2 that catalyze the hydrolysis of fatty acid ester bonds in th e triacylglycerol molecule in aqueous solutions and have many, versatile uses. Triacylglycerol lipases are present in animals, plants and microorganisms and are widely used in the food, detergent, pulp and paper, and pharmaceutical industries. The proposed use of tr iacylglycerol lipase is in sanitizi ng cleaners used in food and food preparation and/or proc essing environments.

The proposed limitation of triacylgly cerol lipase in pesticide products follows the limit in commercial products set by the detergent industry, a maximum of 500 parts per million (ppm) in final commercial products. Triacylglycerol lipases are used in many laundry and cleaning products. The enzyme concentration in these products typically ranges from 0.007 % to 0.05% (500 ppm).

The acute toxicity studies of Humi cola lanuginose lipase artificially expressed in Aspergillus oryzae suggest the toxicity of tri acylglycerol lipase would be low. The test material is not ac utely toxic by the

oral or inhalation routes. It is also not a dermal irritant, ey e irritant or dermal sensitizer.

The test material was also not toxic in short-term studies. In a 2 week stu dy, Sprague-Dawley rats were dosed once daily by gavage at dose levels of 0, 0.2, 2 or 10 grams per kilogram per day (g/kg/day) and in a second 13 week study, Sprague-Daw ley rats were administered the sa me test material at dose levels of 0, 0.2, 1 and 5 g/kg/day. There were no treatment relate d clinical signs nor any to xicity seen in either study.

In a two-generation reproductive toxi city study in Sprague-Dawley rats, a triacylglycerol lipase known as SP 225 (batch PPW 1798) was administered oral ly to 5 treatment groups of rats. Each group

contained 24 males and 24 females and received diets containing 0, 0.5, 1.5 or 5.0% SP 225 by weight in the diet (equivalent to 0, 500, 1500 or 5000 milligra ms per kilogram body weight per day (mg/kg bw/day)). There were no effects of treat ment with the test material on either F 0 or F 1 fertility, general reproductive performance and systemic toxicity at exposure levels of up to 5000 mg/kg bw/day SP 225 by weight in the diet.

There is no data available on the ca rcinogenicity of triacy lglycerol lipase. Howe ver, carcinogenicity is not expected since there is no indi cation in the public literature that lipase enzymes possess carcinogenic properties. In addition, triacylglyce rol lipase was negative for mutageni city in the Ames test and also negative for clastogenicity in the in vitro human lymphocytes assay. Lipase enzymes are normal constituents of the human body. Systemic bioavailability of ingested enzy mes is expected to be very low and toxicologically insignificant as these enzymes ar e readily biodegraded in the gastrointestinal tract resulting in negligible bioavailability.

No developmental toxicity studies are available in the database. Ho wever, no systemic toxicity was observed in a two-generation reprod uctive toxicity study in rats at doses up to 5% (equivalent to 5000 mg/kg bw/day). There was also no systemic toxicity observed at doses up to 10 g/kg in a 2-week, subacute oral toxicity study in rats and at doses up 5 g/ kg/day in a 13 week oral t oxicity study in rats. In terms of hazard, there are low concerns and no residua l uncertainties regarding prenatal and/or postnatal toxicity. Based on this information, there is no concern at this time fo r increased sensitivity to infants and children to triacylglycerol lipase when used as an inert ingredient in pesticide formulations and a safety factor analysis has not been used to assess risk. For the same reason, EPA has determined that an additional safety factor is not needed to protect the safety of infants and children.

Residues of concern are not antici pated for dietary exposure (food and drinking water) or for residential (dermal and inhalation) exposure from the use of triacy lglycerol lipase for the proposed use pattern as an inert ingredient in pesticide products. EPA expects aggregate exposure to tri acylglycerol lipase to pose no appreciable dietary risk given that the data on tri acylglycerol lipase triacylglycerol show a lack of any3 systemic toxicity and a lack of any apparent re productive or developmental effects. Any potential adverse effects regarding respiratory sensitization and dermal irritation are mitigated by the proposed limit of triacylglycerol lipase in pe sticide formulations of 500 ppm.

