1 October 16, 2009

MEMORANDUM

SUBJECT: Decision Document for Petition Number 8E7387; Choline Chloride, CAS Reg. No 67-48-1

FROM: Deirdre Sunderland, Industrial Hygienist Inert Ingredient Asse ssment Branch (IIAB) Registration Division (7505P)

TO: PV Shah, Chief Inert Ingredient Asse ssment Branch (IIAB) Registration Division (7505P)

EXCUTIVE SUMMARY

Choline chloride is a quaternary ammonium salt which dissociates in water resulting in a positively charged quater nary hydroxyl alkylammonium ion and a negatively charged chloride ion. Choline is an essential compone nt of the human diet and acts as a precursor to acet ylcholine, phospholipids, and the methyl donor betaine. It is import ant for the structural in tegrity of cell membranes, cholinergic neurotransmissi on, transmembrane signaling, methyl metabolism, and lipid and cholesterol transport and metabolism.

Choline is found naturally in foods in both esterified forms and as free choline. Historically, it has been used as a food additive for animal husbandry, in plant growth regulator formulations , and as food supplements for humans. Choline was officially made an “essent ial nutrient” in 1998 and adequate intake (AI) level were established (women- 425 mg/day, pregnant women- 450 mg/day, men and lactating women- 550 mg/day). The Daily Upper Intake Level for choline is 3.5 grams for adults. Research indicate s that many individuals are not getting enough choline, with daily intake levels far below the AI.

Chloride is a binary compound of chlori ne; a salt of hydrochloric acid. Chloride is the major extracellular anion an d contributes to many body functions UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF PREVENTION, PESTICIDE, AND TOXIC SUBSTANCES2 including the maintenance of osmotic pressure, acid-base balance, muscular activity, and the movement of water between fluid compartments. The World Health Organization has performed two assessments which det ermined that from a toxicological point of view, there were no concerns for the chloride ion. It was considered to be naturally-occurring and a normal participant of animal and

human metabolism.

Choline chloride has demonstrated a low acute oral toxicity with LD 50 values for rats ranging from 3150 to ≥ 6000 mg/kg and LD 50 for mice in the range of 3900 to 6000 mg/kg. Alt hough appropriate animals studies are lacking for acute dermal toxicity, an in vitro percutaneous absorption study performed under

occluded and unoccluded conditions showed that choline chloride is expected to have a low potential for percutaneous absor ption. Acceptable acute inhalation

studies are not available.

Studies were conducted in the early 1960’s which resulted in only slight transient irritation of the skin and ey e; however, these studies were not performed under OECD guidelines and therefore the degree of irritation is not classifiable under the Globally Harmoniz ed System of Classif ication and Labeling of Chemicals (GHS). Choline chloride is not expected to cause skin sensitization.

Repeat dose animal studies on choline chloride are limited. One study in mice, evaluated the impact of 200 mg/kg/day choline chloride given orally or intranasally for 28 days. No adverse effect s were observed with regards to body weight, food and water consumption, hem atology, clinical biochemistry (Urea, BUN, HDL, TC, ALT, AST, and creatinine), or histopathology of various organs (lung, heart, liver, spleen, and kidney). Re sults from intranasal exposure to choline chloride were comparable with t heir respective controls and to other treatment groups. The NOAEL for oral and intranasally administered choline

chloride is ≥ 200 mg/kg/day.

A 72-week feeding study in rats admi nistered 500 mg/kg/day of choline chloride and observed the animals for 30 weeks post exposure. There were no significant difference between the contro l and treated group in relation to body weights, relative liver weight, survival rates, and the number of neoplastic liver nodules, hepatocellular carcinomas, lung tumo rs, leukemia, or other tumors. This study resulted in a NOAEL of 500 mg/kg.

Choline is a precursor to the vital neurotransmitter acetylcholine. Studies

show that choline has beneficial effe cts on the nervous system and memory. Choline is necessary to promote proper development in the fetus and infant and prevent cognitive problems. Choline chloride not expected to cause neurotoxicity.

Choline chloride is not a known endoc rine disruptors nor is it or its metabolites related to any class of known endocrine disruptors. Based on the3 results of the in vitro and in vivo st udies the Agency concluded that choline cloride is not expected to be carcinogenic or mutagenic.

Since the 1930’s choline chloride has been used as a widespread nutrient in animal feed without adverse effects repor ted on fertility or te ratogenicity. The Food and Drug Administration (FDA) requi res choline be added to non-milk

based infant formulas at a minimum concentration of 7 mg for every 100 kilocalories (21 CFR 107.100). Although one study did show developmental effects, they were only seen at very high doses ( ≥ 4,160 mg/kg/day) and only in the presence of maternal toxicity. Ther e were no observed adverse effects for both mothers and pups exposed to 1250 mg/kg/day. Based on this information the Agency concluded that choline chlori de, when used as an inert ingredient, will not cause reproductive or developmental toxicity and therefore, does not anticipate an increased risk to infants and children.

In addition to its low toxicity, exposure to choline chloride will be limited. Humans are currently exposed to c holine on a daily basis through commonly consumed foods (both naturally occu rring and when added as a nutrient) and through the bodies natural ability to synthes ize the nutrient. It is unlikely that the exposure from choline chloride, when used as an inert ingredient applied preharvest to food commodities, will signific antly increase the current concentration

of choline in foods. Choline chloride is readily biodegradable and because of its high water solubility it is expected that mo st of the inert will be washed from the plant prior to consumption. Once in wate r, its preferred medi a, it will be broken into in a quaternary hydroxyl alkyl ammonium ion and a chloride ion.

