XYLAZINE First draft prepared by Dr Pamela L. Chamberlain Center for Veterinary Medicine Food and Drug Administration, Rockville, Maryland, USA 1. Explanation 2. Biological data 2.1 Biochemical aspects 2.1.1 Pharmacodynamics 2.1.2 Absorption, distribution and excretion 2.1.3 Biotransformation 2.2 Toxicological studies 2.2.1 Acute toxicity studies of xylazine and 2,6-xylidine 2.2.2 Short-term toxicity studies 2.2.3 Long-term toxicity/carcinogenicity studies 2.2.4 Special studies on teratogenicity 2.2.5 Special studies on genotoxicity 2.2.6 Special studies on methaemoglobin and haemoglobin adduct formation with 2,6-xylidine 2.3 Observations in humans 3. Comments 4. Evaluation 5. Acknowledgements 6. References 1. EXPLANATION Xylazine is a clonidine analogue that acts on presynaptic and postsynaptic receptors of the central and peripheral nervous systems. It is an alpha2-adrenergic agonist used in animals, including cattle, horses, dogs, cats and deer, for its tranquillizing, muscle relaxant and analgesic effects, but it has numerous other pharmacological effects. It inhibits the effects of postganglionic cholinergic nerve stimulation. Xylazine is administered by the intramuscular, intravenous or subcutaneous (in cats) routes, often in combination with other anaesthetic agents, e.g., barbiturates, chloral hydrate, halothane and ketamine. Xylazine had not been previously evaluated by the Committee. The molecular structure of xylazine is shown below.2. BIOLOGICAL DATA 2.1 Biochemical aspects 2.1.1 Pharmacodynamics With respect to xylazine's sedative effect, there are marked species differences in the dose rates required to achieve this state. Table 1 illustrates dosages required for various animal species (Gross & Tranquilli, 1989). Table 1. Dosage of xylazine in various animal species Xylazine (mg/kg bw) Species Intravenous Intramuscular Horses 0.5 to 1.1 1 to 2 Cattle 0.03 to 0.11 0.1 to 0.21 Sheep 0.05 to 0.11 0.1 to 0.31 Goats 0.01 to 0.51 0.05 to 0.51 Swine 2 to 3 Dogs 0.5 to 1 1 to 2 Cats 0.5 to 1 1 to 2 Birds 5 to 10 1 Lower end of dose range should be used if sedation without recumbency is desired (Gross & Tranquilli, 1989). Mydriasis is a feature of xylazine-induced sedation in the cat. The mechanism has been determined as central inhibition of parasympathetic tone in the iris due to xylazine's activation of post-synaptic alpha-2 receptors (Hsu et al., 1981). Thermoregulatory control is impaired in cats administered xylazine. They become more susceptible to hyper- and hypothermia both during and after recovery from the sedative effects of the drug. Foals have demonstrated a hypothermic response to xylazine. Thermoregulatory effects in cattle have been variable (Ponder & Clark, 1980; Booth, 1988; Robertson et al., 1990). Cardiovascular effects of xylazine include decreased heart rate and variable effects on blood pressure. Xylazine-induced arrhythmia is common in the horse due to sinoatrial and atrioventricular blocks. Arrhythmias have also been recorded in dogs, but could not be induced in sheep. The induction of cardiovascular effects may be influenced by route of administration, e.g., xylazine administered epidurally to horses produced no cardiovascular changes, whereas cattle injected by this route experienced decreases in heart rate and arterial blood pressure (Sagner et al., 1969; Holmes & Clark, 1977; Freire et al., 1981; Hsu et al., 1981; Wasak, 1983; Singh et al., 1983; Leblanc & Eberhart, 1990; Skarda et al., 1990). The effects of xylazine on respiration, acid-base balance and blood gas values vary according to species and anaesthetic combination. In cattle, xylazine causes a slowing of the respiratory rate. This is accompanied by an increase in pH and metabolic acidosis. Respiratory rate is also slowed in dogs administered xylazine, but arterial pH, pO2 or pCO2 are not significantly affected. The literature contains conflicting reports on the effect of xylazine on the respiratory rate of horses. Tachypnoea is characteristic of the ovine response to xylazine. Hypoxaemia induced by xylazine in sheep can be life-threatening (DeMoor & Desmet, 1971; Klide et al., 1975; Holmes & Clark, 1977; Hsu et al., 1989; Carter et al., 1990; Wagner et al., 1991). Hyperglycaemia is induced by xylazine in adults of all target species. Increased blood glucose concentrations are accompanied by a decrease in insulin levels. In adult horses, hyperglycaemia is accompanied by increased urine volume without glycosuria. Xylazine administered to neonatal foals did not result in hyperglycaemia. The hyperglycaemic effect of xylazine is thought to be due to its direct effect on alpha-2-adrenoceptors of pancreatic islet beta cells resulting in an inhibition of insulin release (Symonds, 1976; Feldberg & Symonds, 1980; Hsu & Hummel, 1981; Thurmon et al., 1982, 1984; Benson et al., 1984). Serum chemistry and cerebral spinal fluid alterations were observed in adult female goats administered intramuscularly with 0.2 mg xylazine/kg bw. Significant elevations of urea nitrogen, total protein and total cholesterol were found in serum. Glucose and urea nitrogen levels were significantly increased (P<0.01) and chloride levels were significantly decreased (P<0.05) in the cerebral spinal fluid (Amer & Misk, 1980). Erythrocyte counts, haematocrit values and haemoglobin concentrations in cattle and dogs have shown significant but reversible decreases following xylazine administration (Eichner et al., 1979; Wasak, 1983). Gastrointestinal effects in ruminants include decreased gut motility, prolongation of gastrointestinal transit time and inhibition of reticulorumen contractions. Xylazine causes decreased muscle tone of the colon and rectum which facilitates rectal examination. Xylazine inhibition of rumen contractions can lead to tympany, which is a potential cause of death in xylazine-sedated ruminants. Ruminants are fasted prior to sedation and maintained in sternal recumbency during sedation to reduce the risk of xylazine-induced tureen tympany. Because xylazine also impairs deglutition, the head and neck of xylazine-sedated ruminants are lowered to avoid aspiration of saliva or ruminal fluid. Tolazoline (an alpha-2-adrenergic antagonist) has shown effectiveness in reversing recumbency, gastric paresis and loss of voluntary lingual control caused by xylazine in cattle (Swift, 1977; Bolte & Stupariu, 1978; Ruckebusch & Toutain, 1984). Gastrointestinal effects in dogs and cats include decreased transit time and vomiting. The mechanism for induction of vomiting is thought to involve the effect of xylazine on alpha-2-adrenoceptors in the area postrema (the chemoreceptor trigger zone for vomiting) in the medulla oblongata (Cullen & Jones, 1977; Colby et al., 1981; Hsu & McNeel, 1983; Hikasa et al., 1987, 1989). 2.1.2 Absorption, distribution and excretion 2.1.2.1 Rats Male Sprague-Dawley rats (170 g bw) were administered xylazine at dosages of 0.02 to 10 mg/kg bw (i.v.) or 0.02 to 100 mg/kg bw (oral). The drug was labelled with both 35S and 14C on the thiazine ring. Following oral administration, absorption was > 95% with a half-life of approximately 5 minutes. After i.v. administration, the drug was distributed within a few minutes to almost all organs but primarily to the kidneys and central nervous system. Relatively high activity concentrations occurred in the pancreas, thyroid glands, liver and cranial glands (e.g., extraorbital, sublingual). Several hours following i.v. administration of 2 mg/kg bw, only small concentrations (< 0.3 µg/g tissue) were present in the musculature. Following oral or i.v. administration, approximately 70% of the administered dose was eliminated in urine and 30% in faeces. Renal elimination following oral or i.v. administration was associated with a half-life of 2 to 3 hours. High oral doses (100 mg/kg bw) were associated with a delay in renal elimination. Faecal elimination was comparable to biliary elimination after oral or i.v. administration. Enterohepatic circulation did not occur to a notable extent (Duhm et al., 1968, 1969). 