomyces chattanoogensis. Natamycin has a non-toxic mode of action. As an antifungal it binds to ergosterol, a sterol found in plants, fish liver oil, egg yolk, dairy products and in the cell membranes of many strains of fungi. In fungi, ergosterol helps regulate fluidity, permeability, stability, and resistance to physical stress of cell membranes similar to the function of cholesterol in animal cells. By binding to ergosterol, natamycin alters the cell membrane of the targeted fungus to the degree that the cell cannot grow, thus preventing germination of fungal spores. Natamycin is active against a broad variety of yeast and molds. However, natamycin  has no antibacterial properties and therefore is not expected to cause resistance by human pathogenic bacteria. 
Natamycin has been used as a food preservative worldwide for over 40 years and is approved as a food additive/preservative by the European Union, the World Health Organization, and by 70 individual countries including New Zealand and Australia for use as a fungistat to suppress mold on cheese, meats and sausage. In the United States, natamycin is approved by the Food and Drug Administration (FDA) as a direct food additive/preservative for the inhibition of mold and yeast on the surface of cheeses (21CFR 172.155) and as an additive to the feed and drinking water of broiler chickens to retard the growth of specific molds (21CFR 573.685). Natamycin is also FDA approved for use as a treatment to suppress fungal eye infections such as blepharitis, conjunctivitis, and keratitis (21CFR 449.40). In 2012, the EPA and Health Canada's Pesticide Management Regulatory Agency (PMRA) jointly established a tolerance exemption for natamycin when used as a fungistat to prevent the germination of fungal spores on mushrooms produced in enclosed production facilities (FR vol. 77, no. 97, p. 29543). PMRA has also registered natamycin under the active ingredient name "pimaricin" for use in laboratories for preserving milk samples (PMRA Reg. Nos. 22612 and 28530). On December 17, 2014, the EPA amended the tolerance by extending use to post-harvest pineapples when used as a fungistat in enclosed packinghouse facilities to prevent certain fungal diseases. (FR 79, no 242, p. 75065). Three products are currently registered containing natamycin for use as a biopesticide fungistat (87485-1, Natamycin TGAI; 87485-2 Natamycin L, (mushrooms), and 87485-3 Zivion P (pineapples)). 

     Biochemical Pesticide Toxicology Data Requirements

      All applicable mammalian toxicology data requirements supporting the petition to amend the existing tolerance exemption for natamycin to include use on citrus, pome, stone fruit crop groups, avocado, kiwi, mango and pomegranate as a post harvest use have been fulfilled by bridging from existing data on file that supported the tolerance exemption for natamycin on pineapples.   No significant toxicological effects were observed in the acute toxicity studies, the Tier I subchronic toxicity studies, or other information that was used to address the toxicity data requirements. Natamycin is not a contact dermal sensitizer and does not cause chromosomal aberration and is not a mutagen. Relative to Natamycin use, non-occupational exposure will be primarily dietary (consumption of treated citrus, pome, and stone fruit crop groups, avocado, kiwi, mango, and pomegranates).  The following is a summary of EPA's review of the toxicity profile of this biochemical.  

Acute Toxicity: Natamycin has a non-toxic mode of action as is categorized as follows: Acute Oral LD50 > 3,000 mg/kg (Toxicology Category III); Acute Dermal LD50 > 5,050 mg/kg (Toxicology Category IV); Acute Inhalation LC50 > 2.39 mg/L (Toxicology Category IV); Primary Eye Irritation was severely irritating but no positive effects after 24 hours (Toxicology Category III); Primary Dermal Irritation was slightly irritating (Toxicology Category IV); and natamycin is not a contact dermal sensitizer.  

The acute toxicity studies on natamycin (98.17% and 98.27% pure), confirm a low toxicity profile. The acute toxicity data show virtual nontoxicity for all routes of exposure. Therefore, it can be concluded that any dietary risks associated with this biochemical would be negligible. 

 Acute Oral Toxicity OSCPP 870.1100) Natamycin was evaluated for acute oral toxicity to albino female rats with the administration via gavage dose at a level of 2,000 mg/kg. There were no clinical signs of toxicity in survivors (one rat died, polyuria was observed). There were no effects on body weight gain in animals surviving to termination. The acute oral LD50 was estimated at greater than 2,000 mg/kg. (MRID 48105505).    

