2

that catalyze the hydrolysis of fatty acid ester bonds in the 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 triacylglycerol lipase is in sanitizing cleaners used in food and food 

preparation and/or processing environments. 

The proposed limitation of triacylglycerol 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 Humicola lanuginose lipase artificially expressed in Aspergillus oryzae 

suggest the toxicity of triacylglycerol lipase would be low. The test material is not acutely toxic by the 

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

The test material was also not toxic in short-term studies. In a 2 week study, 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-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 related clinical signs nor any toxicity seen in either 

study. 

In a two-generation reproductive toxicity study in Sprague-Dawley rats, a triacylglycerol 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 containing 0, 0.5, 1.5 or 5.0% SP 225 by weight in 

the diet (equivalent to 0, 500, 1500 or 5000 milligrams per kilogram body weight per day (mg/kg 

bw/day)). There were no effects of treatment with the test material on either F0 or F1 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 carcinogenicity of triacylglycerol lipase. However, carcinogenicity is 

not expected since there is no indication in the public literature that lipase enzymes possess carcinogenic 

properties. In addition, triacylglycerol lipase was negative for mutagenicity 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 enzymes is expected to be very low 

and toxicologically insignificant as these enzymes are readily biodegraded in the gastrointestinal tract 

resulting in negligible bioavailability. 

No developmental toxicity studies are available in the database. However, no systemic toxicity was 

observed in a two-generation reproductive 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 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 anticipated for dietary exposure (food and drinking water) or for residential 

(dermal and inhalation) exposure from the use of triacylglycerol lipase for the proposed use pattern as an 

inert ingredient in pesticide products. EPA expects aggregate exposure to triacylglycerol lipase to pose 

no appreciable dietary risk given that the data on triacylglycerol lipase triacylglycerol show a lack of any 
3

systemic toxicity and a lack of any apparent reproductive or developmental effects. Any potential 

adverse effects regarding respiratory sensitization and dermal irritation are mitigated by the proposed 

limit of triacylglycerol lipase in pesticide formulations of 500 ppm. 

There is limited environmental fate and ecotoxicity data available for tricylglycerol lipase. 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. 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 determined 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 allergenic 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 final pesticide formulations. 

Taking into consideration all available information on triacylglycerol lipase, EPA has determined that 

there is a reasonable certainty that no harm to any population subgroup will result from aggregate 

exposure to triacylglycerol lipase when used as an inert ingredient in pesticide formulations when 

considering dietary exposure and all other non-occupational sources of pesticide exposure for which 

there is reliable information. Therefore, the establishment 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 contact surfaces in public eating places, dairy-processing 

equipment, and food-processing equipment and utensils 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 aid in the removal of lipids 

in pesticide formulations under 40 CFR 180.950, tolerance exemptions for minimal risk active and inert 

ingredients. EPA published the Notice of Filing for this petition in the Federal Register on February 25, 

2011 (76 FR 10584). There were no comments received in response to the notice of filing. 

This document provides an assessment of the risk to human health and the environment for 

triacylglycerol lipase when used as an inert ingredient in pesticide formulations and was based on 

information provided by the submitter and information 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 preparation, breweries and beverage manufacturing plants, 

produce processing facilities, food-canning facilities and bakeries. Surface cleaning use patterns include 

food contact surfaces, food-processing equipment and dairy processing equipment. 

Triacylglycerol lipases are a class of lipase enzymes that catalyze the hydrolysis of fatty acid ester bonds 

in the triacylglycerol molecule in aqueous solutions and have many, versatile uses. Enzymes are 

proteins that act as catalysts to increase the rate of chemical reactions and are produced by all living 

cells. Only minute quantities of enzymes are needed to catalyze chemical reactions. Triacylglycerol 
4

lipases are present in animals, plants and microorganisms and are widely used in the food, detergent, 

pulp and paper, and pharmaceutical industries. 

Triacylglycerol lipases decompose triglycerides to hydrophilic, mono- and diglycerides, free fatty acids 

and glycerol. Lipase preparations are used for removal of stains in powder laundry detergents. The 

typical concentration of lipase in these types of products is less than 0.01%. 

Enzymes are not simple synthetic chemicals. They are complex organic macromolecules isolated from 

living organisms. Most detergent enzymes are produced using genetically engineered microorganisms. 