2.1.2.2 Cattle Three male calves (200-250 kg) and one dairy cow (450 kg) were injected intramuscularly with a 0.33 mg/kg dose of 14C-xylazine labelled in the thiazine ring. Radioactivity in blood plasma reached its peak in the first 1.5 hours after injection. Total excretion of radioactivity in urine and faeces was 68, 86, 83 and 100% at 10, 24, 48 and 72 hours, respectively (Murphy & Jacobs, 1975). In another study, five 2-month old calves and four lactating cows were administered a single intramuscular dose (0.3 or 0.6 mg/kg bw) of xylazine hydrochloride. Maximum concentrations of xylazine were achieved in blood 20 minutes after dosing. These were 0.04 mg/litre for the 0.3 mg/kg bw dose and 0.06 mg/litre for the 0.6 mg/kg bw dose. No xylazine was found in blood 8 hours after administration (Takase et al., 1976). Three lactating cows were administered an i.m. dose of 0.2 mg xylazine/kg bw and two others were administered an i.m. dose of 0.4 mg xylazine/kg bw. Milk was analysed for the presence of xylazine at 5 and 21 hours following administration. No xylazine was found at either time point for either dose. The limit of detection was 0.06 mg/litre (Pütter & Sagner, 1973). Urinary excretion of xylazine was studied in three cows. Two were administered an i.m. dose of 0.2 mg xylazine/kg bw and one was administered an i.m. dose of 0.5 mg xylazine/kg bw. Less than 1% of the dose was excreted unchanged in the urine. Unchanged xylazine was no longer detectable 6 hours following administration. Metabolites were no longer detected in urine 10 hours after administration. The limit of detection for unchanged xylazine was 1-5 µg/litre (Pütter & Sagner, 1973). 2.1.2.3 Comparative pharmacokinetics in dogs, sheep, cattle and horses The comparative pharmacokinetics of xylazine in dogs, sheep, cattle and horses are summarized in Table 2. Pharmacokinetic parameters do not vary greatly between species following intravenous administration. The rapid elimination of xylazine is attributed to extensive metabolism, and not to rapid renal excretion of unchanged xylazine. Significant amounts of parent xylazine were not found in the urine of sheep collected at 10-minute intervals after dosing. The pharmacokinetics of xylazine were unmodified when it was administered to rabbits with occluded renal arteries. The lack of correlation between pharmacokinetic parameters and clinical effects of xylazine in cattle suggests that clinical effects in cattle are due to a rapidly produced long-acting metabolite(s) and not due to an increased sensitivity to xylazine (Garcia-Villar et al., 1981). Table 2. Single-dose pharmacokinetics of xylazine in domestic species (Garcia-Villar et al., 1981) Species Dog Sheep Cattle Horse Body weight range (kg) 14-24 42-65 240-440 415-550 Dose rate (mg/kg bw)1 1.4 1.0 0.2 0.6 Number 4 6 4 4 Intravenous2 Distribution half-life (min) 2.57 1.89 1.21 5.97 Volume of distribution (l/kg) 2.52 2.74 1.94 2.46 Elimination half-life (min) 30.13 23.11 36.48 49.51 Body clearance (ml/min/kg) 81 83 42 21 Intramuscular2 Absorption half-life (min) 3.44 5.45 ND 2.72 Elimination half-life (min) 34.65 22.36 ND 57.7 Cmax (mg/ml) 0.43 0.13 ND 0.17 Tmax (min) 12.7 14.68 ND 12.92 Bioavailability: mean (%) 73.9 40.8 ND 44.6 standard deviation (%) 17.89 23.81 4.16 range (%) 52-90 17-73 40-48 1 Dosage expressed as xylazine-base 2 Blood sampling times after injection: 1, 2, 4, 8, 16, 30 and 120 minutes. ND = Not determined (assay was not sensitive enough to determine xylazine plasma concentrations lower than 0.01 mg/litre) 2.1.3 Biotransformation 2.1.3.1 Rats Studies were conducted with urine and bile of rats administered 2 mg xylazine (35S or 14C)/kg bw intravenously. Approximately 20 metabolites were detected and quantified as xylazine equivalents. Approximately 8% of the dose was eliminated as unchanged compound in the urine 24 hours after dosing. The major metabolite comprised 35% of the administered dose. Final products of metabolism were inorganic sulfate and carbon dioxide (Duhm et al., 1968). Specific metabolites of xylazine were identified following incubation of xylazine with rat liver microsomes. Those metabolites were 2-(4'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3- thiazine, 2-(3'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H- 1,3-thiazine, N-(2,6-dimethylphenyl)thiourea and 2-(2',6'- dimethylphenylamino)-4-oxo-5,6-dihydro-1,3-thiazine. N-(2,6-dime- thylphenyl)thiourea was the major metabolite produced in vitro. Figure 1 shows the proposed metabolic pathways of xylazine based on these findings (Mutlib et al., 1992). 2.1.3.2 Horses One mare was administered a 1 g dose of xylazine (route not stated) and urine was collected over 24 hours. Metabolites were recovered from horse urine only after the urine was hydrolysed with beta-glucuronidase. The major urinary metabolites detected were the same as those produced by incubating xylazine with rat liver microsomes, described in section 2.1.3.1 (Mutlib et al., 1992). 2.1.3.3 Cattle Urine from three cows administered an i.m. dose of 0.2 mg xylazine/kg bw (two cows) or 0.5 mg xylazine/kg bw (one cow) was examined for metabolites. One urinary metabolite, identified as 2,6-xylidine1, was found in both free and conjugated forms. The authors concluded that xylazine was essentially eliminated in cattle by rapid biotransformation. Breakdown of the thiazine ring, resulting in formation of 2,6-xylidine, was proposed as the primary biotransformation pathway (Patter & Sagner, 1973). 1 2,6-xylidine is also known as 1-amino-2,6-dimethylbenzene and as 2,6-dimethylaniline
2.2 Toxicological studies Because 2,6-xylidine is also a chemical intermediate used in dyes, a component of tobacco smoke and a degradation product of aniline-based pesticides, its toxicology has been studied extensively. Toxicological studies conducted with this compound were also reviewed and will be presented in addition to the review and results of toxicological studies of xylazine. 2.2.1 Acute toxicity studies of xylazine and 2,6-xylidine The acute systemic toxicity of xylazine has been investigated in both laboratory and domestic species. It is generally recognized that ruminants are much more sensitive than most other species to the pharmacological and toxicological effects of xylazine. Results of LD50 studies of xylazine and 2,6-xylidine are summarized in Table 3. 