1

**Federal Food, Drug, and Cosmetic Act (FFDCA) Considerations for Choline Chloride** 

**Docket ID Number: EPA**  **- HQ**  **- OPP**  **- 20**  **15 -**  **0023**

**Date: August 11**  **, 2015** Section 408(c)(2)(A)(i) of FFDCA allows the U.S. Environmental Protection Agency ( EPA ) 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 or tolerance exemption, and to “ensure that there is a reasonabl e certainty that no harm will result to infants and children from aggregate exposure to th e 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 analy ses to determine the risks from aggregate exposure to pesticide residues. First, the EPA determines the toxicity of the pesticide . Second, the EPA examines exposure to the pesticide through fo od, drinking water, and other exposures that occur as a result of pesticide use in residential settings. **I. Summary of Petitioned -**  **for Tolerance Exemption** In the Federal Register of March 4 , 2015 ( Volume 80, Number 42, FR page numbers 11611

- 11614 ), the 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 4F8287 ) by CP Bio, In c.; 4802 Murrieta Street, Chino, CA 91710 . The petition requested that 40 CFR part 180 be amended b y establishing an exemption from the requirement of a tolerance for residues of Choline Chloride in or on all food commodities (when applied preharvest)

. Th at document referenced a summary of the petition prepared by the petitioner CP Bio, Inc. , which is available in D ocket ID Number EPA

- HQ

- OPP

- 20 15

- 0023 via http://www.regulations.gov

. **II .**  **Toxicological Profile** Consistent with FFDCA section 408(b)(2)(D), the EPA reviewed the available scientific data and other relevant information on Choline Chloride , and considered its validity , completeness, and reliability , as well as the relationship of this information to human risk. The EPA also considered available information concerning the variability of the sensitivities of major identifiable subgroups of consumers, including infants and children. **A. Overview of Choline Chloride**  Choline C hloride is a quaternary ammonium salt consisting of a choline cation with a chloride anion. As with many

salts, it presents as a white cry stalline solid that is odorless. It can readily dissociate into two constituents

– Choline and Chloride

– particularly in water

. Each constitu ent is ubiquitous in the2

environment , constitutes a regular part of the human diet, and serves man y critical functions in the human body

. Choline is an essential human dietary component and is found in foods such as egg yolk, vegetables and animal fat as f ree Choline and as phophatidyl cholines, such as leci thin. Choline is a precursor for the production of the neurotransmitter acetyl choline , and is important for the structural integrit y of cell membranes and various phy siological pathway s such as methylatio n metabolism, transmembrane signaling , and lipid and cholesterol transport and metabolism. Choline is also endogenousl y produced in the pancreas. Chloride is also a regular part of the human diet, particularly as a constituent of edible salt, and serves many functions in human biology

. Chiefly , Chloride is an essential electrol yt e located in all body fluids and is responsible for maintaining acid/base balance, transmitting nerve impulses and regulating fluid in and out of cells

. Choline C hloride is designated as GRAS (Generally Recognized as Safe) and is approved by the Food and Drug Administration (FDA) as a human nutrient under 21 CFR 182.8252. It is also considered a GRAS substance as a nutrient and or dietary supplement in animal feeds under 21 CFR 582.5252. It has been used as a food additive in animal feed since the earl y 1930s (OECD, 2004) **.** As a b iopesticide, Choline Chloride is considered a plant growth regulator (PGR) and is intended for use to increase growth and decrease stress in growing crops . ( The crops listed in the End

- use Product (EP) associated with this new active ingredient include fru iting vegetables, cereal grains, stone fruit, tree nuts and cotton. ) The product is to be applied at application rates ranging from 0.0004 to 0.282 lb ai/A to foliage. The most active constituent of the active ingredient is considered to be Choline but the Chloride Salt of Choline compound (Choline Chloride) provides joint activity as the salt compound is more stable and effective as a delivery vehicle. As with most PGRs, Choline Chloride is necessarily applied at low concentrations because use at high co ncentrations results in detrimental effects to the plant. Choline Chloride is already approved for use by EPA as an inert ingredient in pesticide products without numerical limitation for pre

- harvest food (as a solvent under 40 CFR 180.920) and nonfood use s. Based on the data submitted in support of this petition (summarized in Unit II. B., below) and the comprehensive risk assessment conducted b y the Agency (References 1