Similar to humans, choline is a di etary requirement for many animals including avian and aquatic species. Based on this information, the biodegradability of the chemical, and t he available toxicity data, the Agency

concluded that choline chloride is unlik ely to be harmful to mammalian, aquatic, and avian organisms.

In light of the prevalence of choline in the diet, the body’s natural ability to synthesis it, the natural occurrence of both choline and chloride, and their low toxicity the Agency conclude d that there is no increased risk to humans from the

use of choline chloride as an inert i ngredient in pesticide products. Therefore, choline chloride has been approved as an inert ingredient in pesticide products

used on all food commodities under 40 CFR 180.920.

I. BACKGROUND AND USES

In October 2008 Loveland Products, Inc. submitted a petition to amend 40 CFR 180.920 by establishing an exemption from the requirement of tolerance for

the use of choline chloride (Ethanaminium, 2-hydroxy-N, N, N-trimethyl-, chloride; CAS Reg. No. 67-48-1) as an inert ingredi ent in pesticide products when used as a solvent on all pre-harvest food commodities.4 Choline chloride is a quaternary ammonium salt which dissociates in water resulting in a positively charged quater nary hydroxyl alkylammonium ion and a negatively charged chloride ion. (OECD, 2004) Choline is an essential component of the human diet and acts as a precursor to acetylcholine, phospholipids, and the methyl donor betaine. It is important fo r the structural integrity of cell membranes, choli nergic neurotransmission, transmembrane signaling, methyl metabolism, and lipid and cholesterol transport and

metabolism.

Choline is found naturally in foods (e .g. eggs, liver meats, fungi, soybeans, fish, nuts, beans, and wheat germ) in both esterified forms (e.g. phosphocholine, glycerophosphocholine, sh ingomyeline, and phosphatid ylchoine) and as free choline. Historically, it has been used as a food additive for animal husbandry, in plant growth regulator formulations , and as food supplements for humans. Choline was officially made an “essential nutrient” in 1998.

An adequate intake (AI) level fo r choline has been established by the Food and Nutrition Board (FNB) of the National Academy of Science’s Institute of Medicine at 550 mg/day for adult men and la ctating women. The AI for women is 425 mg/day and 450 mg/day for pregnant wom en. This AI was set based on the amount necessary to prevent liver abnormalit ies as indicated by serum levels of the liver enzyme alanine aminotransferase (ALT). Daily Upper Intake Levels (UL, the highest level of intake that is not lik ely to cause harm) for choline are: 1 gram daily for children 1-8 years, 2 grams for children 9-13 years, 3 grams for children 14-18 years, and 3.5 grams fo r adults over 18 years of age”.(National Academy of Sciences (NAS), 1998)

Chloride is a binary compound of chlori ne; a salt of hydrochloric acid. Chloride is the major extracellular anion an d contributes to many body functions including the maintenance of osmotic pressure, acid-base balance, muscular activity, and the movement of wa ter between fluid compartments.

II. PHYSICAL AND CHEMICAL PROPERTIES

Choline chloride is a quaternary ammonium salt which dissociates in water resulting in a positively charged quater nary hydroxyl alkylammonium ion and a negatively charged chloride ion. Based on it s chemical structure choline chloride is not expected to undergo hydrolysis. T he Mackay Level I V2.11 model indicates that for pure choline chloride water is the preferred medium with almost 100% distribution. Other compartments such as air, soil, sediment, fish, and aerosol are expected to only receive a negligible amount of choline chloride (<0.00000005%).5 The Mackay model results are cons istent with the PCKOCWIN model which resulted in a logK oc of 0.37 indicating that cho line chloride will not adsorb to soil and sediment or suspended solids. According to OECD-criteria, choline chloride is readily biodegradable (MITI I-Test: 93% biode gradation within 14 days). A BOD 5 test performed on choline chloride obtained a BOD 5 /ThOD 5 ratio of 75% which confirms the biodegradabili ty of choline chloride. (OECD, 2004 citing MITI, 1992 and BASF AG 1984) Alth ough estimated bioconcentration factors (BCF) for choline chloride vary between models, all study results indicate that the potential for bioaccumulation is low

.

Using the HENRYWIN model to calcul ate Henry’s Law Constant resulted

in 2.06 X10 -11 Pa*m 3 /mole indicating that choli ne chloride will not rapidly evaporate into the atmosphere (OECD, 2004 citing BASF AG 2003c). In the

event that choline chloride does enter the atmosphere it is expected to be rapidly degraded based on the AOP v1.90 model calculation for OH-radicals (t 1/2 of approximately 6.9 hours based on a 12 hour day). (OECD, 2004 citing BASF AG 2004a)6 Table 1: Summary of physico-chemical properties 1

Property Value Remarks and Secondary

Reference Citations

Physical state white crystalline solid

Liquid, colorless, amine-like odor Pure choline chloride

Solution 75% w/w in water; BASF AG (2002)

Physical structure SCCNFP, 2003

Molecular formula C 5 H 14 NO.Cl

Synonyms Ethanaminium, 2-hydr oxy-N, N, N-trimethyl-, chloride; Biocoline; Cholinium chloride; Hepachloride; Lipotril; Luridin chloride; Paresan; Trimethyl (2-hydroxyethyl) ammonium chloride Confirmed with CAS Online

