United States Department of Agriculture Agricultural Marketing Service | National Organic Program

Document Cover Sheet https://www.ams.usda.gov/rules-regulations/organic/national-list/petitioned

Document Type:

☐ National List Petition or Petition Update

A petition is a request to amend the USDA National Organic Program’s National List of Allowed and Prohibited Substances (National List). Any person may submit a petition to have a substance evaluated by the National Organic Standards Board (7 CFR 205.607(a)). Guidelines for submitting a petition are available in the NOP Handbook as NOP 3011, National List Petition Guidelines. Petitions are posted for the public on the NOP website for Petitioned Substances.

☒ Technical Report

A technical report is developed in response to a petition to amend the National List. Reports are also developed to assist in the review of substances that are already on the National List. Technical reports are completed by third-party contractors and are available to the public on the NOP website for Petitioned Substances. Contractor names and dates completed are available in the report.

Xylazine/Tolazoline Livestock

___________________________________ January 4, 2019 Technical Evaluation Report Page 1 of 17

Compiled by Savan Group for the USDA National Organic Program

1 Identification of Petitioned Substance 2

3 Chemical Names: 4 Xylazine 5 Xylazine 6 Xylazine Hydrochloride 7 Xylazine Monohydrochloride 8 N-(2,6-Diphenylmethyl)-5,6-dihydro-4H-1,3-9 thiazin-2-amine 10 N-(2,6-Diphenylmethyl)-5,6-dihydro-4H-1,3-11 thiazin-2-amine hydrochloride 12 13 Tolazoline 14 Tolazoline 15 Tolazoline hydrochloride 16 2-Benzylimidazoline 17 2-Benzyl-4,5-dihydro-1H-imidazole 18 2-Benzyl-4,5-dihydro-1H-imidazole 19 hydrochloride 20 21 Other Names: 22 Xylazine 23 Xylazine HCl 24 Xylaxine 25 Xylazin 26 Xilazina 27 Xylazinium 28 Xylazine Chloride 29 30 Tolazoline 31 Tolazoline HCl 32 Tolazoline Chloride 33 Imidaline Hydrochloride 34 Benzidazol 35 Benzalolin 36 Benzazoline Hydrochloride 37 Pridazole 38 Phenylmethylimidazoline 39 Tolazolinum 40 Tolazolina 41 Peripherin 42

43 Trade Names: 44 Xylazine 45 AnaSed® LA 46 Rompun® 47 TranquiVed Injection 48 X-Ject SA 49 XylaMed™Celactal 50 51 Tolazoline 52 Tolazil™ 53 Tolazoline® Injection 54 Priscoline 55 Priscoline Hydrochloride 56 Divascol 57 Olitensol 58 Lambril 59 Arterodyl 60 Priscol 61 62 CAS Numbers: 63 7361-61-7 (Xylazine) 64 23076-35-9 (Xylazine Hydrochloride) 65 59-98-3 (Tolazoline) 66 59-97-2 (Tolazoline Hydrochloride) 67 68 Other Codes: 69 EC No. 230-902-1 (Xylazine) 70 NSC No. 758142 (Xylazine) 71 UNII No. 2KF9TP5V8 (Xylazine) 72 EC No. 245-417-0 (Xylazine Hydrochloride) 73 UNII No. NGC3S0882S (Xylazine Hydrochloride) 74 EC No. 200-448-9 (Tolazoline) 75 NSC No. 35110 (Tolazoline) 76 UNII No. CHH9H12AQ3 (Tolazoline) 77 EC No. 200-447-3 (Tolazoline Hydrochloride) 78 NSC No. 757353 (Tolazoline Hydrochloride) 79 UNII No. E669Z6S1JG (Tolazoline Hydrochloride)80

81 Summary of Petitioned Use 82

83 The United States Department of Agriculture (USDA)’s National Organic Program (NOP) has approved xylazine 84 and tolazoline for medicinal applications in organic livestock production. Both xylazine and tolazoline are 85 restricted to “use by or on the lawful written or oral order of a licensed veterinarian,” and must be followed by “a 86 meat withdrawal period of at least 8 days after administering to livestock intended for slaughter; and a milk 87 discard period of at least 4 days after administering to dairy animals,” at Title 7 of the Code of Federal 88 Regulations (CFR) Section 205.603. Tolazoline is further restricted for “use only to reverse the effects of sedation 89

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and analgesia caused by Xylazine,” at §205.603. This technical report outlines the veterinary applications of 90 xylazine for organic livestock production and serves to update a previous technical report from 2002 (USDA 91 2002b). 92 93

Characterization of Petitioned Substance 94 95 Composition of the Substance: 96 97 Xylazine 98 99 Xylazine (Figure 1) is a synthetic α2-adrenergic agonist, which Farbenfabriken Bayer developed in 1962 to 100 treat hypertension (Kreeger et al. 1986a, Kreeger et al. 1986b, Greene and Thurmon 1988, EMEA 1999, 101 Lester et al. 2012, Thies et al. 2017). Xylazine is a molecule in the clonidine family, and the basic sites on the 102 molecule (nitrogen and sulfur atoms with electron pairs) provide a basic structure important for the 103 biological absorption and distribution of the substance (Garcia-Villar et al. 1981, Greene and Thurmon 104 1988). Xylazine’s neurological activity has resulted in veterinary medicinal uses. The substance is 105 commonly administered as both the neutral compound (Figure 1) and its hydrochloride salt (PubChem 106 5707, PubChem 68554, Kreeger et al. 1986a, Kreeger et al. 1986b, Lester et al. 2012, Sigma-Aldrich 2014b, 107 Flecknell 2016, Sigma-Aldrich 2017). 108 109 Tolazoline 110 111 Tolazoline (Figure 1) is a synthetic α2-adrenergic antagonist, which also interacts with histamine and 112 cholinergic receptors (Goetzman and Milstein 1979, McIntosh and Waters 1979, Pawson 2008, Greenough et 113 al. 2012, Ebert 2013). Like xylazine, tolazoline is in the clonidine family and shares the basic attributes 114 (nitrogen atoms with electron pairs) that enable its rapid biological absorption and distribution 115 (Garcia-Villar et al. 1981, Greene and Thurmon 1988). Structural similarities with xylazine allow tolazoline 116 to compete with xylazine for biological binding sites. This provides the mode of action for its approved use 117 in organic livestock production as a reversal agent for xylazine (Levy et al. 1977, Goetzman and Milstein 118 1979, Kreeger et al. 1986a, Kreeger 1986b, Samanta et al. 1990, JECFA 1998a, JECFA 1998b, Pawson 2008). 119 Tolazoline’s neurological activity has resulted in its use in veterinary medicine and is commonly 120 administered as both the neutral compound (Figure 1) and its hydrochloride salt (PubChem 5504, 121 PubChem 6048, Kreeger et al. 1986a, Kreeger et al. 1986b, Sigma-Aldrich 2006, Pawson 2008, Rotta et al. 122 2011, Ebert 2013, Coleman and Cox 2014, Sigma-Aldrich 2014a). 123 124

HN

S

N

HN

N

Xylazine Tolazoline 125 126

Figure 1 127 128 Source or Origin of the Substance: 129 130 Xylazine 131 132 Xylazine is a synthetic substance produced by the multi-step reaction of 2,6-dimethylaniline, as shown in 133 Scheme 1 on the next page (Elliot and Ruehle 1985). 134