Protein-engineered variants are also produced. Lipases can be isolated from bacteria and fungi. Most 

commercial enzymes, including lipases, are derived from microorganisms. The production of enzymes 

is a multi-step process including fermentation and 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 triacylglycerol lipase provided by Novozymes North 

America, Inc., this lipase is manufactured by submerged 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/feed facility according to 21 CFR. All equipment used in the 

manufacturing process is carefully maintained to prevent contamination by foreign microorganisms. 

During all stages of the fermentation and recovery process, quality control measures are taken to ensure 

the absence of foreign microorganisms 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 respiratory irritation that may 

occur as a result of the use of enzymes 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 Administration (FDA) has reviewed Generally Recognized as Safe (GRAS) 

Notifications (GRNs) for the use of lipase enzymes 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 interesterification 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, the hydrolysis of 

lecithin, and the modification of egg 

yolks as a processing aid at a minimum 

level necessary in accordance with 
5

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 specified 
6

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/m3) 0.6-1.3 

1 

Granulates and liquid 

preparations, crystalline 

enzyme 

Melting point (oC) Not relevant Heating leads to destruction 

Boiling pt. (oC) Not relevant Heating leads to destructions 

Vapour Pressure at 25 

oC (Pa) 

1 x 10-6 Minimum value acc. To TGD 

Water Solubility at 25 

oC (g/L) 

< 1.3 (at pH 7, 21 oC) Henkel 2005 

Octanol-water partition 

coefficient: log Kow

-2.95 at 21 oC, 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 

(*) Table 3 adopted from Human and Environmental Risk Assessment on Ingredients of Household Cleaning Products (alphaamylases, 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 LD50>5g/kg IV Stavnsbjerg, 

1988a(*)

Acute 

inhalation 

SpragueDawley 

rats 

LC50 > 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. Greenough,, et.al, Safety Evaluation of a Lipase Expressed in Aspergillus oryzae (MRID 483732011) 

B. Sub-acute Oral Toxicity 

In a 2-week study, groups of 10 male and 10 female Sprague-Dawley rats were 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 Sprague-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 clinical 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 and organ weight analysis gave no indication of 

adverse responses to treatment. (MRID 48373201) 

C. Carcinogenicity 

There is no data available on the carcinogenicity of triacylglycerol lipase. However, carcinogenicity is 

not expected since there is no indication in the public literature that lipase enzymes possess carcinogenic 

properties. In addition, triacylglycerol lipase was negative for mutagenicity in the Ames test and also 

negative for clastogenicity in the in vitro human lymphocytes assay. Triacylglycerol lipase enzymes are 

normal constituent of the human body. Systemic bioavailability fo these enzymes is expected to be very 

low and toxicologically insignificant as these enzymes are readily biodegraded in the gastrointestinal 

tract following ingestion. (MRID 48373201) 

D. Reproductive Toxicity 

In a two-generation reproductive toxicity study in Sprague-Dawley rats, a triacylglycerol 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 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 contained a constant concentration 

of test material throughout the study and was available continuously to the animals. 

The F0 males received the treated diet for ten weeks prior to mating and the F0 females received the 

same diet for two and a half weeks prior to mating. Treatment for both sexes continued throughout 

mating, gestation and lactation, following which the F0 animals were sacrificed. The F1 animals selected 

for rearing to maturity and breeding received treated diets for ten weeks prior to mating and continued 

throughout gestation and early lactation. Shortly after Day 4 of lactation, the F1 animals and F2 litters 

were sacrificed. 

Clinical observations for all animals were conducted twice daily. The animals were checked for 

viability and examined for reaction to treatment. The onset, intensity and durations of any signs were 
8

recorded. Body weights, food consumption and mating and littering performance were also recorded 

throughout the study. Following completion of the treatment period, F0 and F1 parent animals were 

necropsied. This consisted of an external examination, followed by a macroscopic examination of 

tissues and organs of the cranial, thoracic and abdominal cavities. 

F1 and F2 juveniles found dead on or after Day 12 of lactation were necropsied. Offspring found dead 

prior to Day 12 were examined for externally visible abnormalities and for the presence of milk in the 

stomach. 

Clinical observations for the F0 generation were considered to be incidental and not related to the test 

material. There was a sporadic incidence of other minor observations common to this strain of animals, 

mild alopecia and eye encrustations. These incidences were not treatment related. 