There is limited environmental fate and ecotoxicity data available for tricylglycer ol lipase. The available

data indicate that this compound is expected to be readily biodeg radable in the environment and of low toxicity to fish and aquatic organisms. The Agency believes that triacylglycerol lipase will not cause

unreasonable adverse effects on the environment.

Based upon the review of the data supporting the petition, EPA has dete rmined an exemption from the requirement of a tolerance under 40 CFR 180.950 is not appropriate given that enzymes such as lipase are known for the potential to cause respiratory irritation and allerg enic responses. Therefore, EPA modified the exemption requested by granting an exemption under 40 CFR 180.940 and limiting the use of triacylglycerol lipase, to a maximum of 500 ppm in fina l pesticide formulations.

Taking into consideration all available information on triacylglycerol lipase, EPA has determined that
there is a reasonable certainty th at no harm to any population s ubgroup will result from aggregate
exposure to triacylglycerol lipase when used as an inert ingredie nt in pesticide formulations when
considering dietary exposure and all other non-occupational sources of pesticide exposure for which

there is reliable information. Therefore, the es tablishment of an exemp tion from tolerance under 40
CFR 180.940(a) for residues of triacylglycerol lipase when used as a component of food contact
sanitizing solutions applied to all food contact surfaces in public eating places, dairy-processing
equipment, and food-processing equipment and ut ensils at a maximum level in the end-use
concentration of 500 parts per million (ppm) is safe under FFDCA section 408.

I. Background

Novozymes North America, Inc. submitted a petition (0E7697) requesting an exemption from tolerance for residues of lipase, triacylglycerol when used as an inert ingredient as an ai d in the removal of lipids in pesticide formulations under 40 CFR 180.950, tolerance exemptions fo r minimal risk active and inert ingredients. EPA published the Notice of Filing for this pe tition in the Federal Register on February 25, 2011 (76 FR 10584). There were no comments recei ved in response to th e notice of filing.

This document provides an assessment of the ri sk to human health and the environment for triacylglycerol lipase when used as an inert ingr edient in pesticide form ulations and was based on information provided by the submitter and informa tion available in the public literature.

The proposed use of triacylglycerol lipase as an inert ingredient in food contact surface sanitizer pesticide products. Sanitizing cleaners are used in food and food preparation and/or processing environments. These use sites include meat prepar ation, breweries and beverage manufacturing plants, produce processing facilities, food-ca nning facilities and bakeries. Surf ace cleaning use patterns include food contact surfaces, food-processing equipm ent and dairy processing equipment.

Triacylglycerol lipases are a class of lipase enzymes that cataly ze the hydrolysis of fatty acid ester bonds in the triacylglycerol molecule in aqueous solu tions and have many, versatile uses. Enzymes are proteins that act as catalysts to increase the rate of chemical r eactions and are pr oduced by all living cells. Only minute quantities of enzymes are needed to catalyze chemical reactions. Triacylglycerol4 lipases are present in animals, pl ants and microorganisms and are wide ly used in the food, detergent, pulp and paper, and pharmaceu tical industries.

Triacylglycerol lipases decompose triglycerides to hydrophilic, mono- an d diglycerides, free fatty acids and glycerol. Lipase preparations are used for re moval of stains in powder laundry detergents. The typical concentration of lip ase in these types of pr oducts is less than 0.01%.

Enzymes are not simple synthetic chemicals. They are complex organic macromolecules isolated from living organisms. Most detergent enzymes are produ ced using genetically engineered microorganisms. Protein-engineered variants are al so produced. Lipases can be isolat ed from bacteria and fungi. Most commercial enzymes, including lipases, are derived fr om microorganisms. Th e production of enzymes is a multi-step process including fermentation a nd recovery (purification, concentration and standardization) stages. During the fermentation process, the enzyme is manufactured by submerged batch-fed pure culture fermentation of the microorganism, the production host.

According to the manufacturing process for tria cylglycerol lipase provided by Novozymes North America, Inc., this lipase is manufactured by subm erged fed-batch pure culture fermentation of the genetically modified strain of Aspergillus oryzae. The starting materials meet the standards required by the Toxic Substances Control Act (TSCA). It is also manufactured in accordance with current good manufacturing practices of a food/fe ed facility according to 21 CF R. All equipment used in the manufacturing process is carefully maintained to prevent contamin ation by foreign microorganisms. During all stages of the fermentation and recovery process, quality control measures are taken to ensure the absence of foreign microorgani sms and confirm strain identity.