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O

O O HN

O

Nao

N

O

SCS

NC

S

H2N OH

HCl

HN NH

O

OH

HN N

S

135 136

Scheme 1 137 138 Tolazoline 139 140 Tolazoline is a synthetic substance that is produced by a one-pot process (i.e., no intermediates are isolated) 141 via the reaction of phenylacetaldehyde with ethylene diamine, with the incorporation of an iodine-based 142 oxidation process, as shown in Scheme 2 (Gogoi and Konwar 2006). 143 144

OH2N

NH2N

NH2

I2 / KIBase

HN

N

I

HN

N

145 146

Scheme 2 147 148 Properties of the Substance: 149 The properties xylazine, xylazine hydrochloride, tolazoline, and tolazoline hydrochloride are summarized in 150 Table 1. 151 152 153 154 155 156

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157 Table 1. Properties of xylazine, xylazine hydrochloride, tolazoline, and tolazoline hydrochloride 158

Compound Xylazine Xylazine Hydrochloride

Tolazoline Tolazoline Hydrochloride

CAS No. 7361-61-7 23076-35-9 59-98-3 59-97-2 Molecular Weight 220.33 g/mol 256.79 g/mol 160.22 g/mol 196.68 g/mol

Appearance White powder Solid White powder Solid Melting Point 141 oC N/A 66-69 oC 174-176 oC

Solubility 0.5 g/L (methanol) N/A 373 mg/L (water) N/A Sources: PubChem 5504, PubChem 5707, PubChem 6048, PubChem 68554, Sigma-Aldrich 2006, 159 Sigma-Aldrich 2014a, Sigma-Aldrich 2014b, Sigma-Aldrich 2017. 160 161 Specific Uses of the Substance: 162 163 Xylazine 164 165 The synthetic substance xylazine was originally developed to treat hypertension in humans (Kreeger et al. 166 1986a, Kreeger et al. 1986b, Greene and Thurmon 1988, EMEA 1999, Lester et al. 2012, Thies et al. 2017). 167 However, the prevalence of its side effects, including the onset of significant cardiac arrythmias, has 168 prohibited its use in human medicine (Green et al. 1981, EMEA 1999, Reyes et al. 2012). 169 170 Despite negative side effects in humans, xylazine is widely used in veterinary medicine, where the 171 frequency and danger of side effects are reduced. Xylazine, due to depression of the central nervous system 172 and cardiac outputs, is commonly used as a sedative and analgesic in veterinary medicine (EMEA 1999, 173 Lorenz et al. 2010, Lester et al. 2012, Otto and von Thaden 2012, Ruiz-Colon et al. 2014, Silva-Torres et al. 174 2014, Thies et al. 2017). Xylazine is the most common co-treatment for ketamine anesthetic applications 175 (Green et al. 1981, Kreeger et al. 1986a, Kreeger et al. 1986b, Saha et al. 2005, Reyes et al. 2012). 176 177 The United States Food and Drug Administration (FDA) has restricted the veterinary application of 178 xylazine with the designation “do not use in domestic food-producing animals,” at 21 CFR 522.2662. 179 Xylazine is also used as an emetic for cats, as an alternative to surgery to recover foreign objects (Thies et 180 al. 2017). 181 182 Tolazoline 183 184 Tolazoline is used in both human and veterinary medical applications. In human medicine, tolazoline is 185 used to treat hypotension, hypertension, newborn respiratory distress, and congenital heart disease 186 through interactions with α2-adrenergic and histamine receptors (Grover et al. 1961, Cotton 1965, Korones 187 and Eyal 1975, Yellin et al. 1975, Levy et al. 1977, Goetzman and Milstein 1979, McIntosh and Walters 1979, 188 Kreeger et al. 1986a, Greenough et al. 2012). These treatments rely on the ability of the substance to induce 189 vasodilation, increasing arterial oxygenation (Levy et al. 1977, Kreeger et al. 1986a, Kreeger et al. 1986b). 190 191 Tolazoline is commonly used in veterinary medicine as a reversal agent for xylazine sedation (Levy et al. 192 1977, Goetzman and Milstein 1979, Kreeger et al. 1986a, Kreeger 1986b, Samanta et al. 1990, JECFA 1998a, 193 JECFA 1998b, Pawson 2008). Tolazoline competes with xylazine for α2-adrenergic binding sites; once 194 bound, it blocks xylazine from interacting with the receptor (Goetzman and Milstein 1979, McIntosh and 195 Waters 1979, Pawson 2008, Greenough et al. 2012, Ebert 2013). Tolazoline’s side effects, such as increased 196 cardiac and respiratory responses (e.g., tachycardia), work to reverse xylazine’s common side effects (e.g., 197 bradycardia, respiratory depression) (Yellin et al. 1975, Levy et al. 1977, Goetzman and Milstein 1979, 198 Kreeger et al. 1986a, Kreeger et al. 1986b). 199 200

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Approved Legal Uses of the Substance: 201 202 Xylazine 203 204 USDA allows xylazine for veterinary applications within organic livestock production at 7 CFR 205.603, 205 restricting it for use “by or on the lawful written or oral order of a licensed veterinarian,” and must be 206 followed by “a meat withdrawal period of at least 8 days after administering to livestock intended for 207 slaughter; and a milk discard period of at least 4 days after administering to dairy animals.” 208 209 The FDA has approved the use of xylazine as an animal drug administered through implantation or 210 injection “to produce sedation, as an analgesic, and as a preanesthetic to local or general anesthesia,” the 211 application of which is restricted “to use by or on the order of a licensed veterinarian,” at 21 CFR 522.2662. 212 213 The FDA also gives the following species-specific conditions for xylazine’s use, at §522.2662: 214 215

Dogs and cats – amount 0.5 mg/pound (lb) intravenously or 1.0 mg/lb subcutaneously; horses – 0.5 mg/lb 216 intravenously or 1.0 mg/lb intramuscularly; Elk and deer – administer intramuscularly, by hand syringe, or 217 by syringe dart, in the heavy muscles of the croup or shoulder as follows: Elk (Cervus canadensis) 0.25 to 0.5 218 mg/lb; mule deer (Odocoileus hemionus), sika deer (Cervus nippon), and white-tailed deer (Odocoileus 219 virginianus): 1 to 2 mg/lb; Fallow deer (Dama dama): 2 to 4 mg/lb. 220