2.2.1.1 Acute toxicity of xylazine in dogs Adult dogs (four males, four females) and cats (two males, four females) were administered a single i.m. or i.v. dose of 22 mg xylazine/kg bw (10 times the recommended therapeutic dose). One cat out of three receiving the i.v. dose died, and two dogs out of four receiving the i.m. dose died. All others recovered from convulsions, unconsciousness and respiratory depression with no apparent after-effects. The authors concluded that xylazine was slightly toxic in this study (Crawford et al., 1970a). Table 3. Results of acute toxicity studies on xylazine and 2,6-xylidine Species Sex1 Route LD50 Reference (mg/kg bw) Xylazine Rat NA p.o. 130 Sagner, 1967 Cat male & female2 s.c. 100-110 Bauman & Nelson, 1969 Dog male & female3 i.m 47 Nelson et al., 1968b Dog 4 male & 3 female i.v 20-25 Nelson et al., 1968b Horses NA4 i.m. 60-705 Nelson et al., 1968a i.v. 15-285 2,6-Xylidine Mouse male p.o. 710 Vernot et al., 1977 Rat p.o. 2042 Lindstrom et al., 1969 male p.o. 840 Jacobson, 1972 male p.o. 630 Short et al., 1983 male p.o. 1230 Vernot et al., 1977 female p.o. 1160 & 1270 US National Toxicology Program, 1990 male p.o. 620-1250 & 1310 1 NA = Information not available 2 Number per sex not stated; 10 animals were used 3 Number per sex not stated; 17 animals were used 4 Sex of test animals was not stated; 5 animals were used 5 Minimum lethal dose 2.2.1.2 Acute toxicity of xylazine in horses Adult horses were administered 11 mg xylazine/kg bw, i.v. (three mares, one gelding) or 22 mg xylazine/kg bw, i.m. (two mares, two geldings). One mare died following i.v. administration. All other test animals recovered from treatment-related effects 24 hours following i.v. administration and 48 hours following i.m. administration. The authors concluded that the i.v. dose was slightly toxic and that the i.m. dose produced no apparent toxicity in this study (Crawford et al., 1970b). 2.2.2 Short-term toxicity studies 2.2.2.1 Xylazine a) Rats Xylazine was administered in the diet to Wistar rats (10/sex/ group) for 32 weeks. Dosages administered were 0, 50, 100, 250 or 500 mg/kg diet (equal to 0, 3, 6, 21 or 41 mg/kg bw per day for males and 0, 4, 8, 19 or 45 mg/kg bw per day for females). Haematology, urinalysis and gross and histopathological evaluations were performed. Decreases in body weight observed in females in the two highest-dose groups (statistically significant (p<0.02) at 500 mg/kg diet) were considered by the author to be treatment-related. Microscopic examination of livers, lungs and kidneys revealed that animals in all groups were diseased but no treatment-related pathology was identified. Based on the dose-related decrease in weight gain observed in females at 250 and 500 mg/kg diet, the NOEL in this study was 100 mg/kg diet, equal to 6 mg/kg bw per day. The author regarded the dose of 250 mg/kg diet as the non-toxic application rate. The reliability of any NOEL derived from this study should be considered questionable, owing to the presence of infection in all groups (Tettenborn & Hobik, 1968a; Trossmann & Hobik, 1970). b) Dogs Dogs of undetermined breed or source were given xylazine orally in gelatin capsules for 14-16 weeks at dose levels of 25, 50 or 100 mg/kg bw per day, 5 days/week. The low-dose group consisted of one male and one female, the mid-dose group of two males and the high-dose group of two males and two females. Haematology, clinical chemistry, blood coagulation, urinalysis and postmortem gross and microscopic evaluations were performed. During week 8 of the test, one animal in the high-dose group died and was replaced with a new animal. Postmortem gross findings in this animal included diffuse reddening of the stomach and intestinal mucous membrane. Histopathological findings in livers (fatty degeneration and necrosis) and kidneys (tubular epithelial necrosis and fat accumulation) of the high-dose group were considered treatment- related. Fatty deposits were noted in the liver and kidneys of the low-dose female. These findings were attributed to parturition, which occurred 3 weeks before the animal was killed. The author concluded that the NOEL for this study was 50 mg/kg bw per day. The reliability of any NOEL derived from this study should be considered questionable due to the lack of a control group and small numbers of animals in each test group (Tettenborn & Hobik, 1968b). Beagle dogs (two/sex/group) were administered xylazine orally by dietary admixture for 13 weeks. Dosages administered were 0, 10, 30 or 100 mg/kg diet (equal to 0, 0.3, 0.9 or 3 mg/kg bw per day). At the beginning of the test the animals were approximately 8.5 months old and weighed 7-10 kg. Parameters evaluated included general appearance, ophthalmology, electrocardiography, haematology, clinical chemistry, urinalyses and gross and microscopic pathology. No treatment-related adverse effects were observed in any of the parameters evaluated. The NOEL for this study was 3 mg/kg bw per day (Tettenborn, 1969; Mawdesley-Thomas, 1970). 2.2.2.2 2,6-Xylidine a) Rats Three groups (nine or ten/group) of male Fischer-344 rats were given oral (gavage) doses of 160 mg 2,6-xylidine/kg bw per day for 5, 10 or 20 days. The dosage administered was 25% of the estimated LD50 determined by the investigator. A significant increase in splenic haemosiderosis (indicative of erythrocyte damage) after 20 days was noted as a treatment-related effect in this study. Splenic congestion and evidence for increased erythropoiesis were minimal (Short et al., 1983). Three groups of Sprague-Dawley rats (five/sex/group for controls, low and mid dose; four/sex/group for high dose) were administered 0, 20, 100 or 500-700 mg 2,6-xylidine/kg bw per day by gavage for 4 weeks. Treatment-related effects included decreased weight gain, decreased haemoglobin levels and hepatomegaly. In this study, the rat appeared to be about 10 times less susceptible to hepatotoxicity of 2,6-xylidine than the dog (see section 2.2.2.2b) (Magnusson et al., 1971; IARC, 1993). Two groups of 8-week-old Sprague-Dawley rats (5/sex/group) were orally administered (gastric intubation) a dose of 0 or 400 mg 2,6-xylidine per kg bw per day for 1 week immediately followed by a daily dose of 0 or 500 mg/kg bw for 3 weeks. Decreased body weight gain and hepatomegaly (most pronounced in centrilobular regions) were noted as treatment-related effects. Electron microscopy of liver tissue showed proliferation of hepatic smooth endoplasmic reticulum, which was deemed responsible for the observed hepatomegaly in treated rats. An increase in microsomal glucuronyltransferase was observed in males while aniline hydroxylase levels were increased in females. Decreases in liver glycogen and glucose-6-phosphatase activity were also observed in the centrilobular regions of treated animals (Magnusson et al., 1979). Male Osborne-Mendel rats were administered up to 10 000 mg 2,6-xylidine per kg in the diet for 3-6 months. Treatment-related effects included 25% weight reduction, anaemia, hepatomegaly with no associated microscopic changes, splenic congestion and renal toxicity (Lindstrom et al., 1963). Groups of F-344/N rats (five/sex/group) were administered doses of 0, 80, 160, 310, 620 or 1250 mg/kg bw of 2,6-xylidine in corn oil by gavage 5 days/week for 2 weeks. Parameters evaluated included clinical observations, body weight, urinalysis, haematology, blood pH and carbon dioxide determinations, and gross postmortem findings. Treatment-related deaths occurred at and above 620 mg/kg bw. All animals in the highest dose group died before the end of the study. A decrease of more than 10% in body weight was observed in males at and above 310 mg/kg bw and in females at and above 160 mg/kg bw. Generalized leukocytosis and an increase in the number of nucleated red blood cells were observed in male rats administered 310 or 620 mg/kg bw. Slight anisocytosis, poikilocytosis and polychromasia of the red blood cells occurred more frequently in dosed animals than in vehicle