The proposed limitation of lipase in pesticide products follows the limit in commercial products set by the detergent industry, a maximum of 500 parts per million (ppm) in final commercial products. This limit is imposed to be protective against the potential of lipases to cause respir atory irritation that may occur as a result of the use of enzy mes in these types of applications . Lipases are used in many laundry and cleaning products. The enzyme concentration in these products typically ranges from 0.007 % and 0.05% (500 ppm).

The U.S. Food and Drug Administra tion (FDA) has reviewed Generall y Recognized as Safe (GRAS) Notifications (GRNs) for the use of lipase enzyme s in food in accordance with Section 21 USC 321 (s) of the Federal Food, Drug and Cosmetic Act (FFDCA).

Table 1. U.S. Food and Drug Administration Generally Recognized as Safe Listings for Lipase

GRN.

Number Substance Intended Use Basis Source

43 Lipase derived

from Aspergillus

oryzae carrying a gene encoding lipase from Thermomyces lanuginosus Use in dough, baked goods, and the fats and oil industry as a processing aid for

the interesterificati on or hydrolysis of glycerides at minimum levels necessary to achieve the desired effect; use as an enzyme in brewing beer (per additional correspondence of April 21, 2010)

Scientific procedures U.S. FDA

GRAS Notice Inventory http://www.ac 75 Lipase derived

from Aspergillus

oryzae carrying a gene encoding lipase from Use in the fats and oils industry, the baking industry, th e hydrolysis of lecithin, and the modification of egg yolks as a processing aid at a minimum level necessary in accordance with5 Fusarium

oxysporum good manufacturing practices cessdata.fda.g ov/scripts/fcn/ fcnNavigation .cfm?rpt=gras Listing 103 Lipase enzyme preparation from Aspergillus

oryzae carrying a gene constructed

from a modified Thermomyces lanuginosus lipase gene and a portion of the

Fusarium

oxysporum lipase

gene Use in bakery products, egg yolks, whole eggs, and fats and oils as an enzyme at minimum levels necessary in accordance with good manufacturing practice

113 Lipase enzyme preparation from Aspergillus

oryzae Use in dairy-based flavoring preparations, cheeses , liquid and dried egg white, bread, flour, unstandardized bakery products, modified triglycerides, hydrolyzed lecithin, edible fats and oils, and modified egg yolk as an enzyme

II. Substance Identity

TABLE 2. Substance identity Lipase triacylglycerol Chemical abstracts service registry number (CAS RN) 9001-62-1

Other names Triacylglycerol lipase, Lipozyme, Lipex, Liposlase

PC Code 908800 Chemical formula Not specified6 IV. Physical and Chemical Properties

TABLE 3. Physico-chemical properties of detergent amylases, cellulases and lipases (*)

Parameter Value Remark

Macromolecular Descriptions Physical

State/Particle Size White crystals or powder (pure enzyme) In detergents added as 0.2-3% preparation: stabilized liquid, slurry or coated granule Bulk density (kg/m 3 ) 0.6-1.3

1 Granulates and liquid preparations, crystalline

enzyme Melting point ( o C) Not relevant Heating leads to destruction
Boiling pt. ( o C) Not relevant Heating leads to destructions
Vapour Pressure at 25

o C (Pa) 1 x 10 -6 Minimum value acc. To TGD

Water Solubility at 25

o C (g/L) < 1.3 (at pH 7, 21 o C) Henkel 2005

Octanol-water partition coefficient: log Kow -2.95 at 21 o C, Henkel 2005

Koc (l/kg) < 1.3 Calculated acc. to TGD Henry coefficient (unit less): log H -4 Minimum value acc. To TGD

pH 7-10

(*) Ta bl e 3 a d opte d f rom Human an d Env i ronmenta l R i s k Assessment on Ingre di ents o f House h o ld C l ean i ng Pro d ucts ( a l p h aamylases, cellulases and lipases) (MRID 483732011)

V. Human Health Assessment

A. Acute Toxicity

Acute toxiciological data on triacylglycerol lipase is summarized in Table 4.