221 For the application of xylazine in horses, the regulation stipulates that it must not be used “in horses 222 intended for human consumption.” The limits on xylazine’s use in agricultural settings are extended 223 provided that xylazine is not for “use in domestic food-producing animals,” at §522.2662. 224 225 Tolazoline 226 227 USDA allows tolazoline for veterinary applications within organic livestock production at 7 CFR 205.603, 228 with the restriction to “use only to reverse the effects of sedation and analgesia caused by xylazine, by or 229 on the lawful written or oral order of a licensed veterinarian,” and must be followed by “a meat 230 withdrawal period of at least 8 days after administering to livestock intended for slaughter; and a milk 231 discard period of at least 4 days after administering to dairy animals.” 232 233 The FDA has approved tolazoline as an animal drug administered to horses through implantation or 234 injection “when it is desirable to reverse the effects of sedation and analgesia caused by xylazine,” at 21 235 CFR 522.2474. The FDA has stipulated that tolazoline be administered to horses “slowly by intravenous 236 injection 4 mg per kilogram of body weight or 1.8 mg per pound (4 milliliters (mL) per 100 kilograms or 4 237 mL per 220 pounds).” Like xylazine, tolazoline may be administered, provided it not be used in “horses 238 intended for human consumption. Federal law restricts this drug to use by or on the order of a licensed 239 veterinarian,” at §522.2474. 240 241 Action of the Substance: 242 243 Xylazine 244 245 Xylazine stimulates the α2-adrenergic receptors in the presynaptic space, which inhibits the release of the 246 neurotransmitters norepinephrine and dopamine (Starke 1977, Hsu 1981, Kreeger et al. 1986a, Ruiz-Colon 247 et al. 2014, Silva-Torres et al. 2014). The inhibited release of these neurotransmitters produces a range of 248 physiological responses, including muscle relaxation, pain relief (analgesia), neural transmission inhibition 249 (giving a general depression of activity to the central nervous system), respiratory system depression 250 (transient hypotension and hypertension, bradycardia, cardiac arrythmias) (Garcia-Villar et al. 1981, Green 251 et al. 1981, Kreeger et al. 1986a, Kreeger et al. 1986b, JECFA 1998a, Delehant et al. 2003, Lester et al. 2012, 252 Otto and von Thaden 2012, Ruiz-Colon et al. 2014, Thies et al. 2017). Xylazine has also been documented to 253 produce transient hyperglycemia due to reduction of insulin levels (Greene and Thurmon 1988, Samanta et 254 al. 1990, JECFA 1998a, JECFA 1998b, Saha et al. 2005). 255 256

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Xylazine is administered through several methods including intravenously, intramuscularly, or 257 subcutaneously (Garcia-Villar et al. 1981, Reyes et al. 2012). Upon administration, xylazine is rapidly 258 absorbed and distributed throughout the body (Garcia-Villar et al. 1981, JECFA 1998a, JECFA 1998b). The 259 substance is also readily metabolized to approximately 20 metabolites, depending on the species in 260 question (JECFA 1998a, EMEA 1999). Xylazine is also rapidly removed from the body—mostly through 261 metabolism but also through excretion—with biological half-lives of 1-58 (Samanta et al. 1990, JECFA 262 1998a, EMEA 1999). 263 264 Due to the rapid distribution and metabolism of xylazine, the physiological effects of xylazine begin to 265 subside shortly after administration of the drug (15-90 minutes), although the effects (e.g., sedation, 266 bradycardia, respiratory depression) are not completely reversed until the bulk of the substance has been 267 metabolized (2-36 hours) (Garcia-Villar et al. 1981, Green et al. 1981, Samanta et al. 1990, EMEA 1999). The 268 substance and its metabolites are excreted in urine and feces (EMEA 1999). The effective dosage and 269 resulting physiological effects are species-dependent, with ruminants (cattle, sheep) exhibiting elevated 270 sensitivity to xylazine; therefore, their veterinary doses are adjusted to approximately 1/10th that of other 271 species (Garcia-Villar et al. 1981, Greene and Thurmon 1988, JECFA 1998a, EMEA 1999). 272 273 Tolazoline 274 275 Tolazoline is most commonly used as a reversal agent for sedatives, including xylazine, by competing for 276 α2-adrenergic receptors, blocking binding events for xylazine (Levy et al. 1977, Goetzman and Milstein 277 1979, Kreeger et al. 1986a, Kreeger 1986b, Samanta et al. 1990, JECFA 1998a, JECFA 1998b, Pawson 2008). 278 Tolazoline is a broad-spectrum α2-adrenergic antagonist with cholinergic and histamine interactions that 279 are not well-defined (Grover 1961, Goetzman and Milstein 1979, McIntosh and Walters 1979, Pawson 2008, 280 Rotta et al. 2011, Greenough et al. 2012, Ebert 2013, Coleman and Cox 2014). Therefore, the physiological 281 responses resulting from tolazoline administration do not have well-defined mechanistic biochemical 282 explanations, with some effects being attributed to α2-adrenergic and histamine receptor interactions 283 (Grover 1961, Yellin et al. 1975, McIntosh and Walters 1979, Kreeger et al. 1986a, Pawson 2008, Ebert 2013, 284 Coleman and Cox 2014). 285 286 Tolazoline affords several physiological effects, including vasodilation (increasing arterial oxygenation), 287 transient hypotension, and histaminic gastrointestinal effects (Cotton 1965, Korones and Eyal 1975, Yellin et 288 al. 1975, Levy et al. 1977, McIntosh and Walters 1979, Greenough et al. 2012). The α2-adrenergic 289 antagonistic effects of tolazoline result in increased central nervous system and respiratory activity, directly 290 reversing xylazine-induced responses (Yellin et al. 1975, Pieter et al. 1982, Kreeger et al. 1986a, Kreeger et 291 al. 1986b, JECFA 1998a, Pawson 2008, Coleman and Cox 2014). However, the effective dosage for xylazine 292 reversal is dependent on species and the applied xylazine dosage (Kreeger et al. 1986a, Kreeger et al. 293 1986b). 294 295 Tolazoline is administered intravenously, via inhalation (Greenough et al. 2012, Ebert 2013, Coleman and 296 Cox 2014). Tolazoline is rapidly absorbed and distributed throughout the body following administration 297 (Ebert 2013). Tolazoline metabolism is species-dependent; however, in many species, the administered 298 tolazoline is excreted intact or with only minor metabolism (Ebert 2013). Tolazoline has a biological half-life 299 of 313 hours and is largely excreted in urine 2-4 weeks following its administration (Ebert 2013). 300 301 Combinations of the Substance: 302 303 Xylazine 304 305 Xylazine is used as an anesthetic and analgesic in veterinary medicine, both as an individual substance, 306 and as a co-treatment in combination with ketamine (Green et al. 1981, Kreeger et al. 1986a, Kreeger et al. 307 1986b, JECFA 1998a, Saha et al. 2005, Lester et al. 2012, Otto and von Thaden 2012, Reyes et al. 2012, 308 Flecknell 2016). Studies have shown that, when the substance is used in combination with ketamine, the 309 resulting sedation/analgesia is more effective than applications of ketamine alone (Green et al. 1981, 310 Kreeger et al. 1986a, Otto and von Thaden 2012). Moreover, the incorporation of xylazine as a co-treatment 311

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for ketamine-induced sedation and analgesia has been shown to reduce the ketamine’s side effects (Green 312 et al. 1981, Saha et al. 2005, Otto and von Thaden 2012). 313 314 Tolazoline 315 316 Tolazoline is not commonly used in combination with other substances (USDA 2002b). Studies have shown 317 that tolazoline is effective for the reversal of xylazine and xylazine/ketamine–combined induced sedations 318 (Kreeger et al. 1986a). 319 320