control animals. Moderate poikilocytosis occurred at 310 mg/kg bw and moderate polychromasia at 310 and 620 mg/kg bw. Slightly macrocytic erythrocytes were observed at the two highest doses. Slight anisocytosis, poikilocytosis and polychromasia were observed in female rats at 310 and 620 mg/kg bw. The NOEL for this study was 80 mg/kg bw per day (US National Toxicology Program, 1990). Groups of F-344/N rats (10/sex/group) were given doses of 0, 20, 40, 80, 160 or 310 mg/kg bw of 2,6-xylidine in corn oil by gavage, 5 days/week for 13 weeks. Parameters evaluated included clinical observations, haematology, urinalysis, serum chemistry and enzyme analyses, gross and histopathological postmortem examinations. A decrease in body weight gain of more than 10% occurred in males and females in the highest dose group and in females at 40 and 160 mg/kg bw per day. In the highest dose group, relative liver weights were significantly (P=0.003) increased for males and females. Relative liver weight was also increased for males in the 160 mg/kg bw group. The liver weight to brain weight and kidney weight to brain weight ratios were significantly increased in females at 310 mg/kg bw per day. Treatment-related effects on haematology included significantly decreased total leukocyte counts in males at doses of 40 mg/kg bw or more. These were accompanied by decreases in the percentage of lymphocytes and increases in the percentage of segmented neutrophils at doses of 80 mg/kg bw or more. In males, haemoglobin levels were significantly decreased at 160 and 310 mg/kg bw and erythrocyte and haematocrit levels were decreased at 310 mg/kg bw. The NOEL for this study was 20 mg/kg bw per day (US National Toxicology Program, 1990). b) Dogs Four groups of beagle dogs (one/sex/group) were given an oral (gelatin capsule) dose of 0, 2, 10 or 50 mg 2,6-xylidine/kg bw per day for 4 weeks. Treatment-related effects included vomiting (mid- and high-dose groups), poor condition and decreased body weights (high-dose group), hyperbilirubinaemia (mid- and high-dose groups), hypoproteinaemia (mid-and high-dose groups) and fatty degenerative changes in the liver that increased in severity with increasing dose (Magnusson et al., 1971; IARC, 1993). 2.2.3 Long-term toxicity/carcinogenicity studies 2.2.3.1 Xylazine No carcinogenicity studies have been performed with xylazine 2.2.3.2 2,6-Xylidine Four groups of Charles River CRL:COBS CD (SD) BR rats (56/sex/group) were fed diets containing 2,6-xylidine (99.06% pure) at concentrations of 0, 300, 1000 or 3000 mg/kg diet (equivalent to 0, 15, 50 or 150 mg/kg bw per day) for 102 weeks. The animals assigned to this study were F1a, generation weanlings from a multigeneration study in which animals were fed diets containing 0, 300, 1000 or 3000 mg/kg 2,6-xylidine beginning at 5 weeks of age. Parameters evaluated in the carcinogenicity study included clinical observations, haematology, blood urea nitrogen, glucose, SGOT, alkaline phosphatase and gross and microscopic postmortem examinations. Treatment-related clinical effects included a decrease in mean body weight gain in high-dose males and females (>10%). Mortality was significantly (P < 0.001) increased (relative to controls) in males in the high-dose group. Mortality was also increased for mid-dose males. Survival at 105 weeks was 43/56, 40/56, 33/56 and 14/56 for males in the control, low-, mid- and high-dose groups, respectively. For females, survival was 33/56, 25/56, 32/56 and 24/56 for the controls, low-, mid- and high-dose groups, respectively. Microscopically, a significant increase in carcinoma of the nasal cavity was observed in high-dose males (26/56; P < 0.001, life table test). For females, the incidence of carcinomas of the nasal cavity were 0/56, 0/56, 1/56 and 24/56 in the low-, mid- and high-dose groups, respectively (P<0.001, life table test). Two adenocarcinomas were diagnosed in high-dose males. The incidence of papillary adenomas in males was 0/56 in controls, 0/56 in low-dose, 2/56 in mid-dose and 10/56 in high-dose rats (P=0.001, incidental tumour test). For females, nasal adenomas occurred in 0/56 in controls, 0/56 in low- dose, 1/56 in mid-dose and 6/56 in high-dose rats (P=0.02, incidental tumour test). Several unusual neoplasms of the nasal cavity were also considered to be related to treatment. These included one undifferen- tiated sarcoma identified in one high-dose female, rhabdomyosarcomas which occurred in two high-dose male and two high-dose females and malignant mixed tumours having features associated with both adenocar- cinoma and rhabdomyosarcoma were observed in one high-dose male and one high-dose female rat. Non-neoplastic nasal cavity lesions included acute inflammation (rhinitis), epithelial hyperplasia and squamous metaplasia. These occurred at increased incidence (relative to controls) in high-dose male and female rats. The incidence of subcutaneous fibromas and fibrosarcomas combined in males was 0/56, 2/56, 2/56 and 5/56 for the control, low-, mid- and high-dose groups, respectively (P=0.001, life table test; P<0.001 life table trend test). For females, the incidence of these tumours combined was 1/56, 2/56, 2/56 and 6/56 for controls, low-, mid- and high-dose groups, respectively ((P=0.01, life table trend test). Neoplastic nodules occurred in livers of female rats with a significant positive trend. The incidence was 0/56, 1/56, 2/56 and 4/55 for the controls, low-, mid-, and high-dose groups, respectively (P=0.03, incidental test; P=0.012, incidental trend test). Treatment-related effects on haematology included decreases in erythrocyte counts and haemoglobin levels at 18 months in the high-dose males. Decreases in these parameters were also observed in the mid- and high-dose females at 12 months. The author remarked that these changes were not severe enough to be considered indicative of anaemia. The author concluded that under the conditions of this study, 2,6-xylidine was dearly carcinogenic for male and female Charles River CD rats. This was based on the observed significant increases in the incidence of adenomas and carcinomas of the nasal cavity. Additionally the author stated that the increased incidence of subcutaneous fibromas and fibrosarcomas in male and female rats and increased incidence of neoplastic nodules of the liver in female rats could have been treatment-related (US National Toxicology Program, 1990). The International Agency for Research on Cancer (IARC) has evaluated the carcinogenic risk of 2,6-xylidine to humans. The Working Group concluded that there was inadequate evidence in humans but sufficient evidence in experimental animals for the carcinogenicity of 2,6-xylidine. The IARC classified 2,6-xylidine as Group 2B (possibly carcinogenic to humans) (IARC, 1993). 2.2.4 Special studies on teratogenicity Xylazine was administered by gavage to groups of pregnant rats (22 animals/group) on gestation days 6 to 15, then killed on day 20 for examination of uterine contents. Dosages administered were 0, 1, 4 or 16 mg/kg bw per day. The study was conducted in accordance with the principles of Good Laboratory Practice Standards and Guidelines of the OECD, United Kingdom, FDA and Japan. Treatment-related maternal effects included partial closing of the eyelids, underactivity, ataxia, flat posture and slightly reduced body weight gain in the high-dose group only. Fetal effects included a decrease in mean fetal weight in the high-dose group only. A teratogenic potential of xylazine was not evident at levels up to and including 16 mg/kg bw per day. The NOEL in this study was 4 mg/kg bw per day (Reynolds, 1994). 