TABLE 4. Acute toxicity studies

Study Type Chemical Species/

strain Results Tox. Category Reference

Acute oral

triacylglycerol lipase Wistar rats LD 50 >5g/kg IV Stavnsbjerg,

1988a (*)

Acute

inhalation SpragueDawley

rats LC 50 > 0.74 mg/L III McDonald,

1988 (*)

Skin

irritation Albino

rabbits Primary Irritation Index (PII)=0 and 0.27 (0=non-irritant and 0.27=mild irritant) IV Berg, 1988b,d

and 1990 (*)

Eye Irritation Albino

rabbits Not irritating IV Berg, 1988a (*)7 TABLE 4. Acute toxicity studies

Study Type Chemical Species/

strain Results Tox. Category Reference

Skin

Sensitization Guinea pigs Not sensitizing N/A Berg, 1988c (*)

(*)All references as cited in R. J. Gr eenough,, et.al, Safety Evaluation of a Lipas e Expressed in Aspergillus oryzae (MRID 4837 32011)

B. Sub-acute Oral Toxicity

In a 2-week study, groups of 10 male and 10 female Sprague-Dawley rats we re dosed once daily by gavage at dose levels of 0, 0.2, 2 or 10 g/kg/day. Humicola lanuginose lipase artificially expressed in Aspergillus oryzae was suspended in distilled water and administered at a dose volume of 10 ml/kg. In a second 13-week study with 20 male and 20 female Sp rague-Dawley rats were administered the same

test material at dose levels of 0, 0.2, 1 and 5 g/kg/day in distilled water at a dose volume of 10 ml/kg.

There were no treatment related clin ical signs seen in either study. There was no effect on body weight gain or food and water consumption. Haematology and clinical chemistry investigations did not reveal any changes and gross and microscopic pathology a nd organ weight analysis gave no indication of adverse responses to treatment. (MRID 48373201)

C. Carcinogenicity

There is no data available on the ca rcinogenicity of triacy lglycerol lipase. Howe ver, carcinogenicity is not expected since there is no indi cation in the public literature that lipase enzymes possess carcinogenic properties. In addition, triacylglyce rol lipase was negative for mutageni city in the Ames test and also negative for clastogenicity in the in vitro human ly mphocytes assay. Triacylg lycerol lipase enzymes are normal constituent of the human body. Systemic bioavailab ility fo these enzymes is expected to be very low and toxicologically insignificant as these enzymes are readily biod egraded in the gastrointestinal tract following ingestion. (MRID 48373201)

D. Reproductive Toxicity

In a two-generation reproductive toxi city study in Sprague-Dawley rats, a triacylglycerol lipase known as SP 225 (batch PPW 1798) was administered oral ly to 5 treatment groups of rats. Each group

contained 24 males and 24 females and received diets containing 0, 0.5, 1.5 or 5.0% SP 225 by weight in the diet (equivalent to 0, 500, 1500 or 5000 mg/kg bw/day). The diet c ontained a constant concentration of test material throughout the study and was available c ontinuously to the animals.

The F 0 males received the treated diet for ten weeks prior to mating and the F 0 females received the same diet for two and a half weeks prior to ma ting. Treatment for both sexes continued throughout mating, gestation and lactation, following which the F 0 animals were sacrificed. The F 1 animals selected for rearing to maturity and breedi ng received treated diets for ten w eeks prior to mating and continued throughout gestation and early lactation. Shortly after Day 4 of lactation, the F 1 animals and F 2 litters

were sacrificed.

Clinical observations for all animals were conduc ted twice daily. The animals were checked for viability and examined for reaction to treatment. The onset, intensity and durations of any signs were8 recorded. Body weights, food consumption and mati ng and littering performance were also recorded throughout the study. Following comple tion of the treatment period, F 0 and F 1 parent animals were necropsied. This consisted of an external exam ination, followed by a macr oscopic examination of tissues and organs of the cranial, thoracic and abdominal cavities.

F 1 and F 2 juveniles found dead on or afte r Day 12 of lactation were n ecropsied. Offspring found dead prior to Day 12 were examined for externally visibl e abnormalities and for the presence of milk in the

stomach.