Status 321 322 Historic Use: 323 324 Xylazine 325 326 Xylazine has been historically used in veterinary surgical applications as an anesthetic and analgesic 327 (Green et al. 1981, Kreeger et al. 1986a, Kreeger et al. 1986b, JECFA 1998a, JECFA 1998b, Saha et al. 2005, 328 Lester et al. 2012, Otto and von Thaden 2012, Reyes et al. 2012, Flecknell 2016). The interaction of the 329 substance with α2-adreneceptors results in the inhibition of norepinephrine, producing a physiological 330 response including depression activity of the central nervous system (CNS) and resulting in sedation 331 (EMEA 1999, Lorenz et al. 2010, Lester et al. 2012, Otto and von Thaden 2012, Ruiz-Colon et al. 2014, 332 Silva-Torres et al. 2014, Thies et al. 2017). As previously mentioned, the substance is also used as an emetic 333 for cats as a veterinary alternative to surgical procedures to remove foreign objects from the intestinal tract 334 (Thies et al. 2017). 335 336 Tolazoline 337 338 Tolazoline has been historically used in veterinary medicine as a reversal agent for xylazine sedation in 339 post-surgery applications (Levy et al. 1977, Goetzman and Milstein 1979, Kreeger et al. 1986a, Kreeger 340 1986b, Samanta et al. 1990, JECFA 1998a, JECFA 1998b, Pawson 2008). Tolazoline competes with xylazine 341 for α2-adrenergic binding sites; once bound, it blocks xylazine from interacting with the receptor 342 (Goetzman and Milstein 1979, McIntosh and Waters 1979, Pawson 2008, Greenough et al. 2012, Ebert 2013). 343 344 Organic Foods Production Act, USDA Final Rule: 345 Neither xylazine nor tolazoline are listed in the Organic Foods Production Act of 1990 (OFPA). Both 346 xylazine and tolazoline are restricted to “use by or on the lawful written or oral order of a licensed 347 veterinarian,” and must be followed by “a meat withdrawal period of at least 8 days after administering to 348 livestock intended for slaughter; and a milk discard period of at least 4 days after administering to dairy 349 animals” at 7 CFR 205.603. Tolazoline has the additional restriction for “use only to reverse the effects of 350 sedation and analgesia caused by Xylazine,” at §205.603. 351 352 International 353 354 Canadian General Standards Board Permitted Substances List 355 Xylazine is listed in the CAN/CGSB-32.311-2015 — Organic production systems - permitted substances list 356 in Table 5.3 “health care products and production aids,” as a “sedative.” 357 358 Tolazoline is not listed in the CAN/CGSB-32.311-2015 — Organic production systems - permitted 359 substances list. 360 361 CODEX Alimentarius Commission, Guidelines for the Production, Processing, Labelling and Marketing 362 of Organically Produced Foods (GL 32-1999) 363 Neither xylazine nor tolazoline are listed in the CODEX. 364 365 European Economic Community (EEC) Council Regulation, EC No. 834/2007 and 889/2008 366

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Neither xylazine nor tolazoline are listed in the EEC EC No. 834/2007 or 889/2008. 367 368 Japan Agricultural Standard (JAS) for Organic Production 369 Neither xylazine nor tolazoline are listed in the JAS for Organic Production. 370 371 International Federation of Organic Agriculture Movements (IFOAM) 372 Neither xylazine nor tolazoline are listed in IFOAM. 373 374

Evaluation Questions for Substances to Be Used in Organic Crop or Livestock Production 375 376 Evaluation Question #1: Indicate which category in OFPA that the substance falls under: (A) Does the 377 substance contain an active ingredient in any of the following categories: copper and sulfur compounds, 378 toxins derived from bacteria; pheromones, soaps, horticultural oils, fish emulsions, treated seed, 379 vitamins and minerals; livestock parasiticides and medicines and production aids including netting, 380 tree wraps and seals, insect traps, sticky barriers, row covers, and equipment cleansers? (B) Is the 381 substance a synthetic inert ingredient that is not classified by the EPA as inerts of toxicological concern 382 (i.e., EPA List 4 inerts) (7 U.S.C. § 6517(c)(1)(B)(ii))? Is the synthetic substance an inert ingredient which 383 is not on EPA List 4, but is exempt from a requirement of a tolerance, per 40 CFR part 180? 384 385

(A) Xylazine and tolazoline are active ingredients for veterinary medicines. Xylazine is primarily used 386 as a sedative, tranquilizer, and an analgesic (Garcia-Villar et al. 1981, Kreeger et al. 1986a, Kreeger 387 et al. 1986b, JECFA 1998a, JECFA 1998b, EMEA 1999, Saha et al. 2005, Lorenz et al. 2010, Lester et 388 al. 2012, Otto and von Thaden 2012, Flecknell 2016, Thies et al. 2017). The interactions between the 389 substance and α2-adrenceptors results in the depression of the central nervous and respiratory 390 systems, inducing sedation and analgesia (EMEA 1999, Lorenz et al. 2010, Lester et al. 2012, Otto 391 and von Thaden 2012, Ruiz-Colon et al. 2014, Silva-Torres et al. 2014, Thies et al. 2017). 392

393 Tolazoline is used in veterinary medicine as a reversal agent for xylazine (Levy et al. 1977, 394 Goetzman and Milstein 1979, Kreeger et al. 1986a, Kreeger 1986b, Samanta et al. 1990, JECFA 395 1998a, JECFA 1998b, Pawson 2008). 396

397 (B) Neither xylazine nor tolazoline are listed by the EPA as an inert ingredient of toxicological concern. 398

399 Evaluation Question #2: Describe the most prevalent processes used to manufacture or formulate the 400 petitioned substance. Further, describe any chemical change that may occur during manufacture or 401 formulation of the petitioned substance when this substance is extracted from naturally occurring plant, 402 animal, or mineral sources (7 U.S.C. § 6502 (21)). 403 404 Xylazine is a synthetic substance produced from the multi-step process outlined in Scheme 1 (Source or 405 Origin of the Substance). 2,6-dimethylanaline is initially employed as a nucleophile, reacting with the 406 electrophilic acetic anhydride to form the resulting amide. The amide is reduced in the presence of sodium 407 metal (Nao), to form a reactive intermediate that readily reacts with carbon disulfide to yield 408 2,6-dimethylisothiocyanate. The electrophilic nature of the central carbon on the isothiocyanate moiety 409 undergoes nucleophilic attack to yield N-(2,6-Dimethylphenyl)-5,6-dihydro-4H-1,3-thiazin-2amine; this 410 undergoes subsequent intramolecular ring-closure under the acidic reaction conditions to give the final 411 xylazine product, which is collected in solid form via filtration (Elliot and Ruehle 1985). 412 413 Tolazoline is a synthetic substance that is produced by a one-pot process (i.e., no intermediates are isolated) 414 by the reaction of phenylacetaldehyde with ethylene diamine, with the incorporation of an iodine-based 415 oxidation process as shown in Scheme 2 (Source or Origin of the Substance) (Gogoi and Konwar 2006). 416 417 In the synthesis of tolazoline phenylacetaldehyde undergoes nucleophilic attack by ethylene diamine to 418 produce a Schiff base. The Schiff base undergoes an intramolecular ring-closure via electrophilic attack of 419 the remaining amine functionality at the electrophilic imine carbon; this in turn undergoes subsequent 420 oxidation with an iodine/potassium iodide mixture (I2/KI). The final product is formed by the elimination 421