2.2.5 Special studies on genotoxicity The results of genotoxicity studies with xylazine and 2,6-xylidine are summarized in Table 4. The results of bacterial mutagenicity testing of xylazine were considered to be negative by the author. Reviewing the data, the Committee concluded that a more than two-fold reproducible increase in revertant colonies in tester strains TA1535 and TA1538 represents weak mutagenic activity, even in the absence of a clear dose-response. 2.2.6 Special studies on methaemoglobin and haemoglobin adduct formation with 2,6-xylidine 2.2.6.1 Cats and dogs Cats and dogs (numbers not specified) were administered an i.v. dose of 30 mg 2,6-xylidine/kg bw or an oral dose of 164 mg N-acetyl 2,6-xylidine/kg bw. 2,6-Xylidine induced a 10% methaemoglobinaemia in cats, and N-acetyl 2,6-xylidine induced a 5% methaemoglobinaemia in cats. Haemoglobin was unaffected in dogs in this study (McLean et al., 1967). Five adult cats (>24 months old) were administered an i.v. dose of 30 mg 2,6-xylidine/kg bw. Blood samples were drawn at 1, 2, 3, 4 and 5 hours after dosing and analysed for methaemoglobin formation. The mean methaemoglobin concentration determined from these sampling intervals was 7% (range = 4.8% to 8.7%). Prior to treatment the mean methaemoglobin concentration of the 152 cats used in this study was approximately 1% (Mclean et al., 1969). 2.2.6.2 Humans 2,6-Xylidine-haemoglobin adduct levels have been found to be elevated in human patients receiving lidocaine treatment for local anaesthesia (1 mg/kg bw) or cardiac arrhythmias (up to 50 mg/kg bw, i.v.). 2,6-Xylidine-haemoglobin adducts are also found in humans with no known exposure to lidocaine. This is attributed to the 120-day lifespan of the erythrocyte and chronic exposure to environmental or iatrogenic sources of aromatic amines, e.g., cigarette smoke. The levels of 2,6-xylidine-haemoglobin adducts found correspond to at an estimated daily exposure (from iatrogenic and environmental sources) of 23 µg (IARC, 1993; Bryant et al., 1994). Methaemoglobinaemia induced by i.v. administration of lidocaine was studied in 40 human cardiac patients. Treatment consisted of a 1 mg/kg bw i.v. bolus followed 15 minutes later with a 0.5 mg/kg bw i.v. bolus. Patients were maintained between and after bolus doses with an infusion rate of 1-4 mg lidocaine/min. Blood samples were drawn before treatment and 1 and 6 hours after treatment. Although the investigators found methaemoglobin levels in these patients to be significantly elevated, the increase was not large enough to be of clinical concern. The highest methaemoglobin level attained was 1.2%. The author did not address the possible role of the 2,6-xylidine metabolite in the observed increases in methaemoglobin levels in treated patients (Weiss et al., 1987). Table 4. Genotoxicity assays with xylazine and 2,6-xylidine Test system Test object Concentration Results Reference Xylazine In vitro Reverse S. typhimurium mutation1 TA1535, 0.4-12 mg/plate Weak positive Herbold, 1984 (-S9) TA1538 0.4-12 mg/plate Weak positive Herbold, 1984 (-S9) TA100, 0.4-12 mg/plate Negative Herbold, 1984 TA98, 0.4-12 mg/plate Negative Herbold, 1984 TA1537 0.4-12 mg/plate Negative Herbold, 1984 Mammalian V79/HGPRT 62-1250 µg/ml Negative Brendler- cell forward (-S9) Schwaab, mutation1 2-40 µg/ml (+S9) 1994 In vivo Cytogenetic Mouse bone 50 mg/kg bw, Negative Herbold, 1995 assay marrow i.p.2 Table 4. Genotoxicity assays with xylazine and 2,6-xylidine (cont'd). Test system Test object Concentration Results Reference 2,6-Xylidine In vitro Reverse S. typhimurium mutation1 TA1535 100-9900 Negative US National µg/plate Toxicology Program, 1990 3 µmol/plate Negative Florin et al., 19804 0.1-10 mg/plate Negative Zeiger et al., 1988 TA100 100-9900 Negative US National µg/plate Toxicology Program, 1990 360 µg/plate Negative Florin el al., 19804 0.1-10 mg/plate Neg (-S9), Zeiger et al., Pos (+S9) 19883 480-4000 Negative Kugler- µg/plate Steigmeier et al., 1989 Table 4. Genotoxicity assays with xylazine and 2,6-xylidine (cont'd). Test system Test object Concentration Results Reference TA1537 100-9900 Negative US National µg/plate Toxicology Program, 1990 360 µg/plate Negative Florin et al., 19804 0.1-10 mg/plate Negative Zeiger et al., 1988 TA98 100-9900 Negative US National µg/plate Toxicology Program, 1990 360 µg/plate Negative Florin et al., 19804 0.1 - 10 mg/plate Negative Zeiger et al., 1988 Gene Mouse lymphoma Not given Positive Rudd et al., mutation1 L5178Y cells, 19837 tk locus Sister Chinese 30-1500 µg/ml Positive Galloway et al., chromatid hamster 1987 exchange1 ovary cells Table 4. Genotoxicity assays with xylazine and 2,6-xylidine (cont'd). Test system Test object Concentration Results Reference In vivo Cytogenetic ICR mouse 350 mg/kg bw, Inconclusive6 Parton et al., assay bone marrow p.o. 1988 375 mg/kg bw, Inconclusive6 Parton et al., p.o. 1990 In vivo- Rat primary 40-850 mg/kg Negative Mirsails et al., in vitro DNA hepatocytes bw, p.o. 1989 repair assay Covalent Rats 87.2 µCi Positive Short et al., 1989 DNA 14C-labelled binding 2,6-xylidine/rat, i.p5 1 Both with and without rat liver S9 fraction 2 Cyclophosphamide positive control 3 Weakly positive in two of three laboratories, negative in the third 4 Spot tests only 5 Pretreatment with unlabelled 262.5 mg/kg bw 2,6-xylidine daily for 9 days 6 Results suggest test article may not have reached target tissue (bone marrow) 7 Reference was an abstract and doses were not stated in that reference 2.3 Observations in humans A 34-year-old man self-injected 10 ml of a 100 mg/ml solution of xylazine intramuscularly. The estimated dose was 15 mg/kg bw. The individual was discovered (30 minutes after retiring for bed) in a deeply comatose, apnoeic and areflexic state. An empty Rompun (xylazine) bottle (known to have contained 10 cc earlier) lay by his side. He was immediately admitted to the hospital. Upon admission his pupils were of moderate size and responded slowly to light. Other findings included a blood pressure of 120/70 mmHg and heart rate of 60 bpm with stable sinus rhythm. Lactate dehydrogenase (LDH) activity was elevated, with the LDH-1 isoenzyme predominating. Creatine phosphokinase (CPK) activity was also elevated, particularly in the CPK-3 and CPK-2 isoenzymes. These enzyme changes persisted for 5 to 7 days. Plasma glucose level was also elevated. Two days following hospital admission sinus tachycardia developed, interspersed with runs of multifocal premature ventricular contractions which were controlled with lidocaine infusion. Blood pressure remained approximately 120/80 for the duration of hospitalization. Coma and respiratory depression lasted 60 hours. The patient was discharged from the hospital 17 days after admission. The author noted that the patient might have died if he had not been found shortly after the injection was administered, owing to the marked respiratory depression that occurred. Hypotension, which has been reported as an effect of xylazine in humans, did not occur in this case. According to the author, the enzyme activity increases indicated that myocardial muscle damage had occurred and an intrinsic cardiotoxic effect of xylazine was suspected. Finally, in this case the greatest threat to life was the CNS-depressant effect of xylazine (Carruthers et