Melting point 247 °C The information corresponds to the pure substance; ICSC 0853 (2004) Boiling point Not applicable due to decomposition on heating Relative density 1.1 g/cm 3 at 20°C 70 ± 1% choline chloride. 30% water, less than 0.05% impurities measured; BASF AG (1974). Data refer to a technical data overview, no details of method used and year the study was performed is given. Further, as the value refers to a solution the density of the pure substance may

differ. Viscosity 21 mPa*s at 20°C 70 ± 1% choline chloride. 30% water, less than 0.05% impurities measured; BASF AG (1974). Data refer to a technical data overview, no details of method used and year the study was performed is given. Vapor pressure 6.57 x 10 -8 Pa at 25°C Calculated using MPBPWIN v1.40, refers to the pure substance; BASF AG (2003a) Water solubility Ca. 650 g/l 50% choline chloride powder; pH=6-7; measured; BASF AG (1974); Study reliability was not assignable. A calculated value for the pure substance of 1000g/l was estimated using WSKOW v1.40; BASF AG (2003b)

Partition coefficient n-octanol/water (logK

ow ) -3.77 at 25°C Solution 75% w/w in water; measured; BASF AG (1988a)
Henry’s law constant 2.06* 10E-11 Pa*m 3 /mole at 25°C Calculated using HENRYWIN v3.10 (bond method), refers to the pure substance; BASF AG (2003d) logK

oc 0.37 Calculated using the PCKOCWIN v1.66 calculation program (K oc = 2.3), refers to the pure substance; BASF AG (2003e)

1 Source of all information (except *)- OECD SIDS Initial Assessment Report (2004) for-Choline

Chloride7 III. METABOLISM / PHARMACOKINETICS

Choline is a necessary component of the human diet and acts as a precursor to the neurotrans mitter acetylcholine, phosph olipids, and the methyl donor betaine. The body requires choline wh ich it obtains through direct ingestion of foods/supplements containing choli ne or through a de novo biosynthesis process. According to the OECD SIDS document on cholin e chloride (2004),

There is an endogenous pathway for the de novo biosynthesis of the choline moiety via the sequential methylation of phosphatidyl ethanolamine using S-adenosylmethionine asthe methyl donor. Thus, the demand for dietary choline is modified by metabolic methylexchange relationships between choline and three nutrients: methionine, folate, and

vitamin B12.

This de novo process allows the body to produce choline; however, the body’s natural synthesis of choline is not sufficient to meet its needs and therefore, dietary choline is required. In light of this evidence, choline was recognized as an essential nutrient in 1998. Effects have been observed with regards to the liver, kidney and brain of choline deficient animals/humans in studies where choline has been withheld. (Po litizer Shronts, 1997; Zeisel et al., 1991)

Once ingested, choline is absorbed from the lumen of the small intestine. Prior to adsorption from the gastrointestina l tract some choline is metabolized by

internal bacteria to form betaine and me thylamines. Pancreatic enzymes can also breakdown dietary phosphatidylcholine (a .k.a. lecithin) to form free choline. Choline is important for t he structural integrity of cell membranes, cholinergic neurotransmission, transm embrane signaling, methyl metabolism and lipid and cholesterol transport and metabolism.

Chloride is a binary compound of chlori ne; a salt of hydrochloric acid. Chloride is the major extracellular anion an d contributes to many body functions including the maintenance of osmotic pressure, acid-base balance, muscular activity, and the movement of water between fluid compartments. The World Health Organization (WHO) has perform ed two assessments which determined that from a toxicological poi nt of view, there were no concerns for the chloride

ion. It was considered to be natur ally-occurring and a normal participant of

animal and human metabolism.

IV. TOXICOLOGY

A. Acute Toxicity

Oral

LD 50 levels reported in rats for cho line chloride show a range from 3150

and ≥ 6000 mg/kg. Restlessness, hypoactivity, increased frequency of8 respiration, convulsions, ruffled coa t, staggered gait, and dyspnea were reported. Some animals experienced diarrhea and in o ne study 3 out of 10 rats in the high dose group had inflamed lungs at necrop sy. (OECD, 2004) Si milarly, the LD 50 reported for mice was in t he range of 3900 to 6000 mg/kg.

Dermal

No acute dermal animal studies are available. An in vitro percutaneous absorption study was conducted in compliance with GLP under OECD draft guideline 428, using human epidermal skin. Liquid scintillation counting was used to determine skin absorption of radiolabelled ( 50 mCi of 1,2 -14 C) choline chloride. Skin samples were mounted on Franz-type diffusion cells, and [ 14 C]-choline chloride was applied to the surface of t he skin for 24 hours. Each sample area was dosed with a single dos e of approximately 10 μ l/cm2 of a 5% choline in water solution. Samples were evaluated at 1, 2, 6, 12, and 24 hours under occluded

and non-occluded conditions.

This study showed that 7.42 μ g/cm 2 and 13.86 μ g/cm 2 (1.9 % and 3.43 %) of the applied dose was absorbed under occluded and non-occluded conditions respectively, with most remaining in the epidermis (5.90 μ g/cm 2 and 10.7 μ g/ cm 2 ) and dermis (1.06 μ g/cm 2 and 2.40 μ g/cm 2 ). Only 0.457 μ g/cm 2 (0.127 %) and

0.383 μ g/cm 2 (0.110 %), of the applied dose penetrat ed into the receptor fluid for occluded and unoccluded cells, respectively. Under the conditions of this study, choline chloride is expected to have a low potential for pe rcutaneous absorption (SCCNFP, 2003 and SCCP, 2008).

Inhalation- No valid data available.