Clinical observations for the F 0 generation were considered to be in cidental and not re lated to the test material. There was a sporadic incidence of other mi nor observations common to this strain of animals, mild alopecia and eye encrustations. These incidences were not treatment related.

There were no treatment related eff ects on body weight performance for F 0 males. High dose F 0 females showed a slight increase in weight gain over the treatment period, resultin g in an 8% increase in absolute weight after 9 weeks of treat ment. Intermediate dose F 0 females showed a 6% total increase over the treatment period.

There were no treatment related effects on food consumption for F 0 males and females.

F 0 fertility and F 1 survival were not affected by treatment. In the high dose group, th ere was a change in gestation period from 21 to 22 da ys. This was not considered to be of biological significance.

For the F 1 generation there were no si gnificant treatment related effects on body weight, food consumption and fertility. F 2 survival was also not affected by treatment.

In conclusion, at 5% and 1.5% SP 225 by weight in the diet, there was slight increase in F 0 female body weights. This effect was not observed in the F 1 animals. There were no e ffects of treatment with the

test material on either F 0 or F 1 fertility and general reproductive perf ormance at exposure levels of up to 5% (equivalent to 5000 mg/kg bw/day )SP 225 by we ight in the diet. The NOAEL for systemic, offspring and reproductive toxic ity is 5000 mg/kg bw/day, the highe st dose tested. (MRID 48373201)

E. Mutagenicity

In a gene mutation study, test batch A (Humicola la nuginose lipase artificially expressed in Aspergillus oryzae) was examined for mutagenic activity us ing Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98 and Escherichia coli WP2uvrA (pKM101) . The test was conducted in the presence

and absence of metabolic ac tivation. The sensitivity of the individual bacteria l strains was confirmed by any notable increases in the number of revertant colonies induced in similar liquid conditions by diagnostic mutagens. There were no dose related increases in revertan ts to phototrophs in any of the tests performed. All results were validated in an i ndependent experiment and it was concluded that there was no evidence of mutagenic activity in the pr esence or absence of metabolic activation.

In a chromosomal aberration test, test batch A (H umicola lanuginose lipase artificially expressed in Aspergillus oryzae) was examined in an in vitro cytogenics assay using hu man lymphocyte cultures

from a male and female donor. The tests were c onducted in the presence and absence of metabolic activation by a rat liver post-mito chondrial fraction (S-9) from Aroclo r-1254 induced animals. Negative

control cultures were included in the test system and contained low incidences of chromosomal aberrations within historical solvent control ranges. The presence of lipase did not affect the activity of the S-9 liver preparation. Treatment of cells with the test material in both the presence and absence of S-9 activation resulted in numbers of aberrations, wh ich were similar to those observed in concurrent9 negative controls. It was determined that the test material was unable to induce chromosome aberrations in human lymphocytes when tested up to 5000 μg/ml in either the presence of absence of metabolic activation. (MRID 48373201)

F. Developmental Toxicity No developmental toxicity studies were located, although sub-acute oral toxi city studies indicate developmental effects are unlikely.

G. Neurotoxicity No neurotoxicity studies were loca ted. Acute and sub-acute toxicity studies do not indicate a neurotoxic

effect would occur.

H. Toxicity Endpoint Selection and Levels of Concern

Triacylglycerol lipase is not toxic by the oral or dermal routes. No toxicity endpoint of concern was
identified in the available toxicity studies. There we re also no adverse effects observed in acute toxicity
studies and short-term toxicity st udies at doses up to 10 g/kg/day. No toxicity was observed in a twogeneration reproductive toxic ity study in rats at doses up to 5% (equivalent to 5000 mg/kg bw/day). A
quantitative risk assessment for the dietary and resident ial exposure from the oral and dermal routes is
not necessary since no endpoint of concern was identifie d in the available database. Inhalation exposure
to enzymes, including triacylglycer ol lipase, may lead to potential respiratory (Type 1) allergy.

I. Mammalian Metabolism of Lipase

Lipases are necessary for lipid meta bolism and are found in almost all liv ing organisms, as well as being regularly consumed in foods. As with other enzyme s, lipases are common in fresh and processed foods and are consumed by humans every day. Like ot her dietary proteins, li pases are degraded and metabolized after ingestion. Lipases have not been associated with t oxicity and are considered safe. Any residual lipases from sanitizing solutions are exp ected to be biologically metabolized as any other lipase or protein.

