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

Docket ID Number: EPA-HQ-OPP-2015-0023

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 reasonable certainty that no harm will result to infants and children from aggregate exposure 

to the pesticide chemical residue . . . .” Additionally, FFDCA section 408(b)(2)(D) requires that the 

EPA consider “available information concerning the cumulative effects of [a particular pesticide’s] . . . 

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

The EPA performs a number of analyses to determine the risks from aggregate exposure to pesticide 

residues. First, the EPA determines the toxicity of the pesticide. Second, the EPA examines exposure to 

the pesticide through food, 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, Inc.; 4802 Murrieta Street, Chino, CA 

91710. The petition requested that 40 CFR part 180 be amended by establishing an exemption from the 

requirement of a tolerance for residues of Choline Chloride in or on all food commodities (when 

applied preharvest). That document referenced a summary of the petition prepared by the petitioner CP 

Bio, Inc., which is available in Docket ID Number EPA-HQ-OPP-2015-0023 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 Chloride is a quaternary ammonium salt consisting of a choline cation with a chloride anion.

As with many salts, it presents as a white crystalline solid that is odorless. It can readily dissociate into 

two constituents – Choline and Chloride – particularly in water. Each constituent is ubiquitous in the 
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environment, constitutes a regular part of the human diet, and serves many 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 free Choline and as phophatidylcholines, such as lecithin. Choline is a 

precursor for the production of the neurotransmitter acetylcholine, and is important for the structural 

integrity of cell membranes and various physiological pathways such as methylation metabolism, 

transmembrane signaling, and lipid and cholesterol transport and metabolism. Choline is also 

endogenously 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 electrolyte 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 Chloride 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 early 1930s (OECD, 2004).

As a biopesticide, 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 fruiting 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 concentrations 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 

uses.

Based on the data submitted in support of this petition (summarized in Unit II. B., below) and the 

comprehensive risk assessment conducted by the Agency (References 1-3, below), EPA concludes that 

there is a reasonable certainty of no harm from aggregate exposures to 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 Chloride is not considered to be acutely toxic based on the available data. The chemical is 

classified into toxicity category III 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” toxicology 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)
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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)

LD50 > 1,750 mg/kg (rat)

LD50 = 3,129 mg/kg estimated III 49403804

Acute dermal toxicity

(870.1200) LD50 > 5,050 mg/kg (rat) IV 49403805

Acute inhalation toxicity

(870.1300) LC50 > 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 by 24 hours 

IV 49403806

Primary dermal irritation 

(870.2500)

Nonirritating (rabbit): no irritation noted 

at any 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 studies are sufficient to 

satisfy the data requirement along with the fact that Choline is an important component of the human 

diet and is endogenously produced in humans (as discussed previously in this document). Likewise, 

adverse effects are also not anticipated from the Chloride portion of the substance as Chloride is

regularly consumed and is present in the human body as an electrolyte (as discussed previously in this 

document). Summaries of the two studies are provided below. Based on the available information, 

adverse effects are not anticipated from exposure to Choline Chloride as a biopesticide.

21-day Oral Toxicity Study in the Rat: In a study to evaluate the effects of Choline on microsomal liver 

enzymes in males versus females, rats were administered choline by gavage twice daily for 21 days 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 enzymes 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 enzyme 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. 
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28-day Oral Toxicity Study in the Mouse: In a 28-day oral toxicity study, female and male mice were 

administered Choline Chloride 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 consumption, skin, eye 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 study indicated no adverse effects for any 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 

Choline 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 differently by the dermal 

route of exposure than by 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 Chloride 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 labels will sufficiently mitigate 

any dermal exposure.

90-Day Inhalation

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 based 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 Chloride

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 pattern.

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 assays, strains of Salmonella typhimurium were 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 assays. In an in vitro chromosomal aberration assay using Chinese hamster ovary (CHO) 

cells, cells were 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 related and not reproduced in the second parallel study. In a separate SCE study
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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 Chloride 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 

exposed 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. Observations 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% lethality) and 

10800 (69% lethality) dose groups. No statistically significant fetal malformations were observed. The 

NOAEL for maternal and developmental toxicity was 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 unlikely to be exposed. All of the doses employed in the study were above OCSPP’s 

currently recommended 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 guidelines. 

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. One, 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 biodegrades rapidly, once applied. A MITI-I test demonstrated that Choline Chloride is 93% 

degraded within 14 days. Three, as a salt, Choline Chloride dissociates readily when in contact with 

water, making its persistence as a residue even more unlikely. (OECD, 2004) Altogether, the pesticidal 

use of Choline Chloride is not likely to contribute significantly to human dietary exposure.

Notably, humans are already dietarily exposed to Choline Chloride. It is produced endogenously; and it 

is found naturally in foods in the human diet. Indeed, it is considered an essential human dietary

component, serving critical functions in nerve transmission, cell membrane integrity and lipid 

metabolism. (OECD, 2004) 

Drinking Water 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 terrestrially at 

low concentrations; it is very soluble in water; and it biodegrades rapidly, 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, the
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pesticidal use of choline chloride is not anticipated to contribute significantly to human exposure

beyond existing background levels.

Other Non-occupational Exposure: Non-occupational exposure to Choline Chloride residues are not 

expected. Choline Chloride is not intended for use in residential settings; it 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 any other 

substances, and Choline Chloride does not appear to produce a toxic metabolite produced by other 

substances. For the purposes of this tolerance action, therefore, the EPA has assumed that 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 safety for infants and children in the 

case of threshold effects to account for prenatal and postnatal toxicity and the completeness of the 

database on toxicity and exposure, unless the EPA determines that a different margin of safety will be 

safe for infants and children. This additional margin of safety is commonly referred to as the Food 

Quality Protection Act Safety Factor. In applying this provision, the EPA either retains the default 

value of 10X, or uses a different additional or no safety factor when reliable data are available to 

support a different additional or no safety factor. 

Because there are no threshold effects associated with this biochemical, an additional margin of safety

for infants and children is not necessary. 

EPA has determined that there are no foreseeable dietary risks to the U.S. population, including infants

and children, from the pesticidal use of Choline Chloride. 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 biodegradability. 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 incident.

And the constituents of Choline Chloride are known to be readily metabolized.

VI. Conclusions
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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 

agricultural 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.