Skin Irritation

Minor skin irritation (questionable r eddening after 24 hour only) was noted

on the back of 1 of 2 rabbits treated with an aqueous formulation of the test substance (70% choline chloride and 30% water). Observations were made at 24hrs, 2, 3 or 8 days after exposure. This study, however, was an old non-GLP (Good Laboratory Practices), non-guideli ne study whose degree of irritation

would not be classifiable under the globally harmonized system (GHS). Compared to OECD TG 404, this test was harsher due to the longer exposure time (20 vs. 4 hrs) and the use of an occlusive rather than a semi-occlusive dressing. These factors may lead to an overestimation of the skin irritation capabilities of the test subs tance; therefore, the resu lts are more conservative

then current studies would indica te. (OECD, 2004 citing BASF AG, 1963)

Eye Irritation

An eye irritation study performed on choline chloride was conducted broadly to OECD test guidelin e 405, although, it did not comply with GLP due to9 the age of the study. One eye of a male and female rabbit were exposed to a 70% chlorine chloride aqueous solution. Th e other eye was used as the control. After 10 minutes reddening and tear secret ions were observed. Slight reddening persisted up to three hours after application. Observations were made at 1 and 3 hours post application and 1 and 8 days. At 1 day post application no effects were noted. (OECD, 2004 citing BASF AG, 1963c)

Skin Sensitization

No data are available on t he sensitization of choline chloride in animals. According to the OECD SIDS report ( 2004) on choline chloride, “the skin sensitization potential of chlorine chlori de for humans is regarded as negligible”. Furthermore, the Scientific Committ ee on Cosmetic Products and Non-food Products Intended for Consumers (SCCNFP) is of the opi nion that dermal exposure to choline chloride, in rinse o ff products at 5%, is not anticipated to pose any serious risk. (2003)

B. Repeat Dose Studies

Although studies have been conducted to evaluate the possible medicinal

benefits of choline chloride in humans, acceptable r epeat dose animal studies

are limited. Available animal st udies have been presented below.

Male and female Balb/c mice were treated with 200mg/kg/day orally or intranasally for 28 days. For orally exposed animals, 200 mg/kg/day was administered by oral gavage once daily until day 28. Intranasally exposed animals were given 200mg/kg/day in a 50 μl vehicle every other day for 28 days. Choline chloride administration by both the oral and intranasal routes of exposure

did not cause adverse effects with r egards to body weight, food and water consumption, hematology, clinical biochem istry (Urea, BUN, HDL, TC, ALT, AST, and creatinine), or histopathology of vari ous organs (lung, heart, liver, spleen, and kidney). Results from intranasal exposure to choline chloride were comparable with their respective cont rols and to other treatment groups (intraperitoneal exposure was also assesse d). (Mehta, 2009) The NOAEL for oral and intranasally administer ed choline chloride is ≥ 200 mg/kg/day.

In a limited 72 week feeding study, Fischer 344 rats were given approximately 500 mg/kg/day of choline ch loride. Animals were observed for 30 weeks post exposure during which time the test animals and the controls

received the same untreated diet. Hist opathology performed at week 103 was restricted to the liver and organs that developed gross abnormalities. There were no significant difference between the cont rol and treated group in relation to body weights, relative liver weight, or surv ival rates. The number of neoplastic liver nodules, hepatocellular carcinomas, lung tumo rs, leukemia, or other tumors was not increased in treated animals. This study resulted in a NOAEL of 500 mg/kg/day. (OECD, 2004 ci ting Shivapurkar, 1986)10 C. Neurotoxicity

Choline is a known precursor to a vi tal neurotransmitter, acetylcholine. The peripheral nervous system uses acetyl choline in skeletal muscle movement, as well as in the regulation of smooth muscle and cardiac muscle. In the central nervous system acetylcholine is believed to be involved in learning, memory, and

mood. Choline chloride has been evaluat ed for its medicinal use in treating neurological conditions such as tardiv e dyskinesia, Alzheimers, Huntington’s disease, ataxia, and memory loss in the el derly. (Mohs et al., 1979; Snell et al., 1980)

Two studies which evaluated the effects of choline chloride on memory in rats showed a positive correlation betw een choline and memory functions and a third study showed no effect. The first study supplemented ra ts prenatally by adding choline to the drinking water of their dams. Another treatment group was made deficient of choline during early devel opment by withhold it from the dam’s diet. A third group was used as controls.

After being maintained on a choline-su fficient diet for 120 days postnatally, the rats began 12-arm radial maze traini ng. The results of the study (Meck and Williams, 1999) indicated:

Both choline-supplemented and choline-defic ient rats performed more accurately than control littermates when trials were spaced. These differences in spatial ability did not appear to be a function of differential re sponse or cue-use strategies. Cholinesupplemented rats showed littl e proactive interference when trials were massed; whereas

control rats demonstrated moderate levels an d choline-deficient rats exhibited high levels of proactive interference as a function of massed trials. These data suggest that the behavioral consequences of early dietary availability of choline may involve the

modification of the discriminative abilities us ed to attend to stimuli that demarcate the end

of one trial and the start of another as well as the capacity for remembering the locations that have been visited during a trial.

A second study utilized a T-maze design to investigate the effect of choline supplementation on memory c onsolidation in young female SpragueDawley rats. One group of rats receiv ed choline chloride daily for two weeks and a second group was used as a control. In the dark area (DA) of the maze, which is naturally preferred by the rat, an electric foot-shock (0.1 mA at 60V) was

delivered when the rats entered the area. Both the treated and control groups showed a preference for the DA and15 minutes after passive avoidance both groups avoided entry into the DA. This suggests that both groups had acquired short-term memory of t he negative stimuli; however, when the test was repeated 24 hours after training the control group di d not avoid entry to the DA, whereas the group who received the choline suppl ements either avoided entry or entered after a significantly longer latency peri od. According to the author, “These results suggest that supplementation with choline facilitated the consolidation of short-11 term memory of the avoidanc e learning into intermediat e long-term memory in young rats.” (Gossell-W illiams et al., 2006)