- 3, below), EPA concludes that there is a reasonable certainty of no harm from aggregate exposures to Choline Chloride , including the consumption of food treated with this active ingredient in accordance with label directions and good agricultural practices. EPA has made this determination because available toxicology data indicate that the active ingredient is of low acute toxicity and is not a developmental toxicant, a mutagen, or toxic via repeat oral exposure. **1. Acute Toxicity** Choline C hloride is not considered to be acutel y t oxic based on the available data. The chemical is classified into toxicity

category I II for acute oral toxicity and into toxicity

category IV for dermal and inhalation toxicity

and dermal and eye irritation. The substance is not considered to be a dermal sensitizer. All Tier I mammalian acute “6

- pack” to xicology data requirements were all conducted according to the OCSPP guidelines, and were found to be acceptable. The results are summarized in Table 1 below. **Table 1. Acute Mammalian Toxicology Data for Choline Chloride (40 CFR § 158.2050)**3

**Table 1. Acute Mammalian Toxicology Data for Choline Chloride (40 CFR § 158.2050) Study/OCSPP**  **Guideline No. Results**  **Toxicity Category**  **MRID** Acute oral toxicity

(UDP method) (870.1100) LD 50 > 1,750 mg/kg (rat) LD 50 = 3,129 mg/kg estimated III 49403804 Acute dermal toxicity (870.1200) LD 50 > 5,050 mg/kg (rat) IV 49403805 Acute inhalation toxicity (870.1300) LC 50 > 2.03 mg/L(rat) IV 49403807 Primary eye irritation (870.2400) Minimally irritating (rabbit): minimal irritation observed at one hour post instillation of the test substance with clearance b y 24 hours IV 49403806 Primary dermal irritation (870.2500) Nonirritating (rabbit): no irritation noted at an y point throughout the 72

- hour study

. IV 49403808 Dermal sensitization (Buehler) (870.2600) Not a sensitizer (guinea pig) N/A 49403809
**2. Subchronic Toxicity** 90

-Day Oral In MRID 49403810 and in the unassigned MRID associated with the subchronic toxicity requirement , data were cited from the open scientific literature to satisfy the data requirement. Two studies (21

- day and 28 day

oral toxicity

) were assessed and found adequate for use in the risk assessment . Both studies confirm a lack of adverse effects through the oral exposure . While neither of these studies individually satisfy the 90

- day

oral toxicity

data requirement, the collective results of these studi es are sufficient to satisfy the data requir ement along with the fact that C holine is an important component of the human diet and is endogenousl y produced in humans (as discussed previousl y in this document). Li kewise, a dverse effects are also not anticip ated from the C hlori de portion of the substance as C hloride is regularl y consumed and is present in the human body as an electrol yt e (as discussed previousl y in this document). Summaries of the two studies are provided below. Based on the available informa tion, adverse effects are no t anticipated from exposure to Choline C hloride as a biopesticide. 21

- day Oral Toxicity

Study in the Rat: In a study

to evaluate the effects of C holine on microsomal liver enzy mes in males versus females, rats were administered choline b y gavage twice dail y for 21 day s at doses of 0 and 500 mg/kg/day (daily dose of 1,000 mg/kg/day). Body weights, food consumption, liver weights and several microsomal enzy mes were evaluated. Body weight and food consumption were unaffected in the test animals when compared to the control animals. In the female test animals, relative liver weights were increased (4%) and microsomal liver enzy me activities related to the smooth endoplasmic reticulum were increased (3%). This effect was not observed in males. The effects were considered to be beneficial in that the increase is related to generation of membranous phospholipids. Therefore, the no

- observed

- adverse

- effect

- level (NOAEL) for the study is 1,000 mg/kg, the highest dose tested.4

28

- day Oral Toxicity

Study in the Mouse: In a 28

- day oral toxicity

study , female and male mice were administered Choline C hloride by gavage daily

at doses of 0 and 200 mg/kg/day . As part of the study , other groups of mice were dosed via intranasal and intraperitoneal routes as well; however, for the purposes of this subchronic oral toxicity assessment, only

the part of the study

involving gavage dosing of mice will be discussed as it is the route relevant to the anticipated exposure. Body weight, food and water consump tion, skin, ey e and mucous membranes and behavior of mice were observed daily . At study termination, hematology , clinical chemistry , gross pathology and histopathology

were assessed. The results of the stud y i ndicated no adverse effects for an y of the parameters tested. The NOAEL for the study is 200 mg/kg/day , which was the highest dose tested. 90