VI. Special Considerations for Infants and Children

In general Section 408(b)(2)(c) of FFDCA provides th at EPA shall apply an a dditional tenfold (10x) margin of safety for infants and children in the case of threshold effects to account for prenatal and postnatal toxicity and the complete ness of the database on toxicity and exposure unless EPA determines based on reliable data that a different margin of safety will be safe for infants and children. This additional margin of safety is commonly referred to as the FQPA safety factor (SF). In applying this provision, EPA either retains the default value of 10x or uses a different additional safety factor when reliable data available to EPA support the choice of a different factor.

No developmental toxicity studies ar e available in the database. Howe ver, there were no adverse effects in a two-generation reproductive toxicity study in ra ts at doses up to 5% (equivalent to 5000 mg/kg bw/day). There was also no systemic toxicity ob served at doses up to 10 g/kg/day in a 2-week, subacute oral toxicity study in rats and no systemic toxicity observed at 5 g/kg/day in a 13 week oral toxicity study in rats. No system ic toxicity was observed in laborat ory animals at high doses, indicating10 relatively low hazard potential. There was no eviden ce of clinical signs of neurotoxicity; therefore, developmental neurotoxicity study is not required. In addition, no evidence of immunotoxicity is available in the database; therefor e, immunotoxicity study is not required . In terms of hazard, there are low concerns and no residual uncertainties regarding prenatal and/or postnatal toxicity. Based on this information, there is no concern at this time for increased sensitivity to infants and children to triacylglcyerol lipase when used as an inert ingredie nt in pesticide formulati ons and a safety factor analysis has not been used to assess risk. For th e same reason, EPA has determined that an additional safety factor is not needed to protect the safety of infants and children.

VIII. Exposure Assessment

Oral, dermal and inhalation exposure to triacylglycerol lipas e from its use as an inert ingredient in sanitizing solutions may occur. Oral exposure to enzymes, such as tri acylglycerol lipase, will lead to a

breakdown of the molecule into sm all peptides and amino acids in the same manner as any other ingested protein. Dermal exposure and skin irritation is also possibl e, however, absorption across intact skin is expected to be precluded by the la rge molecular size of the molecule.

The key health concern for enzyme s, including lipase, is inhalati on exposure leading to potential respiratory (Type 1) allergy. Lipa ses are used in many laundry and cl eaning products. According to a 2003 International Associate for Soap s, Detergents, and Maintenance Pr oducts (A.I.S.E.) survey, the enzyme concentration in these products typically ranges from 0.007 % to 0.05% (500 ppm). This standard is set by the detergent i ndustry. In order to limit exposure from sanitizers containing lipase, a maximum concentration of 500 ppm of lipase in r eady-to-use cleaners is recommended. This is consistent with enzyme concentrations typically us ed in detergent and cleaning products and would be protective of any potential respirator y or skin irritation concerns. Although there is no well-defined threshold for the induction of sensitization to the pot ential allergic effects from exposure to enzymes such as triacylglycerol lipase, allergic symptoms have not been observed when inhalation exposure levels are at or below 1 ng/m 3 . A limitation of 500 ppm of triacy lglycerol lipase in final pesticide formulations will result in exposures se veral orders of magnitude below 1 ng/ m 3 . This limitation will ensure that inhalation exposures to triacylglycerol lipase will be below the threshold for adverse respiratory effects and is protective of a ny potential respiratory allergy concerns.

A. Aggregate Exposure

In examining aggregate exposure, the Federal Food, Drug, and Cosmetic Act (FFDCA) section 408

directs EPA to consider available information con cerning exposures from pest icide residue in food and all other non-occupational exposures, including drinking water from gr ound water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses).

The primary route of exposure to triacylglycerol lipase from its use as an iner t ingredient in pesticide products would most likely be through consumption of food which came in contact with food contact surfaces to which pesticide produc ts containing it have been a pplied. Residential (dermal and inhalation) exposures from home uses, such as laundry detergents and food contact surface sanitizing solutions, are also possible.