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of the iodide to form the imidazolidine ring, which is isolated as an oil via extraction and subsequent 422 evaporation (Gogoi and Konwar 2006). 423

424 Evaluation Question #3: Discuss whether the petitioned substance is formulated or manufactured by a 425 chemical process, or created by naturally occurring biological processes (7 U.S.C. § 6502 (21)). 426 427 Xylazine is a synthetic substance that is produced through the multi-step reaction of 2,6-dimethylanaline as 428 shown in Scheme 1 on the previous page (Elliot and Ruehle 1985). Tolazoline is a synthetic substance that is 429 produced by a one-pot process (i.e., no intermediates are isolated) by the reaction of phenylacetaldehyde 430 with ethylene diamine, with the incorporation of an iodine-based oxidation process as shown in Scheme 2 431 on the previous page (Gogoi and Konwar 2006). 432 433 Evaluation Question #4: Describe the persistence or concentration of the petitioned substance and/or its 434 by-products in the environment (7 U.S.C. § 6518 (m) (2)). 435 436 Xylazine was first identified as a potential aquatic contaminant in rivers on the Iberian Peninsula in 437 concentrations of 50–100 ng/L (Fabrega et al. 2013, Pugajeva et al. 2017). Fabrega and his colleagues 438 identified xylazine as “one of the chemicals with the highest contribution to the total IRCAP value in the 439 different river basins” (Fabrega et al. 2013). However, these results may not be indicative of typical use in 440 veterinary medicine, as JSC Grindeks, a global producer of the substance, is also located on the Iberian 441 Peninsula (Pugajeva et al. 2017). 442 443 Studies on the persistence and activity of xylazine in the soil also support the conclusion that xylazine may 444 contribute to water pollution, due to the mobility within soils and the leaching potential of the substance 445 (Choi et al. 2014). Choi and coworkers have reported that xylazine has a slow rate of dissipation and 446 degradation within soil systems that may result in its environmental accumulation, a trait that may be 447 linked to its relatively small size (Choi et al. 2014, Pugajeva et al. 2017). Choi et al. (2014) were limited to 448 artificial soil environments (laboratory tests rather than actual environmental conditions), which are likely 449 to impede dissipation and degradation of the substances examined. 450 451 The by-products of xylazine were not discussed in the above studies by Fabrega et al., Pugajeva et al., and 452 Choi et al. 453 454 Evaluation Question #5: Describe the toxicity and mode of action of the substance and of its breakdown 455 products and any contaminants. Describe the persistence and areas of concentration in the environment 456 of the substance and its breakdown products (7 U.S.C. § 6518 (m) (2)). 457 458 Environmental studies on xylazine are discussed in Question #4 and highlight the possible persistence of 459 the substance and its accumulation in soil systems as well as its role as an aquatic pollutant (Fabrega et al. 460 2013, Choi et al. 2014, Pugajeva et al. 2017). 461 462 There are no reported studies on the environmental toxicity, persistence, or concentration of tolazoline. 463 464 As described in the Characterization of Petitioned Substance section, xylazine is a neurologically active 465 compound that interacts with α2-adreneceptors, histamine, and cholinergic receptors, resulting in sedation 466 and analgesia (Garcia-Villar et al. 1981, Kreeger et al. 1986a, Kreeger et al. 1986b, JECFA 1998a, JECFA 467 1998b, EMEA 1999, Saha et al. 2005, Lorenz et al. 2010, Lester et al. 2012, Otto and von Thaden 2012, 468 Flecknell 2016, Thies et al. 2017). However, the neurological activity of xylazine also results in undesired 469 side effects, including inhibition of neural transmissions, giving a general depression of activity to the 470 central nervous system; depression of the respiratory system (transient hypotension and hypertension, 471 bradycardia, cardiac arrythmias); and transient hyperglycemia due to reduction of insulin levels 472 (Garcia-Villar et al. 1981, Green et al. 1981, Kreeger et al. 1986a, Kreeger et al. 1986b, Greene and Thurmon 473 1988, Samanta et al. 1990, JECFA 1998a, Delehant et al. 2003, Saha et al. 2005, Lester et al. 2012, Otto and 474 von Thaden 2012, Ruiz-Colon et al. 2014, Thies et al. 2017). 475 476

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Signs of xylazine toxicity are typically the result of continued application, with re-administration before the 477 previous doses have been removed from the system (Veilleux-Lemieux et al. 2013). Xylazine has been 478 identified as a substance with moderate toxicity, giving an LD50 of 121–240 mg/kg in mice and rat studies 479 (JECFA 1998a, JECFA 1998b). Across species, studies indicate that rats and mice are less sensitive to 480 xylazine than other, larger species (JECFA 1998a). For instance, studies with cats rendered an LD50 of 100 – 481 110 mg/kg, while dogs and horses have still higher sensitivities with LD50s of 20 -47 mg/kg and 15 – 70 482 mg/kg, respectively (JECFA 1998a). 483 484 Xylazine is readily metabolized to approximately 20 metabolites, depending on the species in question 485 (JECFA 1998a, EMEA 1999). Among the metabolites, 2,6-xylidine has been investigated for toxicological 486 effects (JECFA 1998a). Xylidine is present in tobacco smoke and is a degradation product of aniline 487 derived-pesticides; therefore, the toxicological effects of the substance have been studied (JECFA 1998a). 488 Xylidine has been identified as a substance with slight toxicity, having an LD50 of 600 – 1000 mg/kg in mice 489 and rat studies (JECFA 1998a). The species dependency of xylidine is not been well-developed, with 490 toxicity studies performed on laboratory mice and rat subjects. 491 492 Carcinogenicity studies have been negative or inconclusive, with no significant findings from single-dose 493 exposures, although the JECFA noted that 2,6-xylidine has genotoxic and carcinogenic character (NTP 494 1990, JECFA 1998a, JECFA 1998b). When multiple doses of xylidine were administered to Charles River 495 rats (10 doses per day), xylidine began accumulating within the body, resulting in significant incidences of 496 nasal cavity adenomas and carcinomas (NTP 1990). These results show that multiple doses and 497 bioaccumulation of xylidine reveal a carcinogenic nature; as a result of such multiple-dose studies, xylidine 498 has been classified as a 2B substance, which may be carcinogenic to humans (IARC 1993). 499 500 Tolazoline is a synthetic α2-adrenergic antagonist that also interacts with histamine and cholinergic 501 receptors in a temporary and reversible manner (Goetzman and Milstein 1979, McIntosh and Waters 1979, 502 Kreeger et al. 1986a, Kreeger et al. 1986b, Samanta et al. 1990, Pawson 2008, Greenough et al. 2012, Ebert 503 2013). Tolazoline affords several physiological effects, including vasodilation (increasing arterial 504 oxygenation), transient hypotension, histaminic gastrointestinal effects (Cotton 1965, Korones and Eyal 505 1975, Yellin et al. 1975, Levy et al. 1977, McIntosh and Walters 1979, Greenough et al. 2012). There are no 506 published toxicity or carcinogenicity studies on the toxicity or lethal dosages of tolazoline. 507 508 Evaluation Question #6: Describe any environmental contamination that could result from the 509 petitioned substance’s manufacture, use, misuse, or disposal (7 U.S.C. § 6518 (m) (3)). 510 511 Environmental studies on xylazine are discussed in Question #4 and highlight the possible persistence of 512 the substance and its accumulation in soil systems as well as its role as an aquatic pollutant (Fabrega et al. 513 2013, Choi et al. 2014, Pugajeva et al. 2017). Reports of xylazine environmental contamination on the 514 Iberian Peninsula may be linked with xylazine manufacturing, resulting in high contributions to water 515 pollution in Iberian river systems (Fabrega et al. 2013, Pugajeva et al. 2017). The leaching ability of xylazine 516 and its reported slow degradation in aquatic systems make wastewater pollution a concern in cases of 517 improper use or disposal (Fabrega et al. 2013, Choi et al. 2014, Pugajeva et al. 2017). 518 519 There are no reported studies on the environmental toxicity, persistence, or concentration of tolazoline. 520 521 Evaluation Question #7: Describe any known chemical interactions between the petitioned substance 522 and other substances used in organic crop or livestock production or handling. Describe any 523 environmental or human health effects from these chemical interactions (7 U.S.C. § 6518 (m) (1)). 524 525 Xylazine and tolazoline are synthetic substances with documented reactivity towards α2-adrenergic, 526 histamine, and cholinergic receptors (Goetzman and Milstein 1979, McIntosh and Waters 1979, EMEA 1999, 527 Pawson 2008, Lorenz et al. 2010, Greenough et al. 2012, Lester et al. 2012, Otto and von Thaden 2012, Ebert 528 2013, Ruiz-Colon et al. 2014, Silva-Torres et al. 2014, Thies et al. 2017). Due to their neurological activities, 529 specifically their interactions with cholinergic receptors, they may cause interactions with atropine, a 530 cholinergic antagonist (USDA 2002a). Atropine has been approved for use in organic livestock for the 531