There were no treatment related effects on body weight performance for F0 males. High dose F0 females 

showed a slight increase in weight gain over the treatment period, resulting in an 8% increase in absolute 

weight after 9 weeks of treatment. Intermediate dose F0 females showed a 6% total increase over the 

treatment period. 

There were no treatment related effects on food consumption for F0 males and females. 

F0 fertility and F1 survival were not affected by treatment. In the high dose group, there was a change in 

gestation period from 21 to 22 days. This was not considered to be of biological significance. 

For the F1 generation there were no significant treatment related effects on body weight, food 

consumption and fertility. F2 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 F0 female body 

weights. This effect was not observed in the F1 animals. There were no effects of treatment with the 

test material on either F0 or F1 fertility and general reproductive performance at exposure levels of up to 

5% (equivalent to 5000 mg/kg bw/day )SP 225 by weight in the diet. The NOAEL for systemic, 

offspring and reproductive toxicity is 5000 mg/kg bw/day, the highest dose tested. (MRID 48373201) 

E. Mutagenicity 

In a gene mutation study, test batch A (Humicola lanuginose lipase artificially expressed in Aspergillus 

oryzae) was examined for mutagenic activity using Salmonella typhimurium strains TA1535, TA100, 

TA1537 and TA98 and Escherichia coli WP2uvrA (pKM101). The test was conducted in the presence 

and absence of metabolic activation. The sensitivity of the individual bacterial 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 revertants to phototrophs in any of the 

tests performed. All results were validated in an independent experiment and it was concluded that there 

was no evidence of mutagenic activity in the presence or absence of metabolic activation. 

In a chromosomal aberration test, test batch A (Humicola lanuginose lipase artificially expressed in 

Aspergillus oryzae) was examined in an in vitro cytogenics assay using human lymphocyte cultures 

from a male and female donor. The tests were conducted in the presence and absence of metabolic 

activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor-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, which were similar to those observed in concurrent 
9

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 toxicity studies indicate 

developmental effects are unlikely. 

G. Neurotoxicity 

No neurotoxicity studies were located. 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 were also no adverse effects observed in acute toxicity 

studies and short-term toxicity studies at doses up to 10 g/kg/day. No toxicity was observed in a twogeneration reproductive toxicity study in rats at doses up to 5% (equivalent to 5000 mg/kg bw/day). A 

quantitative risk assessment for the dietary and residential exposure from the oral and dermal routes is 

not necessary since no endpoint of concern was identified in the available database. Inhalation exposure 

to enzymes, including triacylglycerol lipase, may lead to potential respiratory (Type 1) allergy. 

I. Mammalian Metabolism of Lipase 

Lipases are necessary for lipid metabolism and are found in almost all living organisms, as well as being 

regularly consumed in foods. As with other enzymes, lipases are common in fresh and processed foods 

and are consumed by humans every day. Like other dietary proteins, lipases are degraded and 

metabolized after ingestion. Lipases have not been associated with toxicity and are considered safe. 

Any residual lipases from sanitizing solutions are expected 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 that 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 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 are available in the database. However, there were no adverse effects 

in a two-generation reproductive 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, 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 systemic toxicity was observed in laboratory animals at high doses, indicating 
10

relatively low hazard potential. There was no evidence of clinical signs of neurotoxicity; therefore, 

developmental neurotoxicity study is not required. In addition, no evidence of immunotoxicity is 

available in the database; therefore, 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 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. 

VIII. Exposure Assessment

Oral, dermal and inhalation exposure to triacylglycerol lipase from its use as an inert ingredient in 

sanitizing solutions may occur. Oral exposure to enzymes, such as triacylglycerol lipase, will lead to a 

breakdown of the molecule into small peptides and amino acids in the same manner as any other 

ingested protein. Dermal exposure and skin irritation is also possible, however, absorption across intact 

skin is expected to be precluded by the large molecular size of the molecule. 

The key health concern for enzymes, including lipase, is inhalation exposure leading to potential 

respiratory (Type 1) allergy. Lipases are used in many laundry and cleaning products. According to a 

2003 International Associate for Soaps, Detergents, and Maintenance Products (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 industry. In order to limit exposure from sanitizers containing lipase, a 

maximum concentration of 500 ppm of lipase in ready-to-use cleaners is recommended. This is 

consistent with enzyme concentrations typically used in detergent and cleaning products and would be 

protective of any potential respiratory or skin irritation concerns. Although there is no well-defined 

threshold for the induction of sensitization to the potential 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/m3

. A limitation of 500 ppm of triacylglycerol lipase in final pesticide 

formulations will result in exposures several orders of magnitude below 1 ng/ m3

. This limitation will 

ensure that inhalation exposures to triacylglycerol lipase will be below the threshold for adverse 

respiratory effects and is protective of any 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 concerning exposures from 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). 