In the third study, male Long-Evans rats were fed diets cont aining either (i) choline chloride 1250 mg/L, (ii) choli ne chloride 250 mg/L and GD3 24 mg/L, or (iii) choline chloride 250 mg/L on postnat al days 5-18. A fourth group was reared normally. The study found “all rats were equa lly proficient at using spatial shortterm memory, regardless of the choline or ganglioside content of the preweaning diet”. (Mohs et al., 1979)

D. Carcinogenicity/Mutagencity

In vitro studies designed to investigat e the ability of choline chloride to induce gene mutation in bacteria were negative. Three Ames test were conducted with Salmonella typhimurium strains TA 98, 100, 1535, 1537, and/or 1538, up to 10,000 μg/plate and E. coli WP 2 uvrA up to 5,000 μg/plate with and without metabolic activation. Although all of the studies were tested up to at least the limit dose of 5 mg/plate, two of the te sts did not show bacterial toxicity. The third study showed a 50% survival at the highest dose tested. (OECD, 2004 citing Haworth et al., 1984; JETOC, 1997; Litton Bioneti cs, 1977; and NTP, 1983).

NTP reported on three experiments desi gned to look at choline chlorides potential to cause chromosomal aberrations. In one study a small but statistically significant increase in simple aberrations was seen at 50 and 500 μg/ml (the highest dose tested) in the absence of S9 only. Two reliable Chinese Hamster Ovary Cell studies, however, showed that cytotoxicity (50% survival) only

occurred at choline chloride concentra tion of 5000 μg/ml. One hundred cells were examined per dose and treatme nt group and S9 from Aroclor induced rat livers

were used for metabolic activation (OECD, 2004 citing Bloom et al., 1982; Galloway et al., 1985; and NTP, 1984).

Two parallel sister chromatid exchange assays at two different

laboratories were conducted on Chinese Ha mster Ovary cells at concentrations up to 500 and 5000 μg/ml, respectively and gave ambiguous results. According to OECD SIDS (2004),

Cytotoxicity (50% survival) was observed at 5000 μg/ml Metabolic activation was with S9 from Aroclor induced rate livers. Approximately 1000 chromosomes were examined per dose and treatment group. The increase (spor adic and not dose related) in SCEs that was observed with metabolic activation in laboratory two was not reproduced in laboratory one. Laboratory one showed a weak po sitive at the top dose without metabolic activation, but a comparison with laboratory two was not possible due to insufficient numbers of cells analyzed.

A third study using Chinese hamster ov ary cells showed no increase in the

number of sister chroma tid exchanges at concentrations up to 5000 μg/ml with and without metabolic activation. (OECD, 2004 citing NTP 1984) Negative results12 were also obtained from a gene conversion assay using Saccharomyces

cerevisiae strain D4 with choline chloride concentration between 12.5 and 50 mg/ml with and without metabolic activation. (OECD, 2004 citing Litton Bionetics, 1977).

The SIDS document concluded that;

Choline chloride does not have any structural alerts for genotoxicity. It did not produce gene mutations, clastogenicity, or DNA damage when tested in vitro. It can be concluded

from these studies that choline chlori de does not have any mutagenic potential.

An epidemiologic study released in 2007 evaluated the relationship

between choline and betaine intake and t he risk of colorectal adenoma in US

women enrolled in the Nurses’ Health Study. The study evaluated responses from female registered nurses aged 30- 55 years who responded to a baseline questionnaire on lifestyle and major illnesse s in 1976. Every 2 years, a follow-up questionnaire was sent to the women to update information regarding diet and lifestyle and to ascertain new diagnoses of major illnesses. The study found that higher choline intake was associated with an elevated risk of colorectal adenoma; however, the investigators stated that: (Cho et al., 2007)

Our findings do not support an inverse association between choline intake and risk of colorectal adenoma. The positive association between choline intake and colorectal adenoma that we observed could represent effe cts of other components in the foods from
which choline was derived and should be investigated further

.

On the contrary, multiple studies hav e indicated that choline many have an inverse relationship to breast cancer risk. (Xu et al., 2008; Shannon et al., 2005; Frazier et al., 2003)

E. Reproductive/Developmental

Choline has been shown to have positive effects on development. Research suggests that adequate amount s of choline during pregnancy and breastfeeding can help ensure healthy fe tal brain development. Studies have shown that pregnant women consuming a choline deficient diet have a significantly higher rate of neural tube and or al cleft defects in their offspring. (Xu et al., 2008) Adequate prenatal choline leve ls may also have long-lasting positive effects on cognitive function, including memory. However, sufficient research has

not been done on the effects of chol ine on pregnant women and their unborn

babies.

A developmental toxicity study ex posed NMRI mice to 1250, 4160, 10800, or 20000 mg/kg/day every other day from gestation day 1 to 18. In all but the lowest group, maternal body weight gain was reduced. Those exposed to ≥ 4,160 mg/kg/day showed almost no net weight gain and those exposed to the highest tested dose (20,000 mg/kg/day) showed weight loss. Embryonic/fetal lethality13 was recorded at 35% and 69% fo r those tested with 4,160 and 10,800 mg/kg/day, respectively. No resorptions occurred in the lowest dose group; conversely, all fetuses in the highest dose group were resorbed. There was no statistically significant, dose related increases in malformations between the treated and control groups. (OECD, 2004 citing BASF AG, 1966)