 Acute Dermal Toxicity (OCSPP 870.1200) An acute dermal toxicity test was conducted with rats to determine the potential for natamycin to produce toxicity from a single topical application. Under the conditions of this study, the single dose acute dermal LD50 of the test substance is greater than 5,050 mg/kg of body weight in male and female rats. There were no clinical signs of toxicity or signs of dermal irritation at any time throughout the study. The animals were observed for mortality, signs of gross toxicity, and behavioral changes at least once daily for 14 days. Body weights were recorded prior to application and again on Days 7 and 14 (termination). Necropsies were performed on all animals at terminal sacrifice (MRID 48105506).

 Acute Inhalation Toxicity (OCSPP 870.1300) Natamycin, was evaluated for its acute inhalation toxicity potential in albino rats where five males and five females were exposed for four hours to an aerosol generated from the test substance (dry powder) at a level of 2.39 mg/L. There was no mortality during the study. Clinical signs included activity decrease and piloerection, which were no longer evident by Day 3. Animal body weight was unaffected. The gross necropsy revealed no observable abnormalities. As indicated by the data, the acute inhalation LC50 is greater than 2.39 mg/L (MRID 48105507).

 Primary Eye Irritation (OCSPP 870.2400) An acute eye irritation study was conducted on three albino rabbits using test substance natamycin. The undiluted test substance (O.1 mL) was placed into the conjunctival sac of the right eye of each test animal. All treated eyes were washed with room temperature deionized water for one minute immediately after recording the 24-hour observation. The number of animals testing "positive" for each parameter (Table 1.0) over the number of animals tested is presented below.

          Table 1.0. Results of Primary Eye Irritation Test (870.2400) for Natamycin

                             Time After Treatment

                                     1hr.
                                     24hr.
                                     48hr.
                                     72hr.
Cornea
Opacity
                                      3/3
                                      0/3
                                      0/3
                                      0/3
Iritis
                                      3/3
                                      0/3
                                      0/3
                                      0/3
Conjunctivae
Redness
Chemosis
                                       
                                      3/3
                                      3/3
                                       
                                      0/3
                                      0/3
                                       
                                      0/3
                                      0/3
                                       
                                      0/3
                                      0/3





      There were no positive effects exhibited in any eyes at 24 hours after treatment. The test substance is rated severely irritating and assigned to Toxicity Category III (MRID 48105508).

 Primary Skin Irritation (OCSPP 870.2500) A primary dermal irritation study was conducted on three albino rabbits using test substance natamycin. There was one intact test site per animal. Each test site was treated with 500 mg of test substance and covered with a semi-permeable dressing. The test substance was maintained in contact with the skin for 4 hours. Observations for dermal irritation and defects were made at 1, 24, 48, and 72 hours after removal of the dressings. Irritation scores derived from the respective erythema and edema scores through the 72-hour observations for each animal are presented in Table 2.0. 

Table 2.0. Results of Primary Skin Irritation Test for Natamycin

Erythema
Edema
Irritation Scores

Hours after unwrap
Hours after unwrap


1
24
48
72
1
24
48
72

5082-M
0
0
0
0
0
0
0
0
0.00
5084-M
1
0
0
0
0
0
0
0
0.25
5071-F
0
0
0
0
0
0
0
0
0.00

      Based on the PII of 0.1, the test substance is rated slightly irritating. Based on the scores at the 72-hour observation, the test substance is assigned to Toxicity Category IV (MRID 48105509).

 Dermal Sensitization: (OCSPP 870.2600) A skin sensitization study was conducted on 3 groups of 5 female mice to determine if test substance Natamycin produced a sensitizing reaction. Five females were assigned to each of three groups, I-III. Naive control group animals remained untreated during the induction phase of the study. Test group animals were treated with 25 ul on the dorsum of the ear. Test animals were treated once daily for three days. The test substance produced a stimulation index of <3 in all groups of test animals and is not considered a sensitizing agent in mice (MRID 48105510).
      
Subchronic Toxicity: : In a subchronic oral toxicity study using natamycin (98.17% and 98.27% pure) as the test substance, doses of 125 and 500 mg/kg/day showed no treatment related findings. The highest concentration level, 2,000 mg/kg/day, showed reduced weight for both male and female rats (MRID 48105511). The Agency does not consider the temporary decrease in body weight or food intake observed in the 2,000 mg/kg bw/day test group to be an adverse effect, as this is likely due to the palatability of the food containing this high dose of test substance. Therefore, the Agency establishes the NOAEL (No Observed Adverse Effect Level) for this study as 2,000 mg/kg bw/day. A LOAEL (Lowest Observed Adverse Effect Level) was not identified, suggesting that the test animals could have tolerated a higher dose.