No hazard was identified for the acute and chronic dietary assessment (food and drinking water), or for the short, intermediate, and long term resident ial assessments, and therefore no aggregate risk assessments were performed.11 B. Cumulative Exposure

Cumulative effects from substances with a common mechanism of toxicity. Se ction 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to es tablish, modify, or revoke a tolerance, the Agency consider “available information” concerning the cumu lative effects of a partic ular pesticide's residues

and “other substances that have a common mechanism of toxicity.”

EPA has not found triacylglycerol lipase to share a common mechanis m of toxicity with any other substances, and does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has assumed that triacylglycerollipase does not have a common mechanism of toxicity with other substances. For information regarding EPA's efforts to

determine which chemicals have a common mechanis m of toxicity and to evaluate the cumulative effects of such chemicals, see EPA's website at http://www.epa.gov/pesticides/cumulative

.

IX. Environmental Justice Statement

Potential areas of environmental just ice concerns, to the extent possible, were considered in this human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions to Address Environmental Justice in Minority Popul ations and Low-Income Populations," http://www.eh.doe.gov/oepa/ guidance/justice/eo12898.pdf

.

As a part of every pesticide risk assessment, OP P considers a large variety of consumer subgroups according to well-established procedures when a pplicable. In line with OPP policy, ARIA/HED estimates risks to population subgroups from pesticid e exposures that are base d on patterns of that subgroup’s food and water consumption, and activities in and around the home that involve pesticide use in a residential setting. Extensive data on f ood consumption patterns are compiled by the USDA under the Continuing Survey of Food Intakes by Individuals (CSFII) and are used in pe sticide risk assessments for all registered food uses of a pesticide. Thes e data are analyzed and ca tegorized by subgroups based on age, season of the year, ethnic group, and region of the country. Additionall y, OPP is able to assess dietary exposure to smaller, specialized subgr oups and exposure assessmen ts are performed when

conditions or circumstances warrant . Whenever appropriate, non-di etary exposures based on home use of pesticide products and associated risks for adult applicators and fo r children (three to less than six years old), youths, and adults entering or playi ng on treated areas post-app lication are evaluated. Further considerations are currently in developmen t as OPP has committed resources and expertise to the development of specialized software and models that consider exposure to bystanders and farm workers as well as lifestyle and traditi onal dietary patterns among specific subgroups.

X. Environmental Fate and Ecotoxicity

Humicola lanuginose lipase artificiall y expressed in Aspergillus oryzae was tested for biodegradability by recording the dissolved organic carbon (DOC) and in a closed bottl e test. In the Organisation for Economic Co-operation and Development (OECD) DOC test (OECD 1981f), the inoculum was collected from activated sludge from domestic sewage. The test mate rial attained 99 % biodegradation within 28 days. The pass level of 70% was reach ed within 10 days of exceeding the 10% level; therefore the test material can be termed readily bi odegradable. The same test material was not readily biodegradable in the closed bottle te st. At a test material concentr ation of 5 mg/L the biodegradation reached 78% after 28 days. However, 60% biodeg radation was not reached within 10 days of biodegradation exceeding 10%. (MRID 48373201)12 Ecotoxicity findings for Humicola lanuginose lipase artificially expr essed in Aspergillus oryzae are

listed in Table 7.

TABLE 7. Ecotoxicity of Lipase triacylglycerol Organism Test Material Duration/Endpoint References (*)

Fish (Cyprimus carpio) Humicola lanuginose lipase artificially expressed in Aspergillus

oryzae 96-h LC 50 > 1 g/L Bogers, M., 1989

Daphnid 24-h EC 50 > 1 g/L Bogers, M., 1989 Green Algae (Scenedesmus subspicatus) 72-h EC 50 = 97 mg/L

24-72-h EC 50 = 99 mg/L NOEL=40 mg/L Douglas and King,

1993

(*)All references as cited in R. J. Gr eenough,, et.al, Safety Evaluation of a Lipas e Expressed in Aspergillus oryzae (MRID 4837 32011)

XII. Risk Characterization

The acute toxicity studies of Humi cola lanuginose lipase artificially expressed in Aspergillus oryzae suggest the toxicity of lipa se, triacylglycerol would be low. The te st material is not acutely toxic by the

oral or inhalation routes. It is also not a dermal irritant, ey e irritant or dermal sensitizer.