In all but the lowest dosed group, dev elopmental toxicity was observed; however, it was not observed in the absence of maternal toxicity. It is important

to bare in mind that all of the levels test ed in this study are much greater than the

usual limit dose used for a non-toxic compound (i.e. 1000 mg/kg/day). There

were no observed effects for thos e both mothers and pups tested with 1250 mg/kg/day; therefore, the NOAEL for mater nal toxicity and developmental toxicity is 1250 mg/kg/day. Due to a lack of suffi cient pups, a NOEAL for teratogenicity was not determined. (OECD, 2004 citing BASF AG 1966)

Currently, there is a study sponsored by the National Inst itute of Mental Health that will “evaluate the safety and effectiveness of taking choline supplements during pregnancy and whether taking choline during pregnancy will have an effect on infant development”. The study is expected to be completed in March 2010. (NIMH, 2006-2010 proposed)

F. Endocrine

EPA is required under the FFDCA, as amended by FQPA, to develop a screening program to determi ne whether certain substances (including all pesticide active and other ingredients) “m ay have an effect in humans that is similar to an effect produced by a natur ally-occurring estrogen, or other such endocrine effects as the Administrator may designate''. Choline chloride is not a known endocrine disruptors nor is it or it s metabolites related to any class of known endocrine disruptors.

G. Special Consideration for Infants and Children (FQPA Safety Factor)

FFDCA section 408 provides that EPA shall apply an additional tenfold margin of exposure (safety) for infant s and children in the case of threshold effects to account for prenat al and postnatal toxicity and the completeness of the

data base unless EPA determines that a diffe rent margin of exposure (safety) will

be safe for infants and children.

Choline is a natural component of a variety of commonly consumed foods (e.g. (per100g food)- eggs (251mg) , wheat germ (152 mg), bacon (125 mg), dried soybeans (116 mg), pork ( 103 mg), cod (83 mg), beef (80 mg), chicken (70 mg), and salmon (65 mg))(USDA, 2004). It has been added as a supplement to infant formula in the US for decades (Politizer Shronts, 1997). In addition to dietary consumption, c holine is made endogenously in the human body.14 Choline chloride has been used as a widespread nutrient in animal feed

since the 1930’s without adverse effects r eported on fertility or teratogenicity. Although one study in mice did show devel opmental effects, they were only seen at very high doses ( ≥ 4,160 mg/kg/day) and only in the presence of maternal toxicity. There were no observed adverse effects for both mothers and pups exposed to 1250 mg/kg/day. Evidence has shown that choline has beneficial properties in regards to proper growth and development and neurological

function.

Exposure to choline chloride not ex pected to significantly increase the pre-existing levels found in commonly eaten foods. Choline chloride residues on food is expected to be minimal. Because of its high water solubility most of the inert will be washed from the plant prior to consumption. Once in water, it will be broken into in a quaternary hydroxyl al kylammonium ion and a chloride ion.

Due to the low toxicity of the chemical, the bodies need for choline from a dietary source, and the beneficial role c holine plays in fetal development; the safety factor has been reduced to 1 X.

V. ENVIRONMENTAL FATE AND DRINKING WATER

Physical chemical properties coupled with modeling indicate that water is expected to be the preferred media for choline chloride. The Mackay model

showed a 100% water distribution and t he PCKOCWIN model resulted in a log

K oc of 0.37, indicating that choline chlori de will not adsorb to soil and sediment or suspended solids. Once in water choline chloride, a quaternary ammonium salt, dissociates resulting in a positively charged quaternary hydrox yl alkylammonium ion and a negatively charged chloride ion. (OECD, 2004) Cholin e chloride is not expected to undergo hydrolysis.

According to the OECD-criteria, c holine chloride is readily biodegradable (MITI I-Test: 93% biodegradati on within 14 days). A BOD 5 test performed on

choline chloride obtained a BOD 5 /ThOD 5 ratio of 75% which also indicates that choline chloride is readily biodegradabl e. (OECD, 2004 citing MITI, 1992 and BASF AG, 1984) Although estimated bioconcentration factors (BCF) for choline chloride vary between models, all resu lts indicated that the potential for

bioaccumulation is low.

Using the HENRYWIN model to calcul ate Henry’s Law Constant resulted

in 2.06 X10 -11 Pa*m 3 /mole indicating that choli ne chloride will not rapidly evaporate into the atmosphere. (OECD, 2004 citing BASF AG, 2003c) In the atmosphere choline chloride is expect ed to be rapidly degraded based on the AOP v1.90 model calculation for OH-radicals (t 1/2 of approximately 6.9 hours based on a 12 hour day) (OECD, 2004 citing BASF AG, 2004a).15 Choline, an essential dietary nutrient, is present in a variety of foods and is synthesized by the body. Because of its nat ural presence in the environment and its readily biodegradable nature, choline chloride when used as an inert ingredient in pesticide formulations, is not expected to significantly increase the levels of naturally occurring cholin e drinking water or the environment.

VI. EXPOSURE ASSESSMENT

A. Exposure Profile

Humans are currently exposed to choline on a daily basis through commonly eaten foods (both naturally occurring and when added as a nutrient) and through the bodies natural ability to synt hesize the nutrient. It is unlikely that the exposure from choline chloride, w hen used as an inert ingredient applied preharvest to food commodities, will signific antly increase the natural concentration of choline present in foods. Because of it s high water solubility it is expected that

most of the inert will be washed from the plant prior to consumption. Once in water, it will be broken into in a quat ernary hydroxyl alkylammonium ion and a

chloride ion. Choline is an essential nut rient and a requirement in many of the

bodies functions.

B. Aggregate Exposure

Section 408 of the Federal Food, Dr ug, and Cosmetic Act (FFDCA) directs the Agency to evaluate aggregate exposure from “pesticide chemical residue and to other related substances, including dietary exposure under the tolerance and

all other tolerances in effect for th e pesticide chemical residue, and exposure from other non-occupational sources”. The expected exposure pathway for choline chloride is through the oral and the dermal routes of exposure.