90-day Oral Toxicity (OCSPP 870.3100) Dose levels were 0, 125, 500, and 2000 ppm. Test substance was administered daily via diet for at least 90 days. One control group and three treatment groups were tested, each consisting of 10 males and 10 females Wistar rats. The following parameters were evaluated: clinical signs daily; body weight weekly and on the day preceding the first necropsy date, food consumption weekly, food scattered daily in weeks 4,7,8,10 and 12, functional observations during week 12-13, ophthalmoscopic examination at pretest and in week 13, clinical pathology, macroscopic examination and organ weight at termination. At concentrations 125 and 500 mg/kg/day there were no treatment related findings. The highest concentration level, 2000 mg/kg/day showed reduced weight for both male and female rats. Alanine aminotransferase activity increased in individual cases (males), urea and inorganic phosphate values increased for both male and female rats, potassium values increased for males, cholesterol and total protein values reduced (females) (MRID 48105511).

Developmental Toxicity: A developmental toxicity study using natamycin (98.17% and 98.27% pure) as the test substance (MRID 48105512) showed no discernable effects on growth, reproduction, teratological or mutagenic responses, or on gross and microscopic pathology, at concentration levels 0, 5, 15 and 50 mg/kg bw/day. 

Prenatal Developmental Toxicity (OCSPP 870.3700; 870.3800; 870.5450; 870.5915) Natamycin dosages of 5, 15, and 50 mg/kg per day administered by diet had no discernable effect on growth, reproduction, teratological or mutagenic response, or on gross and microscopic pathology (MRID 48105512).       

Mutagenicity:  Two mutagenicity studies, using natamycin (98.17% and 98.27% pure) as the test substance, were performed. These studies are sufficient to confirm that there are no expected dietary or non-occupational risks of mutagenicity with regard to food use of natamycin. 
Bacterial Reverse Mutation Test (OCSPP 870.5100) Natamycin was tested in the Salmonella typhimurium reverse mutation assay with four histidine-required strains (TA1535, TA1537, TA100, and TA96) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. Based on the results of this study, Natamycin is not a mutagen in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (MRID 48105513)

In Vitro Mammalian Cell Gene Mutation (OCSPP 870.5300) This test reported the effects of Natamycin on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix). Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cell with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Natamycin did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and in the presence of S9-mix, in two independently repeated experiments (MRID 48105514).

	Table 3.0. Toxicological Data for Natamycin 
                                  Guideline #
                                     Test
                           Results/Toxicity Category
                                     MRIDs
                               Study Conclusion
Acute Oral Toxicity
OCSPP 870.1100
                             LD50 >3,000 mg/kg
                                      III
48105505
Kuhn, (2008)
                                  Acceptable
Acute Dermal Toxicity
OCSPP 870.1200
                             LD50 > 5,050 mg/kg
                                      IV
48105506
Kuhn, (2008)
                                  Acceptable 
Acute Inhalation Toxicity
OCSPP 870.1300
                              LC50 > 2.39 mg/L
                                      IV
[48105507]
[Crutchfield, (2008)]
                                  Acceptable 
                                       
Primary Eye Irritation
OCSPP 870.2400
            Severely irritating no positive effects after 24 hours.
                                      III
48105508
Kuhn, (2008)
                                  Acceptable
Primary Dermal Irritation
OCSPP 870.2500
                              Slightly irritating
                                      IV
48105509
Kuhn, (2008)
                                  Acceptable 
Dermal Sensitization
OCSPP 870.2600
Not a dermal sensitizer
48105510
Kuhn, (2008)
                                  Acceptable 
                                       
90 day Oral Toxicity-Rat
OCSPP 870.3100
At 125 and 500 mg/kg/day no treatment related findings. At 2000 mg/kg/day showed reduced weight. 
48105511
Otterdijk, (2003)
                                  Acceptable
870.3700, 3800, 5450, 5915.
Prenatal Developmental Toxicity Study - Rat 
Dosages of 5, 15 and 50 mg/kg/day administered by diet had no effect on growth, reproduction, teratology or mutagenic response. 
48105512
Knickerbocker and Re, (1979)
                                  Acceptable 
870.5100
Bacterial Reverse Mutation Test
Not a mutagen
48105513
Verspeek-Rip, (2002)
                                  Acceptable
 870.5375 
In Vitro Mammalian Chromosome Aberration Tests 
No chromosomal aberrations 
48105514
Meerts, (2002)
                                       
                                  Acceptable

 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).

  Dietary Exposure: Dietary exposure to residues of natamycin are expected to be insignificant, even in the event of exposure. Furthermore, the active ingredient is of low acute toxicity and is not a developmental toxicant, a mutagen, or toxic via repeat oral exposure.