-Day Dermal The 90

- day

dermal data requirement is ‘ conditionally required’ ( CR) to support the proposed use of C holine Chloride . The Agency has waived this data requirement at this time. This decision is based in accordance with 40 CFR 158.2050 , which states that data is required when application of a product involves purposeful application to the human skin or uses that would result in prolonged dermal exposure, or if the active ingredient is known or expected to be metabolized differentl y b y the dermal route of exposure than b y the oral route and the metabolite is of toxicological concern. Although purposeful application and prolonged exposure to the human skin is not anticipated the proposed use will result in potential short

- term dermal exposure to occupational mixer/loaders and applicators using aerial and ground equipment. However, Choline C hloride is a PGR and will , therefore, be used at low concentrations with an application rate ranging from 0.00044 to 0.282 lb ai/acre on agricultural crops

. Appropriate Personal Protective Equipment ( PPE ) on end

- use product label s will sufficiently mitigate any dermal exposure. 90

-Day Inhala tion In accordance with 40 CFR 158.2050, the 90

- day inhalation data requirement is ‘ conditionally required ’ if there is a likelihood of significant levels of repeated inhalation exposure to the pesticide as a gas, vapor or aerosol. The Agency has waived this data requirement at this time b ased on the use pattern provided in the proposed products. First , Choline Chloride is applied as a liquid, and has a low vapor pressure and volatility (6.57

- 8 Pa (4.73

- 10 mmHg) at 25°C). Second, as a PGR, Choline C hloride will be applied at low concentrations (0.0004 to 0.282 lb ai/A) approximately 2

- 4 times per season. Significant inhalation exposure is not anticipated based on the character of this PGR and the proposed use patt ern . **3. Mutagenicity**  Mutagenicity

data from the open scientific literature from the open scientific literature (MRID 49403810) satisfy

the mutagenicity

data requirement and support food uses for Choline Chloride. Three separate Ames assay s, strains of Salmonella typhimurium we re exposed to Choline Chloride at up to 10,000 μg/plate with or without metabolic activation. Choline Chloride was not considered mutagenic in any of the assay s. In an in vitro chromosomal aberration assay using Chinese hamster ovary (CHO) cells, cells wer e exposed to Choline Chloride at up to 5,000 μg/ml with or without metabolic activation. The substance was not considered mutagenic in the assay . Ambiguous results were observed in a sister chromatid exchange (SCE) assay consisting of two parallel studies where CHO cells were exposed to Choline Chloride at concentrations of up to 5,000 μg/ml with or without metabolic activation. An increase in SCEs were observed with metabolic activation in one of the studies; however, the increase was sporadic, not dose re lated and not reproduced in the second parallel study . In a separate SCE study5

with CHO cells exposed to concentrations of choline chloride at up to 5,000 μg/ml no increase in the number of SCEs was observed. The available data indicate that that Choline C hloride is not mutagenic. **4. Developmental Toxicity**  A developmental toxicity study on mice from the open scientific literature (MRID 49403810) satisfy the data requirement and support the proposed food uses of Choline Chloride. In the study, mice were e xposed every

other day from gestation day

1 to 18 to Choline Chloride in feed in doses of 1250, 4160, 10800 and 20000 mg/kg/day . Historical control data from 414 pregnant mice served as the control. All mice were sacrificed on gestation day 19. Observation s were made for body weight gain, resorptions, fetal survival, fetal weight and fetal external, visceral and skeletal abnormalities. Maternal body weight gain was reduced in all but the lowest dose group. All fetuses were resorbed in the highest dose group

. No resorptions occurred in the low dose group. Fetal lethality occurred in the 4160 (35% lethalit y) and 10800 (69% lethality) dose groups. No statistically significant fetal malformations were observed. The NOAEL for maternal and developmental toxicity w as 1250 mg/kg. Although adverse effects were observed in this study , the effects were at extremely high doses. These are doses to which humans are extremely unlikel y to be exposed. All of the doses employed in the study were above OCSPP’s currentl y recomme nded top dose, or limit dose of 1,000 mg/kg, for toxicity testing. No adverse effects were observed at the dose of 1250 mg/kg which is above the limit dose. The top dose used in the study was 20 times that recommended in the current OCSPP and OECD test gui delines. **III. Aggregate Exposure**  In examining aggregate exposure, FFDCA section 408 directs the EPA to consider available information concerning exposures from the pesticide residue in food and all other non