Although a method is not require to determine the residues on food when an exemption from tolerance is grant ed it is expected that because of the low oral and dermal toxicity, the rapi d degradation of the chemical, and the natural presence of choline and chlori de in the environment, aggregate exposure will result in minimal risk to all subpopu lation including infants and children. Based on the physical and chemical properties , the inhalation route is not a likely exposure pathway; theref ore, the anticipated risk fr om inhalation exposure is considered minimal. (Cho et al., 2007)

C. Cumulative Exposure

Section 408(b)(2)(D)(v) of FFDCA requires that, when considering whether to establish, m odify, or revoke a tolerance, the Agency consider "available information" concerning the cumulative effects of a particular pesticide's residues and "other subst ances that have a common mechanism of toxicity." Unlike other pesticides for which EPA has followed a cumulative risk16 approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as to choline chloride and any other substances, and these chemicals do not appear to produce toxic metabolites produced by other substances. For the purposes of this tolerance action, therefore, EPA has not assu med that choline chloride has a common mechanism of toxicity with other substances. Fo r information regarding EPA's efforts to

determine which chemicals have a commo n mechanism of toxicity and to

evaluate the cumulative effects of such chemicals, see the policy statements released by EPA's Office of Pesticide Programs concerning common mechanism determinations and procedures for cumulati ng effects from substances found to

have a common mechanism on EPA's website at http://www.epa.gov/pesticides/cumulative/

VII. RISK CHARACTERIZATION

Choline chloride is a quaternary ammonium salt which dissociates in water resulting in a positively charged quater nary hydroxyl alkylammonium ion and a negatively charged chloride ion. (OECD, 2004) Choline is an essential component of the human diet and acts as a precursor to acetylcholine, phospholipids, and the methyl donor betaine. It is important fo r the structural integrity of cell membranes, choli nergic neurotransmission, transmembrane signaling, methyl metabolism, and lipid and cholesterol transport and

metabolism.

Choline is found naturally in foods (e .g. eggs, liver meats, fungi, soybeans, fish, nuts, beans, and wheat germ) in both esterified forms (e.g. phosphocholine, glycerophosphocholine, sh ingomyeline, and phosphatid ylchoine) and as free choline. Historically, it has been used as a food additive for animal husbandry, in plant growth regulator formulations , and as food supplements for humans. Choline was officially made an “essentia l nutrient” in 1998. The adequate intake (AI) level for choline in women is 425 mg/day and 450 mg/day for pregnant women. Men and lactating women have a sl ightly higher AI at 550 mg/day. Daily Upper Intake Level for choline is 3.5 grams for adults. (NAS, 1998) Research on choline in the diets of the US pop ulation using the NHANES, 2003–2004 data indicates that many individuals are not getting enough choline, with daily intake levels far below the AI. (Jensen et al., 2007)

Chloride is a binary compound of chlori ne; a salt of hydrochloric acid. Chloride is the major extracellular anion an d contributes to many body functions including the maintenance of osmotic pressure, acid-base balance, muscular activity, and the movement of water between fluid compartments. The World Health Organization has performed two assessments which det ermined that from a toxicological point of view, there were no concerns for the chloride ion. It was considered to be naturally-occurring and a normal participant of animal and

human metabolism.17 Choline chloride has demonstrated a low acute oral toxicity with LD 50 values for rats ranging from 3150 to ≥ 6000 mg/kg and LD 50 for mice in the range of 3900 to 6000 mg/kg. Alt hough appropriate animals studies are lacking for acute dermal toxicity, an in vitro percutaneous absorption study performed under

occluded and unoccluded conditions showed that choline chloride is expected to have a low potential for percutaneous absor ption. Acceptable acute inhalation

studies are not available.

Studies were conducted in the early 1960’s to evaluate the eye and skin irritation potential of choline chloride. Because their age, the studies were not performed under OECD guidelines and therefore the degree of irritation is not classifiable under the Globally Harmoniz ed System of Classif ication and Labeling of Chemicals (GHS). Although these re sults would not be classifiable under GHS, it is important to note that only slight transient irritation was observed in the skin and eye irritation studies. Choline chloride is not expected to cause skin

sensitization.

Repeat dose animal studies on cholin e chloride are limited. One study using male and female Balb/c mice, evaluated the impact of 200 mg/kg/day choline chloride given orally or intranasally for 28 days. No adverse effects were observed with regards to body weight, food and water consumption, hematology, clinical biochemistry (Urea, BUN, HDL, TC, ALT, AST, and creatinine), or histopathology of various organs (lung, hear t, liver, spleen, and kidney). Results from intranasal exposure to choline ch loride were comparable with their respective controls and to other treatm ent groups (intraperitoneal exposure was also assessed). The NOAEL for oral and intranasally administered choline

chloride is ≥ 200 mg/kg/day.

A 72 week feeding study administered 500 mg/kg/day of choline chloride to Fischer 344 rats and observed the animals for 30 weeks post exposure during

which time the test animals and the controls received the same untreated diet. There were no significant difference bet ween the control and treated group in relation to body weights, relative liver weight, survival rates, and the number of neoplastic liver nodules, hepatocellular carcinomas, lung tumors, leukemia, or other tumors. This study resulted in a NOAEL of 500 mg/kg.

Choline is a precursor to the vital neurotransmitter acetylcholine. Studies

show that choline has beneficial effe cts on the nervous system and memory. Choline is necessary to promote proper development in the fetus and infant and prevent cognitive problems. Choline chlori de not expected to cause neurotoxicity.