Drinking Water Exposure
Exposure of humans to natamycin in drinking water is not expected because natamycin is approved for application indoors only.


Other Non-occupational Exposure: Non-occupational exposure is not expected because natamycin is not approved for residential uses. The active ingredient is applied directly to commodities and degrades rapidly.


 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 natamycin to share a common mechanism of toxicity with any other substances, and natamycin does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, the EPA has assumed that natamycin does not have a common mechanism of toxicity with other substances. Following from this, the EPA concludes that there are no cumulative effects associated with natamycin 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 su   ch chemicals, see the EPA's website at http://www.epa.gov/pesticides/cumulative.

     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 natamycin as a pesticide (fungstat) on mushrooms in enclosed mushroom facilities, pineapples, citrus, pome, stone fruit crop groups, avocado, kiwi, mango and pomegranates when label instructions and good agricultural practices are followed. The available data and information indicate that the chemical is of low 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 natamycin 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 natamycin do not demonstrate significant toxic potential to mammals, including infants and children.

      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 natamycin. Therefore, the tolerance exemption is amended for residues of the biochemical pesticide natamycin when used on citrus, pome, stone fruit crop groups, avocado, kiwi, mango and pomegranates when used in accordance with label directions and good agricultural practices. 

References

 Crutchfield, V. (2008) Natamycin TGAI: Acute Inhalation Toxicity Study in Rats: Final Report. Project Number: 11405/07. Unpublished study prepared by Stillmeadow, Inc. 19 p. (MRID 48105507)
      
 Devine, J.; Maryann C. (2013) Magnitude of Residues of Natamycin in Post-Harvest Treated Pineapples (Decline, Processing). Project Number: 197SRUS12R/1, 029653/1, 029653. Unpublished study prepared by SynTech Research. 204p. (MRID 49292403)

  Knickerbocker, M. (1979) Natamycin TGAI: Teratological Evaluation of Pimaricin in Dutch-Belted Rabbits. Project Number: 5906. Unpublished study prepared by Food & Drug Research Laboratories, Inc. 290 p. (MRID 48105512)

 Kuhn, J. (2008) Natamycin TGAI: Acute Oral Toxicity Study (UDP) in Rats: Final Report. Project Number: 11403/07. Unpublished study prepared by Stillmeadow, Inc. 12 p (MRID 48105505)

 Kuhn, J. (2008) Natamycin TGAI: Acute Dermal Toxicity Study in Rat: Final Report. Project Number: 11404/07. Unpublished study prepared by Stillmeadow, Inc. 13 p. (MRID 448105506)

 Kuhn, J. (2008) Natamycin TGAI: Acute Eye Irritation Study in Rabbits: Final Report. Project Number: 11406/07. Unpublished study prepared by Stillmeadow, Inc. 18 p. (MRID 48105508)

 Kuhn, J. (2008) Natamycin TGAI: Acute Dermal Irritation Study in Rabbits: Final Report. Project Number: 11407/07. Unpublished study prepared by Stillmeadow, Inc. 13 p. (MRID 48105509)

 Kuhn, J. (2008) Natamycin TGAI: Skin Sensitization: Local Lymph Node Assay in Mice: Final Report. Project Number: 11408/07. Unpublished study prepared by Stillmeadow, Inc. 13 p. (MRID 48105510)

 Meerts, I. (2002) Natamycin TGAI: Evaluation of the Ability of Natamycin to Induce Chromosome Aberrations in Cultured Peripheral Human Lymphocytes. Project Number: NOTOX/PROJECT/339356. Unpublished study prepared by Notox B.V. 28 p. (MRID 48105514).
 Otterdijk, F. (2003) Natamycin TGAI: 90 Day Oral Toxicity Study with Natamycin in the Rat. Project Number: NOTOX/PROJECT/339323, 002019. Unpublished study prepared by Notox B.V. 220 p. (MRID 48105511)

 Van der Lee, J, and Jovanovich, A. 2011. 18-month Final Report for Storage Stability and Corrosion Characteristics; Natamycin L. Project No. STAB/SLD/09/008. (MRID 48544501).

 Verspeek, C. (2002) Natamycin TGAI: Evaluation of the Mutagenic Activity of Natamycin in the Salmonella typhimurium Reverse Mutation Assay and the Escherichia coli Reverse Mutation Assay (with Independent Rat). Project Number: NOTOX/PROJECT/339345. Unpublished study prepared by Notox B.V. 29 p. (MRID 48105513).
    
