-----·, EPA REGISTRATION DIVISION COMPANY NOTICE OF FILING FOR 

PESTICIDE PETITIONS PUBLISHED IN THE FEDERAL REGISTER 

EPA Registration Division contact: Linda Hollis, Biochemical Pesticides Branch 

Chief, 703-308-8733 

CP Bio, Inc. 

EPA Reg. # 4F8287 

Choline Chloride 

EPA has received a pesticide petition Choline Chloride from CP Bio Inc. P. 0. Box 

3367 Ontario, CA 91761, proposing, pursuant to section 408(d) of the Federal Food. 

Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a (d), to amend 40 CFR part 180. 

The addition of an exemption from the requirement of a tolerance for Choline 

Chloride in or on the raw agricultural commodity growing crops under 40 CFR. 

EPA has determined that the petition contains data or information regarding the 

elements set forth in section 408 (d) (2) ofFDDCA; however, EPA has not fully 

evaluated the sufficiency of the submitted data at this time or whether the data supports 

granting of the petition. Additional data may be needed before EPA rules on the 

r--- petition. 

A. Residue Chemistry 

The petitioner has provided EPA with sufficient toxicity data to support the 

issuance of this tolerance exemption for this inert ingredient. The data submitted 

will allow EPA to determine the sensitivities of major identifiable subgroups of 

consumers, including infants and children. 

1. Analytical method. The purpose of this study was to confirm a 

chromatographic method as the enforcement analytical method for the analysis of 

the active ingredient, in Choline Chloride 98o/o Technical. The Choline Chloride 

concentration is 98o/o) Technical was determined to be 99.03o/o Choline Chloride 

by high-performance liquid chromatographic (HPLC) analysis. The identity of the 

active ingredient, Choline Chloride, was confirmed by chromatography data and 

spectral scans performed by GC/MS. 

2. Magnitude of residues. CP Bio, Inc. is petitioning the agency to establish an 

exemption from the requirement of a tolerance for this natural class of chemistry 
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that is well known. Therefore information regarding the nature and magnitude of 

chemical re idue · re ulting from the usc of thi product i. not r-equired. 

B. Toxicological Profile 

I. utt toxicity. The test substance, Choline Chloride 98 % Technical, wa. 

evaJuated for it. acute oral toxicity potential in female albino rats when 

administered as a gavage dose at a level of 5000 mg/kg. ince the test substance 

failed the limit te t, the main test was conducted following the up-and-down 

procedure (UDP) at 175, 550, 1750 and 5000 mglkg. The tudy was terminated 

following the stopping rule ofthi procedure. Mortality occurred only at the 5000 

mg/kg level. There were no clinical signs of toxicity during the tudy. AnimaJ 

su_rviving to termination exhibited weekly weight gain, with thee ception of one 

that to t weight between Days 7 and 14. Abnormal necropsy findings that occurred, 

only m the animals dying on te. t, pertained to contents of the to mach and 

intestines. The acute oral LDSO was estimated to be 3129 mglkg. 

Derma) ensitization (OPPT 870.2600) 

A primary dermal irritation , tudy was conducted on three albino r-.tbbits u ing te. t 

sub t-ance Choline Chloride 98o/u Technical. There was one intact te t itc per 

animal. Each test ite wa treated with 500 mg of te t substance moistened witb O.S 

mL of Dl water and covered with a semi-permeable dre. ing. The te. t sub tance wa 

maintained m contact witb the skin for 4 hour • Ob ervation for dermal irritation 

and defects were made at 1, 24, 48 and 72 bours after removal of the dressing • 

Irritation scores derived from there ·pcctive erythema and edema scores through 

the 72-hour observations for each animal are presented below. 

Acute Inhalation Toxicity (OPPT 870.1300) 

Test sub tance Choline Chloride 98% Technical wa. to bee •aluated for acute 

inhalation toxicity potential in albino rats. Two pre-study trial assays were 

conducted to determine if aero olizing the test substance into the e. posure chamber 

wouJd produce an acceptable concentration of at least 2 mgfL with mass median 

aerodynamic diameter (MMAD) between 1 and 4um. Prior to one aero olization, 

test substance was heated and ground. An aerosol was attempted using a Venturi 

A pirator connected to a SOOL e. po ure chamber. ln both attempts, the powder test 

ub ·tance began to liquefy during aerosolization; the delivery method could not be 

adju ted for that occurrence and eoncentratjon information could not be taken. Due 

to the nature of the tc t sub tam~e, it · con idcred that an inhalation exposure to 

provide meaningful acute to icity data could not be done. 

Primary Eye Irritation (OPPT 870.2400) 

An acute e e irritation study a conducted on three albino rabbit using test 

sub tance Choline Chloride 98% Technical. The test substance, 100 mg, was placed 

into the conjunctiva sac of the right eye of each animal elected for testing. All 

treated eyes were wa bed with room teblperature DI water for one minute 

immediately after recording the 24-hour observation. There were no positive effects 

exhibited in any eyes after treatment. The test ubstance i. rated minimall 
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irritating and assigned to Toxicity Category IV. 