al., 1979). A 20-year-old woman ingested 400 mg of xylazine. Approximately 2 hours later she became drowsy, incontinent (urine), difficult to arouse and occasionally unresponsive to verbal commands. She was admitted to the hospital approximately 3 hours after the ingestion. In a similar way to the case of human poisoning described by Carruthers et al. (1979), she experienced a relatively low initial cardiac rate, which later gradually increased, significant central nervous system and respiratory depression, transient hyperglycaemia and ventricular arrhythmias. However there was no evidence of myocardial damage. A sample of this patient's urine was analysed using a gas chromatograph- mass spectrometer computer system. Xylazine was found largely unchanged in the urine as shown by a lack of any structurally related compounds in the basic urine extract. No xylazine was found in a blood sample taken at the time of admission. The author concluded that plasma levels were below the limit of detection of the method (100 ng/ml). The patient was discharged ambulatory and without apparent adverse effects two days following admission to the hospital (Gallanosa et al., 1981). A 36-year-old man died following ingestion of alcohol and clorazepate combined with an injection of approximately 40 ml of xylazine (100 mg/ml). Xylazine was found in the decedent's blood, brain, kidney, liver, lung, fat and urine at concentrations of 0.2, 0.4, 0.6, 0.9, 1.1, 0.05 and 7 ppm, respectively (Poklis et al., 1985). A 29-year-old woman self-injected 40 mg of xylazine intramuscularly. The estimated dose was 0.73 mg/kg bw. Clinical findings included disorientation, miosis, hypotension and bradycardia, but no cardiac arrhythmias were noted. The abnormalities resolved spontaneously (Spoerke et al., 1986). A 37-year-old woman self-injected 24 ml (2400 mg) xylazine intramuscularly. The estimated dose was 22 mg/kg bw. Twenty minutes after the injection her blood pressure was 166/130 mmHg, heart rate was 76 bpm and respirations 18 per minute. The serum glucose level was 175 mg/dl. Blood pressure later decreased to 130/90 mmHg and she became apnoeic. No cardiac arrhythmias were observed during her 3 days of hospitalization. Hypotension and bradycardia occurred two days after the injection. The patient survived (Spoerke et al., 1986). A 29-year-old woman self-injected an unknown amount of xylazine intravenously. She became apnoeic and had an initial blood pressure of 130/90 mmHg with a pulse of 60 bpm. Serum glucose levels did not exceed 90 mg/dl. Twenty-four hours after the injection the patient experienced hypotension and bradycardia. Spontaneous respiration resumed 18 hours after hospital admission. The patient recovered fully (Spoerke et al., 1986). A 19-year-old man accidentally injected himself subcutaneously with 2 ml (100 mg/ml) of xylazine. The dose administered was 3 mg/kg bw. Thirty minutes later he became difficult to rouse and was hospitalized. Clinical findings included miosis, hyporeflexia, hypotension, bradycardia, respiratory and central nervous system depression and hyperglycaemia. He was treated with intravenous fluids and assisted ventilation. Eight hours after hospitalization the patient was alert and responsive. Twenty-four hours later he was released (Samanta et al., 1990). A 39-year-old woman was admitted to the hospital with symptoms of tiredness, faintness and blurred vision. Clinical findings included sinus bradycardia with a blood pressure of 130/90. Xylazine was found in the urine and serum at concentrations of 1674 µg/litre and 30 µg/litre, respectively (Lewis et al., 1983). 3. COMMENTS The Committee considered toxicological data on xylazine, including the results of acute and short-term toxicity studies as well as studies on pharmacodynamics, pharmacokinetics, reproductive and developmental toxicity, genotoxicity and effects in humans. In addition, toxicological studies on 2,6-xylidine, a metabolite of xylazine, were reviewed; these included studies on acute and short-term toxicity, carcinogenicity and genotoxicity. Numerous pharmacological side-effects of xylazine have been observed in treated animals, including mydriasis, impairment of thermo-regulatory control, various effects on the cardiovascular system, acid-base balance and respiration, hyperglycaemia, and haematological and gastrointestinal effects. Cattle and sheep are approximately 10 times more sensitive to xylazine than horses, dogs and cats. Rats were administered radiolabelled xylazine intravenously at doses of 0.02 to 10 mg/kg bw or orally at doses of 0.02 to 100 mg/kg bw. More than 95% of the oral dose was absorbed, with a half-life of approximately 5 minutes. Following oral or intravenous administration, approximately 70% of the administered dose was eliminated in urine and 30% in faeces. Renal excretion following oral or intravenous administration was associated with a half-life of 2 to 3 hours. Enterohepatic circulation did not occur to a notable extent. In cattle administered an intramuscular dose of 0.2 or 0.5 mg xylazine/kg bw, less than 1% of the dose was excreted unchanged in the urine, and the parent compound was detected in the urine up to 6 hours following administration. Metabolites of xylazine were detected in urine from these cattle up to 10 hours following administration. Pharmacokinetic parameters following intravenous administration showed minor variations between species. Xylazine disappeared rapidly from plasma following intravenous administration, with an elimination half-life of approximately 40 minutes in cattle and approximately 20 minutes in sheep. Xylazine could not be detected in the plasma of cattle following intramuscular administration of a single therapeutic dose. In rats administered an intravenous dose of 2 mg/kg bw radio- labelled xylazine, approximately 20 metabolites were quantified as xylazine equivalents in urine and bile. The major metabolite comprised 35% of the administered dose. Approximately 8% of the dose was eliminated as unchanged xylazine 24 hours after dosing. In an in vitro study, 4 metabolites were identified when xylazine was incubated with rat liver microsomes. The same metabolites were identified in the urine of horses treated with xylazine. The major metabolite in both cases was identified as N-(2,6-dimethylphenyl) thiourea. In cattle administered an intramuscular dose of 0.2 mg xylazine/kg bw (two cows) or 0.5 mg xylazine/kg bw (one cow), 2,6-xylidine was identified as a metabolite excreted in urine in both conjugated and unconjugated forms. The acute oral toxicities of xylazine and 2,6-xylidine were tested in mice and rats. Xylazine was determined to be moderately toxic (LD50 = 121-240 mg/kg bw) and 2,6-xylidine to be slightly toxic (LD50 = 600-1000 mg/kg bw). Three studies on the short-term toxicity of xylazine were reviewed. A 32-week dietary study in rats and a 16-week oral (capsules) study in dogs were considered inadequate for the determination of the toxicity of xylazine owing to the use of insufficient numbers, poor quality animals and inadequate study design. The third was a 13-week oral study in beagle dogs fed diets containing 0, 10, 30 or 100 mg/kg xylazine in the feed (equal to 0.3, 0.9 or 3 mg/kg bw per day). No treatment-related effects were observed in any of the treated groups. In a two-week oral (gavage) toxicity study in rats with 2,6-xylidine, rats were dosed with 80, 160, 310, 620 or 1250 mg 2,6-xylidine/kg bw per day, 5 days per