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treatment of organophosphate poisoning and acts via competition with acetylcholine for binding 532 interactions with choline receptors (USDA 2002a). Moreover, reports of atropine interactions with xylazine, 533 as with tolazoline, show some ability to reverse the physiological effects of xylazine sedation (Greene and 534 Thurmon 1988, Ruiz-Colon et al. 2014). 535 536 As Figure 1 depicts in the Characterization of the Petitioned Substances section, the structures of xylazine 537 and tolazoline both feature nitrogen atoms with localized electron pairs; this structure renders them 538 efficient bases. As such, the substances will react with acids (Scheme 3), resulting in xylazine and tolazoline 539 salts, with the cation being supplied by the acid used in the reaction. Due to the basic nature of the 540 substance, it is likely to undergo neutralization reactions with allowed organic acids such as peracetic acid 541 (7 CFR 205.601(a)), ammonium carbonate (7 CFR 205.601(e)), boric acid (7 CFR 205.601(e)), humic acids (7 542 CFR 205.601(j)), sulfurous acid (7 CFR 205.601(j)), phosphoric acid (7 CFR 205.603(a)), and formic acid (7 543 CFR 205.603(b)). 544 545

546 547

Scheme 3 548 549 Due to the ionic nature of the product (xylazine or tolazoline salt), with its identity defined by the acid 550 used in the reaction (identity of R for acids shown in Scheme 3), the effects of potential salts are difficult to 551 predict. Xylazine and tolazoline salts (hydrochlorides) are used for medicinal purposes, and the substances 552 are likely to maintain their medicinal activity in salt forms (PubChem 5504, PubChem 5707, PubChem 6048, 553 PubChem 68554, Sigma-Aldrich 2006, Sigma-Aldrich 2014a, Sigma-Aldrich 2014b, Sigma-Aldrich 2017). 554 However, due to the charged nature of the salt, the ionic form of the substance may be absorbed differently 555 from the neutral form, which could influence the biological delivery mechanisms. 556 557 As discussed throughout this technical report, xylazine and tolazoline have both been approved for 558 veterinary applications within organic livestock production. These substances interact with one another by 559 competition for neurological activity by interactions with α2-adreneceptors, cholinergic, and histamine 560 receptors (Levy et al. 1977, Goetzman and Milstein 1979, Kreeger et al. 1986a, Kreeger 1986b, Samanta et al. 561 1990, JECFA 1998a, JECFA 1998b, Pawson 2008). 562 563 Due to the veterinary applications of the substances for approved organic use, they are unlikely to be 564 combined with any of the above acids. Undesirable chemical reactions are unlikely to occur when used as 565 approved, making environmental and human health concerns unlikely. 566 567 Evaluation Question #8: Describe any effects of the petitioned substance on biological or chemical 568 interactions in the agro-ecosystem, including physiological effects on soil organisms (including the salt 569 index and solubility of the soil), crops, and livestock (7 U.S.C. § 6518 (m) (5)). 570 571 Environmental studies on xylazine are discussed in Question #4 and highlight the possible persistence of 572 the substance and its accumulation in soil systems as well as its role as an aquatic pollutant (Fabrega et al. 573