- occupational exposures, including drinking water from ground water or surface water and exposure through pesticide use in gardens, lawns, or buildings (residential and other indoor uses). Food Exposure : Dietary

exposure to the pesticidal residues of Choline Chloride is expected to be negligible

. O ne, Choline Chloride is a PGR and is necessarily applied at low concentrations. ( As with most PGRs, use of high concentrations can result in detrimental effects to the plant. ) Two, Choline Chloride bio degrade s rapidly , once applied. A MITI

- I test demonstrated that Choline Chloride is 93% degrad ed within 14 day s. Three , as a salt, Choline C hloride dissociate s readily when in contact with water, making its persistence as a residue even more unlikely . (OECD, 2004) Altogether, the p esticidal use of Choline Chlo ride is not likely to contribute significantl y to human dietary exposure. Notably , humans are already dietaril y exposed to Choline Chloride. It is produced endogenously ; and it is found naturally

in foods in the human diet. Indeed, it is considered an esse ntial human dietary component , serving critical functions in nerve transmission, cell membrane integrit y and lipid metabolism. (OECD, 2004) Drinking W

ater Exposure : No significant residues of choline chloride are expected in drinking water when products are used according to label instructions. The active ingredient is applied terrestriall y at low concentrations; it is very soluble in water ; and it biodegrades rapidl y, once applied. As such, any residues of choline chloride in drinking water are anticipated to be negligible. It should be additionally

noted that both choline and chloride, the constituents of choline chloride, are ubiquitous in the environment; and there is a long history of incidental , but minor, exposure through drinking water. For the reasons discussed above

– namely , high solubility and biodegradability , the6

pesticidal use of choline chloride is not anticipated to contribute significantly to human exposure beyon d existing background levels. Other Non

- occupational Exposure : Non

- occupational exposure to Choline Chloride residues are not expected

. Choline Chloride is not intended fo r use in residential settings; i t is intended for agricultural use only

. **IV .**  **Cumulative Effects from Substances with a Common Mechanism of Toxicity** Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, modify , or revoke a tolerance, the EPA consider “available information concerning the cumulative effects of [a particular pesticide’s] . . . residues and other substances that have a common mechanism of toxicity .” The EPA has not found Choline Chloride to share a common mechanism of toxicity with an y othe r substances, and Choline Chloride does not ap pear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, the EPA has assumed that Choline Chloride does not have a common mechanism of toxicity with other substances. For information regarding the EPA’s efforts to determine chemicals that have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the EPA’s website at http://www.epa.gov/pesticides/cumulative

. **V. Determination of Safety for the U**  **.S. 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 safet y 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 safet y will be safe for infants and children. This additional margin of safet y is commonly referred to as the Foo d Quality Protection Act Safet y Factor. In appl yi ng this provision, the EPA either retains the default value of 10X, or uses a different additional or no safet y 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 pesticidal use of Choline C hloride

. Exposure to the residues of Choline Chloride are expected to be negligible due to the low concentrations associated with its use as a PGR and its high solubility and rapid biodegradabilit y. Moreover , any exposure to Choline Chloride residues are not expected to pose a risk

. No toxic endpoints have been established for Choline Chloride. There has been a long history

of significant human dietary and endogenous exposure without documented incide nt
. And the constituents of Choline Chloride are known to be readil y metabolized. **VI. Conclusions**7

The 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 Choline Chloride . Therefore, an exemption from the requirement of a tolerance is established for residues of Choline Chloride in or on all food commodities when applied preharvest and used in accordance with label directions and good agri cultural practices.

**VII . References**  OECD (2004) SIDS Initial Assessment Report for SIAM 19: Choline Chloride. October 19

- 22. < http://www.inchem.org/documents/sids/sids/67481.pdf > Ottenstein, B., Boncoddo, N., Walker, A. and Thurmon, F. (1952) Experiments on the choline content of the skin and sebum. J. Invest. Dermatol. 19:105

- 108.