week. Treatment-related effects included increased mortality (all animals in the high-dose group died), decreased body weight (males at 310 mg/kg bw per day and above and females at 160 mg/kg bw per day and above) and various effects on haematological parameters as indicated by leukocytosis and changes in red blood cell parameters indicative of increased erythropoiesis (males and females at 310 mg/kg bw per day and above). The NOEL in this study was 80 mg/kg bw per day. In a 13-week oral (gavage) toxicity study in rats with 2,6-xylidine, rats were dosed with 20, 40, 80, 160 or 310 mg 2,6-xylidine/kg bw per day, 5 days per week for 13 weeks. Treatment- related effects included decreased body weight gain (males at 310 mg/kg bw per day and females at 40 mg/kg bw per day and above), increased absolute and relative liver weights (females at 160 mg/kg bw per day and above; males at 310 mg/kg bw per day), leukopenia (males at 40 mg/kg bw per day and above), haemoglobinaemia (males at 160 mg/kg bw per day and above) and anaemia (males at 310 mg/kg bw per day). The NOEL was 20 mg/kg bw per day. In a carcinogenicity study, male and female rats were fed diets containing 2,6-xylidine at concentrations of 300, 1000 or 3000 mg/kg food (equivalent to 15, 50 or 150 mg/kg bw per day). Significant increases in the incidences of papillomas and carcinomas of the nasal cavity were observed in high-dose males and females. There was a significant dose-related increase in the incidence of adenomas in the nasal cavity in both males and females. In addition, unusual rhabdo- myosarcomas and malignant mixed tumours of the nasal cavity were observed in the high-dose males and females. There was a dose-related significant increase in the incidence of subcutaneous fibromas and fibrosarcomas in both treated males and females. In females, neoplastic nodules occurred in livers with a significant positive trend and the increase was significant in the high-dose group by the incidental tumour test. The Committee concluded that 2,6-xylidine was carcinogenic in this study. The International Agency for Research on Cancer has evaluated the carcinogenic risk of 2,6-xylidine and has classified it as Group 2B (possibly carcinogenic to humans). In a teratogenicity study, xylazine was administered to pregnant rats at doses of 1, 4 or 16 mg xylazine/kg bw per day on gestation days 6 to 15. Treatment-related maternal effects included partial closing of the eyelids, hypoactivity, ataxia, flat posture and slightly reduced body weight gain in the high-dose group only. A decrease in mean fetal weight was seen in the high-dose group. No teratogenic effects were noted in this study. The NOEL for maternal and fetal effects was 4 mg/kg bw per day. Xylazine has been tested in reverse mutation assays in Salmonella, a forward mutation assay in cultured mammalian cells and in an in vivo cytogenetic assay. In Salmonella, weak positive results were obtained. Negative results were observed in a forward mutation assay on cultured mammalian cells and in a mouse bone marrow micronucleus test. The Committee concluded that xylazine is weakly mutagenic. 2,6-Xylidine was tested in a series of in vitro and in vivo genotoxic assays. It was weakly positive for reverse mutation in Salmonella. In mammalian cells, it induced forward mutation and was positive in a sister chromatid exchange test. Inconclusive results were obtained in a mouse bone marrow micronucleus test because there was no assurance that the bone marrow had been adequately exposed. 2,6-Xylidine was found to be inactive in an in vivo-in vitro rat hepatocyte unscheduled DNA synthesis assay. Covalent binding of the compound to DNA was observed in rats. The Committee concluded that 2,6-xylidine is genotoxic. The potential for 2,6-xylidine to induce methaemoglobinaemia was reviewed by the Committee. Single doses of 30 mg 2,6-xylidine/kg bw intravenously or 164 mg/kg bw N-acetyl-2,6-xylidine orally have been shown to induce methaemoglobinaemia in cats but not in dogs. 2,6-Xylidine has also been shown to be a product of lidocaine metabolism in humans. Methaemoglobin and 2,6-xylidine-haemoglobin adduct levels have been shown to increase in human cardiac patients receiving lidocaine treatment. Effects of xylazine on humans poisoned following accidental or intentional self-injection (0.7-15 mg/kg bw) or ingestion (7 mg/kg bw) included symptoms of central nervous system depression, respiratory depression, hypo- and hypertension, bradycardia, tachycardia, ventricular arrhythmias, and transient hyperglycaemia. 4. EVALUATION The Committee was unable to establish an ADI for xylazine because it concluded that the 2,6-xylidine metabolite was genotoxic and carcinogenic. Annex 4 lists the information that would be required for further review. 5. ACKNOWLEDGMENTS The preparer of the first draft would like to recognize the following individuals for their assistance and contributions to the preparation of the first draft: Ms. Deborah Brooks, information specialist, Center for Veterinary Medicine Dr. Steve Brynes, residue chemist, Center for Veterinary Medicine Dr. Jennifer Burris, veterinary pathologist, Center for Veterinary Medicine Dr. Robert Condon, biostatistician, Center for Veterinary Medicine Dr. Haydee Fernandez, toxicologist, Center for Veterinary Medicine Dr. Devaraya Jagannath, genetic toxicologist, Center for Veterinary Medicine Dr. Alan Pinter, toxicologist, National Institute of Public Health, Budapest, Hungary Dr. Leonard Schechtman, genetic toxicologist, Center for Veterinary Medicine 6. REFERENCES Amer, A.A. & Misk, N.A. (1980). Rompun in goats with special reference to its effect on the cerebral spinal fluid (c.s.f.). Vet. Med. Rev., 2, 168-174. Bauman, E. K. & Nelson, D.L. (1969). Toxicity of BAY Va 1470 to cats. Unpublished report No. 24208 from Chemagro Corporation. Submitted to WHO by Bayer AG, Leverkusen, Germany. Benson, G.J., Thurmon, J.C., Neff-Davis, C.A., Corbin, J.E., Davis, L.E., Wilkinson, B., & Tranquilli, W.J. (1984). Effect of xylazine hydrochloride upon plasma glucose concentrations in adult pointer dogs. J. Am. Anita. Hosp. Assoc., 20, 791-794. Bolte, S. & Stupariu, A (1978). Motility of the rumen in cattle and sheep under the influence of neuroleptics and analgesics, particularly xylazine, propionylpromazine and diazepam. Lucrari Stiintifice Inatitutul Agron. Timisoara, Ser. Med. Vet., 15, 157-167. Booth, N.H. (1988). Nonnarcotic analgesics. In: Booth N.H.& McDonald E. (eds.), Veterinary Pharmacology and Therapeutics, 6th edition, Iowa State University Press, Ames, Iowa, pp. 351-359. Brendler-Schwaab, S. (1994). Rompun hydrochloride. Mutagenicity study for the detection of induced forward mutations in the V79-HGPRT assay in vitro. Unpublished report No. T9049349 from Bayer AG, Fachbereich Toxicology. Submitted to WHO by Bayer AG, Leverkusen, Germany. Bryant, M.S., Simmons, H.F., Harrell, R.E., & Hinson, J.A. (1994). 