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2013, Choi et al. 2014, Pugajeva et al. 2017). However, these studies do not discuss the physiological 574 impacts of the substance on the environment. 575 576 There are no reported studies on the environmental toxicity, persistence, or concentration of tolazoline. 577 578 As discussed in Evaluation Question #5, xylazine and tolazoline are substances with demonstrated 579 neurological activity used in veterinary medicine; therefore, they will likely elicit physiological responses if 580 absorbed by livestock. These responses include inhibition of neural transmissions, giving a general 581 depression of activity to the central nervous system, depression of the respiratory system (transient 582 hypotension and hypertension, bradycardia, cardiac arrythmias), and transient hyperglycemia due to 583 reduction of insulin levels vasodilation (increasing arterial oxygenation), and histaminic gastrointestinal 584 effects (Cotton 1965, Korones and Eyal 1975, Yellin et al. 1975, Levy et al. 1977, McIntosh and Walters 1979, 585 Garcia-Villar et al. 1981, Green et al. 1981, Kreeger et al. 1986a, Kreeger et al. 1986b, Greene and Thurmon 586 1988, Samanta et al. 1990, JECFA 1998a, Delehant et al. 2003, Saha et al. 2005, Greenough et al. 2012, Lester 587 et al. 2012, Otto and von Thaden 2012, Ruiz-Colon et al. 2014, Thies et al. 2017). The degree of expression 588 induced by the substance is species-dependent, with ruminants showing increased sensitivity 589 (Garcia-Villar et al. 1981, Greene and Thurmon 1988, JECFA 1998a, EMEA 1999). 590 591 Evaluation Question #9: Discuss and summarize findings on whether the use of the petitioned 592 substance may be harmful to the environment (7 U.S.C. § 6517 (c) (1) (A) (i) and 7 U.S.C. § 6517 (c) (2) (A) 593 (i)). 594 595 Environmental studies on xylazine are discussed in Question #4 and highlight the possible persistence of 596 the substance and its accumulation in soil systems as well as its role as an aquatic pollutant (Fabrega et al. 597 2013, Choi et al. 2014, Pugajeva et al. 2017). While these studies report the possibility of xylazine 598 environmental contamination, they do not discuss the effects of the substance beyond its persistence in soil 599 and aquatic systems. 600 601 There are no reported studies on the environmental toxicity, persistence, or concentration of tolazoline. 602 603 Evaluation Question #10: Describe and summarize any reported effects upon human health from use of 604 the petitioned substance (7 U.S.C. § 6517 (c) (1) (A) (i), 7 U.S.C. § 6517 (c) (2) (A) (i)) and 7 U.S.C. § 6518 605 (m) (4)). 606 607 Farbenfabriken Bayer developed xylazine, a synthetic α2-adrenergic agonist, in 1962 to treat human 608 hypertension (Kreeger et al. 1986a Kreeger et al. 1986b, Greene and Thurmon 1988, EMEA 1999, Lester et al. 609 2012, Thies et al. 2017). Xylazine is a substance with potent hypnotic and muscle-relaxation properties. The 610 side effects of xylazine include significant cardiac arrythmias, which has resulted in its lack of approval for 611 human medical applications (Green et al. 1981, EMEA 1999, Reyes et al. 2012). Due to the lack of approval 612 for use in human medical applications, information on the mode of action and toxicity of xylazine is 613 limited. 614 615 Reported cases of xylazine in humans have shown physiological effects like those seen in veterinary 616 applications (Samanta et al. 1990, JECFA 1998a). Upon absorption of xylazine, patients were difficult to 617 rouse and showed signs of confusion (indicative of central nervous system and neuropathic depression) 618 and expressed symptoms of bradycardia, hypotension (respiratory depression), and hyperglycemia 619 (Gallanosa et al. 1981, Spoerke et al. 1986, Samanta et al. 1990). These symptoms indicate that xylazine 620 operates through similar biochemical mechanisms in humans as in veterinary species. This is likely due to 621 interactions with α2-adreneceptors and histamine and cholinergic receptors (JECFA 1998a). With regard to 622 human carcinogenicity, no studies of direct effects have been published; however, the IARC has designated 623 the xylazine metabolite xylidine as potentially carcinogenic to humans based on studies with laboratory 624 animals (NTP 1990, IARC 1993, JECFA 1998a). 625 626 The lethal dosage of xylazine in humans is not well known and appears to vary dramatically between 627 individuals (Spoerke et al. 1986, Ruiz-Colon et al. 2014). Fatal doses of xylazine recorded have been as low 628

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as 40 mg, while other individuals have survived exposure to levels as high as 2400 mg (Spoerke et al. 1986, 629 Ruiz-Colon et al. 2014). 630 631 Tolazoline is a synthetic substance that offers broad-spectrum α2-adreneergic antagonism and interacts 632 with histamine and cholinergic receptors (Goetzman and Milstein 1979, McIntosh and Waters 1979, 633 Pawson 2008, Greenough et al. 2012, Ebert 2013). Tolazoline is used in both human and veterinary medical 634 applications. In human medicine, tolazoline is used to treat hypotension, hypertension, newborn 635 respiratory distress, and congenital heart disease through interactions with α2-adrenergic and histamine 636 receptors (Grover et al. 1961, Cotton 1965, Korones and Eyal 1975, Yellin et al. 1975, Levy et al. 1977, 637 Goetzman and Milstein 1979, McIntosh and Walters 1979, Kreeger et al. 1986a, Greenough et al. 2012). 638 These treatments rely on the ability of the substance to induce vasodilation, increasing arterial oxygenation 639 (Levy et al. 1977, Kreeger et al. 1986a, Kreeger et al. 1986b). However, no studies on the toxicity, 640 carcinogenicity, or lethal dosages of tolazoline have been published. 641 642 Evaluation Question #11: Describe all natural (non-synthetic) substances or products which may be 643 used in place of a petitioned substance (7 U.S.C. § 6517 (c) (1) (A) (ii)). Provide a list of allowed 644 substances that may be used in place of the petitioned substance (7 U.S.C. § 6518 (m) (6)). 645 646 Xylazine and tolazoline are active ingredients for veterinary medicines. Xylazine is primarily used as a 647 sedative, tranquilizer, and an analgesic (Garcia-Villar et al. 1981, Kreeger et al. 1986a, Kreeger et al. 1986b, 648 JECFA 1998a, JECFA 1998b, EMEA 1999, Saha et al. 2005, Lorenz et al. 2010, Lester et al. 2012, Otto and von 649 Thaden 2012, Flecknell 2016, Thies et al. 2017). No natural alternatives are common for either substance 650 (i.e., a sedative alternative for xylazine or a xylazine-reversal agent as a tolazoline alternative). Moreover, 651 while there are several synthetic alternatives for both substances, no other synthetic alternatives have been 652 approved by the USDA for use in organic agricultural production. 653 654 Evaluation Question #12: Describe any alternative practices that would make the use of the petitioned 655 substance unnecessary (7 U.S.C. § 6518 (m) (6)). 656 657 As described in Evaluation Question #11, the substances are used only for veterinary applications, with no 658 natural alternatives or USDA-approved synthetic alternatives. No alternative practices that would make 659 the anesthetic agent unnecessary exist. 660 661 Tolazoline may be made unnecessary by allowing the veterinary subject to recover from the effects of 662 xylazine by natural metabolism of the substance, rather than its active reversal. However, the rate of 663 xylazine metabolism is species-dependent; therefore, this may prove problematic in species with slower 664 metabolic rates (e.g., cattle) (Garcia-Villar et al. 1981, Green et al. 1981, Samanta et al. 1990, EMEA 1999). 665 The active reversal of xylazine sedation also reverses xylazine side effects (e.g., bradycardia, 666 hyperglycemia), which may be necessary to treat unexpected medical conditions and responses to xylazine 667 administration (Yellin et al. 1975, Pieter et al. 1982, Kreeger et al. 1986a, Kreeger et al. 1986b, JECFA 1998a, 668 Pawson 2008, Coleman and Cox 2014). 669 670

Report Authorship 671 672 The following individuals were involved in research, data collection, writing, editing, and/or final 673 approval of this report: 674 675

• Philip Shivokevich, Visiting Assistant Professor of Chemistry, University of Massachusetts 676 Amherst 677

• Samantha Olsen, Technical Writer, Savan Group 678 679 All individuals are in compliance with Federal Acquisition Regulations (FAR) Subpart 3.11—Preventing 680 Personal Conflicts of Interest for Contractor Employees Performing Acquisition Functions. 681 682