The test material was also not toxic in short-term studies. In a 2-week stu dy, Sprague-Dawley rats were dosed once daily by gavage at dose levels of 0, 0.2, 2 or 10 g/kg/day and in a second 13 week study, Sprague-Dawley rats were administered the same test material at dose levels of 0, 0.2, 1 and 5 g/kg/day.

There were no treatment re lated clinical signs se en in either study.

In a two-generation reproductive toxicity study in Sprague-Dawley rats, a lipase known as SP 225 (batch PPW 1798) was administered orally to 5 treatment groups of rats. Each group contained 24 males and

24 females and received diets cont aining 0, 0.5, 1.5 or 5.0% SP 225 by weight in the diet (equivalent to 0, 500, 1500 or 5000 mg/kg bw/day). There were no effects of treatment with the te st material on either

F 0 or F 1 fertility and general reproductive performance at exposure levels of up to 5% SP 225 by weight

in the diet.

No developmental toxicity studies are available in the database. Ho wever, no systemic toxicity was observed in a two-generation reprod uctive toxicity study in rats at doses up to 5% (equivalent to 5000 mg/kg bw/day). There was also no systemic toxicity observed at doses up to 10 g/kg/day in a 2-week, sub-acute oral toxicity study in rats and at doses up 5 g/kg/day in a 13 week oral toxicity study in rats. In terms of hazard, there are low concerns and no residual uncertainties regarding prenatal and/or postnatal toxicity. Based on this information, there is no concern at this time for increased sensitivity to infants and children to triacylglycerol lipase, when used as an inert ingredient in pesticide formulations and a safety factor analysis has not been used to assess risk. For the same reason, EPA has determined that an additional safety factor is not needed to protect the safety of infants and children.

Residues of concern are not antici pated for dietary exposure (food and drinking water) or for residential exposure (dermal and inhalation) from the use of lipas e triacylglycerol for the proposed use pattern as an inert ingredient in pesticide products. EPA expects aggregate exposure to lipas e triacylglycerol to pose no appreciable dietary risk given th at the data on triacylglycerol lipas e show a lack of any systemic toxicity and a lack of any apparent reproductive or developmental effect s. Any potential adverse effects regarding respiratory sensitization is mitigated by the proposed limit in formulation of 500 ppm.13 There is limited environmental fate and ecotoxicity data available for lipas e tricylglycerol. The

available data indicate that this compound is expected to be readily biodegradable in the environment and of low toxicity to fish and aquatic organisms.

Based upon the review of the data supporting the petition, EPA has determined and exemption from the requirement of a tolerance under 40 CFR 180.950 is not appropriate given that enzymes such as lipase are known for the potential to cause respiratory irritation and allerg enic responses. Therefore, EPA modified the exemption requested by granting an exemption under 40 CFR 180.940 and limiting the use of lipase, triacylglycerol to a maximum of 500 ppm in final pesticide formulations.

Taking into consideration all available information on triacylglycerol lipase at a maximum of 500 ppm
in final pesticide formulations, EPA has determined th at there is a reasonable certainty that no harm to
any population subgroup will result from aggr egate exposure to triacylglycerol lipase under reasonably

foreseeable circumstances. Therefore, the establis hment of an exemption from tolerance under 40 CFR
180.940(a) for residues of triacylglycerol lipase when used as a component of food contact sanitizing
solutions applied to all food cont act surfaces in public eating places , dairy-processing equipment, and
food-processing equipment and utensils at a maximu m level in the end-use concentration of 500 parts
per million (ppm) is safe under FFDCA section 408.

References

EPA MRID 48373201, Novozymes North America, Inc. (2011) “Information Submitted to Support Petition Number 0E7697, Petition Proposing as Exempti on from Tolerance for Lipase triacylglycerol, and Inert Ingredients to be Used in Antimicrobial Products.” (Compilation on information available in the public literature)

EPA MRID 48565801, Paschal, J. (2011) ; “Lipase-Manufacturing Process and Product Specifications;” Novozymes North America, Inc.