Primary Skin Irritation (OPPT 870.2500) 

A skin sensitization tudy was conducted on 15 male and 15 female short~haircd 

albino guinea pig to determine if test substance Choline Chloride 98%) Technical 

produced a en itizing reaction. Males and female were assigned to each of two 

groups, de. ignated Nah•e (5/ ex) and Test (10/ ex). aive control group animals 

remained untreated during the induction phase of the study. Test group animals 

were treated with 400 mg of test ub tance moistened with DJ water (selected from 

previou creenjng). Tc t animal were 

treated once weeki for three weeks for a total of three treatments. After a twoweek rest period, all animals (both group ) were challenged at a virgin test ite witb 

an application of 400 mg of test ub tanee moi tencd with Dl water. The te t 

substance produced no reaction m either the Tc t animal. or the Naive control 

animals after the challenge treatment. Therefore, Choline bloride 98% Technical 

is not a ensitizer in guinea pigs. 

2. Geuotoxit'ly. The literature wa extensively searched for studies on the 

genotoxicity of choline chloride. TOXNET, Pubmed, FDA website, TJ and 

Google cholar searche were conducted. Tbi waiver request for the genoto icity 

studies i based on the results Ames abnonella a says and in vitro chromo omaJ 

aberration and si ter chromatid e, change a. ays conducted on choline chloride and 

Ame almonclla and mou. c micronuclcu a ay conducted on PC. All assayi\ 

were negative except for one out of m o i ·ter chromatid exchange as ays which was 

considered positive by TP and "ambiguous' by OE D. A weight of the evidence 

evaluation on the available data on choUne chl.oride indicate it bas little genoto ic 

potential. The literature wa extensively earchcd for tudies on tbe toxicity to 

plants of choline chloride. TOXNET, the internet in general and Google Scholar 

searches were conducted. o stu die meeting the guideline requirement of the 

tudie ; however, tudies were found on the effect of choline chloride on pea 

seedling , choline on rice eedlings, emergence and elongation of radish root and 

algal tudie u ing lobjbition (%)of Pseudokircllerilla ubcpilata and Scenedesmus 

subspicatus were located. In aU cases, choline, choline chloride and acetyl choline 

bad beneficial effect or no effect on the parameters studied. A waiver is requested 

for the above tudie based on the natural occurrence of choline in pJant and its 

lack of adver. e effects described in the cited studies. 

'3. /lr ·,lfluc :ve and develoom 11/a/ toxic:'v. The literature was extensi\'ely 

earched for studies on the developmental/reproductive toxicjty of choline chloride. 
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TOXNET, Pubmed, the FDA website, TIS and Google cbolar earche were 

conducted. A waiver is requested forte ·ting choline chloride in a preliminary 

developmental to . icity crecn based on the existence of informatio.n available on the 

developmental toxicity of choline chloride in mice and of APC in rat , on 

reproductive toxicity of choline in male rats and on the po itive influence of choline 

on learning and memory in young rats. 

Overall, the study authors concluded that the re u!t indicate that prenatal choline 

upplementation facilitate. cognitive function aero . the lifespan, whereas prenatal 

choline deficiency impairs divided attention and accelerate.~ age-related· decline in 

temporal processing (4). In another tudy, the arne laboratory studie the effect of 

perinatal choline upptementation on timing and temporal memory u ing a similar 

treatment regime. Reaction to nicotine e. posur was determined. holine 

enhanced the temporal proce ing domain. The tudy authors related the effect to 

modification in cholinergic function (5). 

4. Subchronic: toxicity. The literature was extensively searched for tudie on 

the subchronk inhalation toxicity of choline chloride. TO ET, Pubmed the FDA 

web itc, TI and Google cbolar earcbes were conducted. o repeated do e 

inhalation to. icity tudies were found; however, a 28-day intranasal tudy was 

located. A waiver is requested for 90-day inhalation toxicity te ting on choline 

chloride based on its low to icity, GRA tatu., its acute inhalation L SO of>1.6 

mg/L (no cUnical sign of to icity reported) and the exi tence of a 28-day intranasal 

toxicity tudy in rat·. The result of the intrana. al tudy are summarized below. 