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References 683 684 Choi JH, Lamshift M, Zuhlke S, El-Aty AMA, Raman MM, Kim SW, Shim JH, Spiteller M. 2014. Analyses of 685 decreasing patterns of veterinary antianxiety medications in soils. Journal of Hazardous Materials. 275: 686 154-165. 687 688 Coleman JJ, Cox AR. 2014. A worldwide yearly survey of new data in adverse drug reaction and 689 interactions. In Side Effects of Drugs Annual. Waltham, MA: Elsevier. 690 691 Cotton EK. 1965. The use of priscoline in treatment of the hypoperfusion syndrome. Pediatrics. 36: 149. 692 693 Delehant TM, Denhart JW, Lloyd WE, Powell JD. 2003. Pharmacokinetics of xylazine, 2,6-dimethylaniline, 694 and tolazoline in tissues from yearling cattle and milk from mature dairy cows after sedation with xylazine 695 hydrochloride and reversal with tolazoline hydrochloride. Veterinary Therapeutics. 4(2): 128-134. 696 697 Ebert TJ. 2013. Autonomic Nervous System Pharmacology. In Pharmacology and Physiology for 698 Anesthesia: Foundations and Clinical Application. Philadelphia, PA: Elsevier Saunders. 699 700 Elliot R, Ruehle PH. 1985. Process for the production of xylazine. U.S. Patent 4,614,798. 701 702 EMEA (The European Agency for the Evaluation of Medical Products Veterinary Medicines Evaluation 703 Unit) 1999. Xylazine Hydrochloride. Summary Report. [October 2018] Available from 704 https://www.ema.europa.eu/documents/mrl-report/xylazine-hydrochloride-summary-report-1-705 committee-veterinary-medicinal-products_en.pdf 706 707 Fabrega F, Marques M, Ginebreda A, Kuzmanovic M, Barcelo D, Schumacher M, Domingo JL, Nadal M. 708 2013. Integrated Risk Index of Chemical Aquatic Pollution (IRCAP): Case studies in Iberian Rivers. Journal 709 of Hazardous Materials. 263: 187-196. 710 711 Flecknell P. 2016. Laboratory Animal Anesthesia. 4th Ed. Waltham, MA: Elsevier Academic Press. 712 713 Gallanosa AG, Spyker DA, Shipe JR, Morris DL. 1981. Human xylazine overdose: A comparative review 714 with clonidine, phenothiazines, and tricyclic antidepressants. Clinical Toxicology. 18(6): 663-678. 715 716 Garcia-Villar R, Toutain PL, Alvinerie M, Ruckebusch Y. 1981. The pharmacokinetics of xylazine 717 hydrochloride: an interspecific study. Journal of Veterinary Pharmacology and Therapeutics. 4: 87-92. 718 719 Goetzman BW, Milstein JM. 1979. Pulmonary Vasodilator Action of Tolazoline. Pediatric Research. 13: 720 942-944. 721 722 Gogoi P, Konwar D. 2006. An efficient one-pot synthesis of imidazolines and benzimidazoles via anerobic 723 oxidation of carbon-nitrogen bonds in water. Tetrahedron Letters. 47: 79-82. 724 725 Green CJ, Knight J, Precious S, Simpkin S. 1981. Ketamine alone and combined with diazepam or xylazine 726 in laboratory animals: a 10 year experience. Laboratory Animals. 15: 163-170. 727 728 Greene SA, Thurmon JC. 1988. Xylazine – a review of its pharmacology and use in veterinary medicine. 729 Journal of Veterinary Pharmacology and Therapeutics. 11: 295-313. 730 731 Greenough A, Murthy V, Milner AD. 2012. Respiratory Disorders in the Newborn. In Kendig & Chernick’s 732 Disorders of the Respiratory Tract in Children. 8th Ed. Philadelphia, PA: Elsevier Saunders. 733 734 Grover RF, Reeves JT, Blount SH Jr. 1961. Tolazoline hydrochloride (Priscoline). An effective pulmonary 735 vasodilator. American Heart Journal. 61: 5-15. 736 737

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Hsu WH. 1981. Xylazine-induced depression and its antagonism by alpha-adrenergic blocking agents. 738 Journal of Pharmacology and Experimental Therapeutics. 218: 188-192. 739 740 IARC (The International Agency for Research on Cancer). 1993. 2,6-Dimethylaniline (2,6-xylidine). IARC 741 Monograph on the Evaluation of Carcinogenic Risks to Humans: Occupational exposures of hairdressers 742 and barbers and personal use of hair colourants; some hair dyes, cosmetic colourants, industrial dyestuffs 743 and aromatic amines. [October 2018] Available from 744 https://monographs.iarc.fr/wp-content/uploads/2018/06/mono57-22.pdf 745 746 JECFA (Joint FAO/WHO Expert Committee on Food Additives). 1998a. 875. Xylazine. International 747 Programme on Chemical Safety (IPCS), Tox Monograph, No. 38. [October 2018] Available from 748 http://www.inchem.org/documents/jecfa/jecmono/v38je03.htm 749 750 JECFA (Joint FAO/WHO Expert Committee on Food Additives). 1998b. Evaluation of Certain Veterinary 751 Drug Residues in Food. WHO Technical Report Series, No. 879. World Health Organization. Geneva. 752 [October 2018] Available from 753 http://apps.who.int/iris/bitstream/handle/10665/42103/WHO_TRS_876.pdf?sequence=1 754 755 Korones SB, Eyal FG. 1975. The pattern of response of pulmonary and systemic arterial pressures with 756 tolazoline. Pediatric Research. 9: 367. 757 758 Kreeger TJ, Del Giudice GD, Seal US, Karns P. 1986a. Xylazine Hydrochloride-Ketamine Hydrochloride 759 Immobilization of Wolves and its Antagonism by Tolazoline Hydrochloride. Journal of Wildlife Diseases. 760 22(3): 397-402. 761 762 Kreeger TJ, Del Giudice GD, Seal US, Karns P. 1986b. Immobilization of White-Tailed Deer with Xylazine 763 Hydrochloride and Ketamine Hydrochloride and Antagonism by Tolazoline Hydrochloride. Journal of 764 Wildlife Diseases. 22(3): 407-412. 765 766 Lester PA, Moore RM, Shuster KA, Myers DD. 2012. Anesthesia and Analgesia. In the Laboratory Rabbit, 767 Guinea Pig, Hamster, and Other Rodents. Oxford, UK: Elsevier’s Science and Technology. 768 769 Levy RJ, Rosenthal A, Freed MD, Smith CD, Eraklis A, Nadas AS. 1977. Persistent Pulmonary 770 Hypertension in a Newborn with Congenital Diaphragmatic Hernia: Successful Management with 771 Tolazoline. Pediatrics. 60(5): 740-742. 772 773 Lorenz MD, Coates JR, Kent M. 2010. Handbook of Veterinary Neurology. St. Lois, MO: Elsevier Saunders. 774 775 McIntosh N, Walters RO. 1979. Effect of tolazoline in severe hyaline membrane disease. Archives of 776 Disease in Childhood. 54: 105-110. 777 778 NTP (United States National Toxicology Program). 1990. Toxicology and Carcinogenesis Studies of 2,6-779 Xylidine (2,6-Dimethylaniline) (CAS No. 87-62-7) in Charles River CD Rats (Feed Studies). Technical 780 Report Series No. 278. [October 2018] Available from 781 https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr278.pdf 782 783 Otto K, von Thaden AK. 2012. Anesthesia, Analgesia and Euthanasia. In the Laboratory Mouse. 2nd Ed. UK: 784 Elsevier’s Science and Technology. 785 786 Pawson P. 2008. α2-Adrenergic antagonists. In Small Animal Clinical Pharmacology. Philadelphia, PA: 787 Elsevier Saunders. 788 789 Pieter BM, Timmermans WM, van Zwieten PA. 1982. Alpha 2-adrenocepters: Classification, localization, 790 mechanisms, and targets for drugs. Journal of Medicinal Chemistry. 25: 1389-1401. 791 792

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Technical Evaluation Report Xylazine/Tolazoline Livestock

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