The primary route of exposure to triacylglycerol lipase from its use as an inert ingredient in pesticide 

products would most likely be through consumption of food which came in contact with food contact 

surfaces to which pesticide products containing it have been applied. 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 residential assessments, and therefore no aggregate risk 

assessments were performed. 
11

B. Cumulative Exposure 

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 Agency 

consider “available information” concerning the cumulative effects of a particular pesticide's residues 

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

EPA has not found triacylglycerol lipase to share a common mechanism 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 mechanism 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 justice 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 Populations and Low-Income Populations," 

http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf. 

As a part of every pesticide risk assessment, OPP considers a large variety of consumer subgroups 

according to well-established procedures when applicable. In line with OPP policy, ARIA/HED 

estimates risks to population subgroups from pesticide exposures that are based 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 food consumption patterns are compiled by the USDA under 

the Continuing Survey of Food Intakes by Individuals (CSFII) and are used in pesticide risk assessments 

for all registered food uses of a pesticide. These data are analyzed and categorized by subgroups based 

on age, season of the year, ethnic group, and region of the country. Additionally, OPP is able to assess 

dietary exposure to smaller, specialized subgroups and exposure assessments are performed when 

conditions or circumstances warrant. Whenever appropriate, non-dietary exposures based on home use 

of pesticide products and associated risks for adult applicators and for children (three to less than six 

years old), youths, and adults entering or playing on treated areas post-application are evaluated. 

Further considerations are currently in development 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 traditional dietary patterns among specific subgroups. 

X. Environmental Fate and Ecotoxicity 

Humicola lanuginose lipase artificially expressed in Aspergillus oryzae was tested for biodegradability 

by recording the dissolved organic carbon (DOC) and in a closed bottle 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 material attained 99% biodegradation 

within 28 days. The pass level of 70% was reached within 10 days of exceeding the 10% level; 

therefore the test material can be termed readily biodegradable. The same test material was not readily 

biodegradable in the closed bottle test. At a test material concentration of 5 mg/L the biodegradation 

reached 78% after 28 days. However, 60% biodegradation was not reached within 10 days of 

biodegradation exceeding 10%. (MRID 48373201) 
12

Ecotoxicity findings for Humicola lanuginose lipase artificially expressed 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 LC50 > 1 g/L Bogers, M., 1989 

Daphnid 24-h EC50 > 1 g/L Bogers, M., 1989 

Green Algae 

(Scenedesmus 

subspicatus) 

72-h EC50 = 97 mg/L 

24-72-h EC50 = 99 

mg/L 

NOEL=40 mg/L 

Douglas and King, 

1993 

(*)All references as cited in R. J. Greenough,, et.al, Safety Evaluation of a Lipase Expressed in Aspergillus oryzae (MRID 483732011) 

XII. Risk Characterization 

The acute toxicity studies of Humicola lanuginose lipase artificially expressed in Aspergillus oryzae 

suggest the toxicity of lipase, triacylglycerol would be low. The test material is not acutely toxic by the 

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

The test material was also not toxic in short-term studies. In a 2-week study, 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 related clinical signs seen 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 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). There were no effects of treatment with the test material on either 

F0 or F1 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. However, no systemic toxicity was 

observed in a two-generation reproductive 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 anticipated for dietary exposure (food and drinking water) or for residential 

exposure (dermal and inhalation) from the use of lipase triacylglycerol for the proposed use pattern as an 

inert ingredient in pesticide products. EPA expects aggregate exposure to lipase triacylglycerol to pose 

no appreciable dietary risk given that the data on triacylglycerol lipase show a lack of any systemic 

toxicity and a lack of any apparent reproductive or developmental effects. 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 lipase 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 allergenic 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 that there is a reasonable certainty that no harm to 

any population subgroup will result from aggregate exposure to triacylglycerol lipase under reasonably 

foreseeable circumstances. Therefore, the establishment 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 contact surfaces in public eating places, dairy-processing equipment, and 

food-processing equipment and utensils at a maximum 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 Exemption 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.