28-day lntr-lna at · tudy. In tbi tudy, Balb/c mice (10/se.x/group)were 

administered choline chloride at a dose of200 mg/kg in 50 ,,L aline very alternate 

day·for 28 day ~ The study wa conducted under OECD GLP regulation . Body 

weight and food consumption were recorded daily. Mice were e amined daily for 

clinical signs of to icity. Hematology (hemoglobin, RBC, WB count, monocytes, 

lymphoc ·tes, neutrophil and eo inophoils) and clinical chemi try (urea, BUN, 

HDL, total chole terol, AL T, A T and creatinine) parameter were evaluated at 

study termination. The pleen was asepticaJJy removed and single ceJJ suspensions 

were made. Viable splenic cells were counted by the trypan blue exclusion test. A 

detailed gross necropsy was performed on all animals and the lungs, heart, liver, 

spleen and kidney were weighed. Microscopic examination wa done on ' vital ' 

organs including tbe lung, heart, liver, spleen and kidney. No mortality occurred 

and no statistically significant effects on body weight gain or food con umption were 

ob enred. o tatistically significant changes were observed in hematology and 

clinical cbemi. try parameters or spleen cell count. Organ weight were comparable 
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between the control and treated group. o treatment-related hi topathology was 

ob erved in the examined organs. The NOAEL was con idered to be >200 

mglkglday in mice (1). 

5. Fmlr>crine disruptio . EPA i required under the FFDCA, a amended by 

FQPA, to develop a creening program to determi.ne whether certain ubstances 

(including all pesticide active and other ingredients) "may ha\•e an effect in human 

that is similar to an effect produced by a naturally occurring estrogen, or other such 

endocrine effect as the dministrator may de. ignate." Following recommendations 

of it Endocrine Disruptor and Testing Advi ory Committe (ED TAC), EPA 

determined that there wa a cientitic basi for includin , a part of the program, 

the androgen and thyroid hormone sy terns, in addition to the c trogen hormone 

system. Then EPA also adopted ED T AC' recommendation that the Program 

include evaluations of potential effect in wildlife. or pe ticidc cbemital , EPA will 

use FIFRA and, to the extent that effects in wildlife ma help determine whether a 

sub tance may have an effect in human , FFD A authority to require the wildlife 

evaluations. As the science del·e)ops and resource allow, creening of additional 

hormone sy terns may he added to the Endocrine Di ruptor creening Program 

(EDSP). 

When additional appropriate screening and/or testing protocols being con ide red 

under the Agency's ED P have been developed, Choline Chloride may be objected 

to further creening and/or testing to better charaderize effect related to endocrine 

disruption. 

C. Aggregate Exposure 

1. c:tary exposure. ln evaluating dietary exposure to Choline Chloride based 

on its u e in food, feed, co metics, drug and in plants CP Bio believe there is no 

ri k form any increase in expo ure from the use as a PGR in food or feed crops. 

EPA e tabUs he exemption from the requirement of a tolerance onl in those ca es 

where it can be demon trated that the ri ks from aggregate e ·posure to pesticide 

chemical residues under rea on ably foreseeable circumstance will po. e no 

appreciable ri ks to human health. In order to determine the ri. ks from aggregate 

exposure to pesticide products, the Agency considers the toxicity of the product in 

conjunction with possible e po ure to re iducs of the product through food, 

drinking water, and through other expo ure that occur a a re ult of pesticide u e 

in residential settings. If EPA is able to determine that a finite tolerance is not 

necessary to ensure that there is a reltsonable certainty that no harm will result 

from aggregate expo ure to the product, an exemption from the requirement of a 

tolerance may be established. 

Section 408(b)(2)(C) ofFFDCA provides that EPA halJ apply an additional tenfold 

(1 OX) margin of afety for infants and children in the case of threshold effect to 
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account for prenatal and postnatal toxicity and the completene of the database on 

toxicity and exposure unle s EPA determines based on reliable data that a different 

margjn of safety will be safe for infants and chiJdren. This additional margin of 

safety is commonly referred to as the FQP A safety factor ( F). In applying this 

provision, EPA either retains the default value of lOX, or use a different additional 

afety factor when reliable data available to EPA support the choice of a different 

factor. 

CP Bio believe that the lOX standard safety factor is unneces ary for Choline 

Chloride a this sugar is used as an additiYe in baby formula a a food supplement. 

D. ( 'umulative t.D.'ecrs 

ufficient data are provided on the chemical identity of Choline Chloride and 

~ ufficient data are available on the metabolism and environmental degradation of 

this compound. CP Bio relied collectively on information provided on the 

representative chemical structure , the ubmitted phy ical chemical data, structure 

similarity information, a wcU a information from. the GRA a es ment and the 

use in food and feed for thi natural sugar. CP Bio ha concluded that a risk 

as essment ba ed on toxicity data for the parent compounds is not likel to 

underestimate risk. 

E. Stifely Determination 

. L. \' popu/ntic'l. Ba ed on these ri~;k as essments, P Bio, Inc. conclude 

that there is a reasonable certainty that no harm will result to the general 

popuJation or to infant and children from aggregate exposure to Choline Chloride. 

:!. /nf(pzts an I · 'ldrt . Based on these risk as e sment , P Bio, Inc. 

concludes that there is a rea onable certainty that no harm will re ult to the general 

population or to infants and children from aggregate expo ore to Choline Chloride. 

F. lnternatwnal Tolerances 

CP Bio, Inc. is not aware of any country requiring 'a tolerance for .Choline 

Chloride nor have any ODEX Maximum Re idue Level been est.ablished for any 

food crops at this time.