Choline chloride is not a known endoc rine disruptors nor is it or its metabolites related to any class of known endocrine disruptors. Based on the results of the in vitro studies the Agen cy feels that choline chloride is not expected to be carcinogenic or mutagenic.18 Since the 1930’s choline chloride has been used as a widespread nutrient in animal feed without adverse effects repor ted on fertility or te ratogenicity. The Food and Drug Administration (FDA) requi res choline be added to non-milk

based infant formulas at a minimum concentration of 7 mg for every 100 kilocalories (21 CFR 107.100). Although one study did show developmental effects, they were only seen at very high doses ( ≥ 4,160 mg/kg/day) and only in the presence of maternal toxicity. Ther e were no observed adverse effects for both mothers and pups exposed to 1250 mg/kg/day. Based on this information the Agency does not feel that choline ch loride, when used as an inert ingredient, will cause reproductive or developmental toxicity and therefore, does not anticipate an increased risk to infants and children.

Choline has demonstrated a beneficia l effect on ischemic cardiac arrhythmias and preventing liver and r enal dysfunction. (Liu, 2008, Canadian journal; Zeisel, 2000). Recent studies sugge sted that cholines anti-inflammatory properties may be an effective treatment for asthma, a chronic inflammatory disease. (Mehta et al., 2007) Other doc umented benefits include a reduction in DNA hypomethylation and tumor development in the liver.

In addition to its low toxicity, exposure to choline chloride will be limited. The expected exposure pathway is via the oral and the dermal routes. Humans are currently exposed to choline on a daily basis through commonly eaten foods (both naturally occurring and when added as a nutrient) and through the bodies natural ability to synthesize the nutrient. It is unlikely that the exposure from choline chloride, when used as an iner t ingredient applied pr e-harvest to food commodities, will significantly increase t he natural concentration of choline in foods. Choline chloride is readily bi odegradable and because of its high water solubility it is expected that most of the inert will be wa shed from the plant prior to consumption. Once in wate r, its preferred media, it will be broken into in a quaternary hydroxyl alkylammoni um ion and a chloride ion.

Similar to humans, choline is a di etary requirement for many animals including avian and aquatic species. Based on this information, the biodegradability of the chem ical, and the available toxicity data, the Agency feels that choline chloride is unlikely to be ha rmful to mammalian, aquatic, and avian organisms.

In light of the prevalence of choline in the diet, the body’s natural ability to synthesis it, and its low toxicity the Agency does not feel that there is an

increased risk to humans from the use of cho line chloride as an inert ingredient in pesticide products. Therefore, choline chloride has been approved as an inert ingredient in pesticide products used on all food commodities under 40 CFR

180.920.19 ECOTOXOCITY

Similar to humans, choline is a di etary requirement for many animals including avian and aquatic species. Based on this information, the biodegradability of the chem ical, and the available toxicity data (Table 2), the Agency feels that choline chloride is unlikely to be harmful to mammalian, aquatic, and avian organisms.

Acute toxicity testing in fish resulted in a LC50 (96h) for oryzias latipes

and limanda limanda at >100mg/L and > 1000mg/L, respectively. For the

invertebrate Daphnia magna the EC50 (48h) is 349 mg/L and the NOEC (21d) is 30.2 mg/L.20 Table 2: Acute toxicity of choline chloride to aquatic organisms 2

2 Table adopted from OECD-SIDS on Choline Chloride (2004) Species Method Effect

Concentration (mg/L) Remark/Reference

Acute Toxicity to Fish

Oryzias latipes (freshwater species) OECD 203 (flowthrough system) LC 50 (96h) > 100 (nominal and measured) purity of test substance: 100.2 %, reliability: 1; MOE Japan (1999a),

KEY STUDY

Leuciscus idus (freshwater species) DIN 38412, part 15,

static LC 50 (96h) > 10,000 (nominal ) no symptoms detectable; two tests with a) 78 % choline chloride watery solution (BASF AG, 1988b) and b) 50 % choline chloride as powder (BASF AG, 1988c) are available, non GLP, no analytics (reliability 2)

Limanda limanda (marine species) according to OECD 203, semistatic LC 50 (96h) > 1,000 (nominal) 75 % choline chloride watery solution; limit test (only 1,000 mg/L tested); ICI (1983) (reliability 2)

Acute and chronic toxicity to aquatic invertebrates

Daphnia magna (freshwater species) OECD 202 (static) EC 50 (48h) = 349 mg/l (nominal and measured) purity of test substance: 100.2 %, reliability: 1; MOE Japan (1999b),

KEY STUDY

Daphnia magna (freshwater species) Directive 79/831 EEC, C2, static LC 50 (48h) > 500 (nominal) NOEC (48hr) = 125 78 % choline chloride watery solution; non GLP, no analytics (reliability 2); BASF AG, 2003g

Daphnia magna (freshwater species) according OECD 211 (renewal system) NOEC (21d) = 30.2 mg/L (nominal and measured) purity of test substance: 100.2 %, reliability: 1; MOE Japan (1999c),

KEY STUDY

Acute toxicity to aquatic plants e.g. algae

Pseudokirchneriella subcapitata (freshwater species) OECD 201 ErC 50 (72h) > 1,000 (nominal and measured), 72h NOEC (growth rate)

= 32 purity of test substance: 100.2 %, reliability: 1; MOE Japan (1999d),

KEY STUDY

Scenedesmus subspicatus (fresh water) DIN 38412, part 9,

static Er(b)C 50 (72h) > 500(nominal), 72h

NOEC (growth rate and biomass) >500 78 % choline chloride watery solution; non GLP, no analytics (reliability 2); BASF AG, 2003h21 Bibliography

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