2,6-Dimethylaniline-hemoglobin adducts from lidocaine in humans. Carcinogenesis, 15(10), 2287-2290. Carruthers, S.G., Nelson, M., Wexler H.R., & Stiller, C.R. (1979). Xylazine hydrochloride (Rompun) iverdise in man. Clin. Toxicol., 15(3), 281-285. Carter, S.W., Robertson, S.A., Steel, C.J., & Jourdenais, D.A. (1990). Cardiopulmonary effects of xylazine sedation in the foal. Equine Vet. J., 22(6), 384-388. Colby, E.D., McCarthy, L.E., & Borison, H.L. (1981). Emetic action of xylazine on the chemoreceptor trigger zone for vomiting in cats. J. Vet. Pharmacol. Ther., 4, 93-96. Crawford, C.R., Nelson, D.L., & Anderson, R.H (1970a). The toxicity of BAY Va 1470 2% injectable to dogs and cats given at ten times recommended dose. Unpublished report No. 28139 from Research Department, Chemagro Corporation. Submitted to WHO by Bayer AG, Leverkusen, Germany. Crawford, C.R., Nelson, D.L., & Anderson, R.H (1970b). The toxicity of BAY Va 1470 10% injectable alone and in combination with (R)NEGUVON* soluble powder (batch No. 0050337) to horses. Unpublished report No. 28136 from Research Department, Chemagro Corporation. Submitted to WHO by Bayer AG, Leverkusen, Germany. Cullen, L.K. & Jones, R.S. (1977). Clinical observations on xylazine/ketamine anaesthesia in the cat. Vet. Rec., 101, 115-116. Demoor, A. & Desmet, P. (1971). Effect of Rompun on acid-base equilibrium and arterial oxygen pressure in cattle. Vet. Med. Rev., 2-3, 163-169. Duhm, B., Maul, W., Medenwald, H., Patzschke, K., & Wegner, L.A. (1968). Experimental tests on animals with radioactive labeled BAY Va 1470. Unpublished report No. 22874 from Bayer Isotope Institute, Elberfeld Branch. Submitted to WHO by Bayer AG, Leverkusen, Germany. Duhm, B., Maul, W., Medenwald, H., Patzschke, K., & Wegner, L.A. (1969). Experiments using radioactively tagged BAY Va 1470 on rats. Berl. Münch. Tierärztl. Wochenschr., 82, 104-109. Eichner, R.D., Prior, R.L., & Kvasnicka, W.G (1979). Xylazine-induced hyperglycemia in beef cattle. Am. J. Vet. Res., 40(1), 127-129. Feldberg, W. & Symonds, H.W. (1980). Hyperglycemic effect of xylazine. J. Vet. Pharmacol. Ther., 3, 197-202. Florin, I., Rutberg, L., Curvall, M., & Enzell, C.R. (1980). Screening of tobacco smoke constituents for mutagenicity using the Ames' test. Toxicology, 15, 219-232. Freire, A.C.T, Gontijo, R.M., Pessoa, J.M., & Souza, R. (1981). Effect of xylazine on the electrocardiogram of sheep. Br. Vet. J., 137, 590-595. Gallanosa, A.G., Spyker, D.A., Shipe, J.R., & Morris, D.L. (1981). Human xylazine overdose: A comparative review with clonidine, phenothiazines, and tricyclic antidepressants. Clin. Toxicol., 18(6), 663-678. Galloway, S.M., Armstrong, M.J., Reuben, C., Colman, S., Brown, B., Cannon C., Bloom, A.D., Nakamura, F., Ahmed, N., Duk, S., Rimpo, J., Margolin, B.H., Resnick, M.A., Anderson, B., & Zeiger, E. (1987). Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals. Environ. Mol. Mutagen., 10(10), 1-175 Garcia-Villar, R., Toutain, P.L., Alvinerie, M., & Ruckenbusch Y. (1981). The pharmacokinetics of xylazine hydrochloride: an interspecific study. J. Vet. Pharmacol. Ther., 4, 87-92. Gross, M.E. & Tranquilli, W.J. (1989). Use of alpha-2-adrenergic receptor antagonists. J. An. Vet. Med. Assoc., 195(3), 378-381. Herbold, B. (1984). Salmonella/microsome test to evaluate for point-mutagenic effects. Unpublished report No. T0016805 from Bayer Sparte Pharma. Submitted to WHO by Bayer AG, Leverkusen, Germany. Herbold, B. (1995). Rompun Hydrochloride. Micronucleus test on the mouse. Unpublished report No. T9058169 + T2058171 from Bayer AG Fachbereich Toxicology. Submitted to WHO by Bayer AG, Leverkusen, Germany. Hikasa, Y. Takase, K., & Ogasawara, S. (1989). Evidence for the involvement of alpha-2-adrenoceptors in the emetic action of xylazine in cats. Am. J. Vet. Res., 50, 1348-1351. Hikasa, Y., Takase, K., Osada, T., Takamatsu, H., & Ogasawara, S. (1987). Xylazine-induced vomiting in dogs: elimination by ablation of the area postrema and blockade by yohimbine. Zent. bl. Vet. med., A34(2), 154-158. Holmes, A.M. & Clark, W.T. (1977). Xylazine sedation of horses. N.Z. Vet. J., 25, 159-161. Hsu, W.H. & Hummel, S.K. (1981). Xylazine-induced hyperglycemia in cattle: a possible involvement of alpha-2-adrenergic receptors regulating insulin release. Endocrinology, 109, 825-829. Hsu, W.H. & McNeel, S.V. (1983). Effect of yohimbine on xylazine-induced prolongation of gastrointestinal transit in dogs. J. Am. Vet. Med. Assoc., 183(3), 297-300. Hsu, W.H., Betts, D.H., & Lee, P. (1981). Xylazine-induced mydriasis: possible involvement of a central postsynaptic regulation of parasympathetic tone. J. Vet. Pharmacol. Ther., 4, 209-214. Hsu, W.H., Hanson, C.E., Hembrough, F.B., & Schaffer, D.D. (1989). Effects of idazoxan, tolazoline and yohimbine on xylazine induced respiratory changes and central nervous system depression in ewes. Am. J. Vet. Res., 50(9), 1570-1573. IARC (1993). 2,6-Dimethylaniline (2,6-xylidine). IARC Monograph on the Evaluation of Carcinogenic Risks to Humans: Occupational exposures of hairdressers and barbers and personal use of hair colourants; some hair dyes, cosmetic colourants, industrial dyestuffs and aromatic amines, 57, 323-335. Jacobson, K.H. (1972). Short communication. Acute oral toxicity of mono- and di-alkyl ring-substituted derivatives of aniline. Toxicol. Appl. Pharmacol., 22, 153-154. Klide, A.M., Calderwood, H.W., & Simen L.R. (1975). Cardio-pulmonary effects of xylazine in dogs. Am. J. Vet. Res., 36(7), 931-935. Kugler-Steigmeier, M.E., Frierich, U., Graf, U., Lutz, W.K., Maier, P., & Schlatter, C. (1989). Genotoxicity of aniline derivatives in various short-term tests. Mutat. Res., 211, 279-289. Leblanc, P.H. & Eberhart, S.W. (1990). Cardiopulmonary effects of epidurally administered xylazine in the horse. Equine Vet. J., 22(6), 389-391. Lewis, S., O'Callaghan, C.L.P., & Toghill, P.J. (1983). Clinical curio: self medication with xylazine. Br. Med. J., 287, 1369. Lindstrom, H.V., Hansen, W.H., Nelson, A.A., & Fitzhugh, O.G. (1963). The metabolism of FD&C Red No. 1. II. The fate of 2,5- para-xylidine and 2,6- meta-xylidine in rats and observations of the toxicity of xylidine isomers. J. Pharmacol. Exp. Ther., 142, 257-264. Lindstrom, H.V., Bowie, W.C., Wallace, W.C., Nelson, A.A., & Fitzhugh, O.G. (1969). The toxicity and metabolism of mesidine and pseudocumidine in rats. J. Pharmacol. Exp. Ther., 167(2), 223-234. McLean, S., Murphy, B.P., Starmer, G.A., & Thomas, J. (1967). Methaemoglobin formation induced by aromatic amines and amides. J. Pharm. Pharmacol., 19, 146-154. McLean, S., Starmer, G.A., & Thomas, J. (1969). Methaemoglobin formation by aromatic amines. J. Pharm. Pharmacol., 21, 441-450. Magnusson, G., Bodin, N.-O., & Hansson, E. (1971). Hepatic changes in dogs and rats induced by xylidine isomers. Acta Pathol. Microbiol. Scand., 79, 639-648. Magnusson, G., Majeed, S.K., Down, W.H., Sacharin, R.M., & Jorgeson, W. (1979). Hepatic effects of xylidine isomers in rats. Toxicology, 12, 63-74. Mawdesley-Thomas, L.E. (1970). Pathology report of the subchronic toxicity for dogs by oral administration of compound BAY Va 1470. Unpublished report No. 26692 from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by Bayer AG, Leverkusen, Germany. Mirsalis, J.C., Tyson, C.K., Steinmetz, K.L, Loh, E.K., Hamilton, C.M., Bakke, J.P., & Splading, J.W. (1989). Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: testing of 24 compounds. Environ. Mol. Mutagen., 13, 155-164. Murphy, J.J. & Jacobs, K. (1975). Residues of ROMPUN and its metabolites in cattle. Unpublished report No. 43814 from Chemagro Agricultural Division, Mobay Chemical Corporation. Submitted to WHO by Bayer AG, Leverkusen, Germany. Mutlib, A.E., Chui, Y.C., Young, L.M., & Abbott, F.S. (1992). Characterization of metabolites of xylazine produced in vivo and in vitro by LC/MS/MS and by GC/MS. Drug Metab. Dispos., 20(6), 840-848. Nelson, D.L., Bauman, E.K., Mosier, J.O., & Allen, A.D. (1968a). A study of the effect of large doses of BAY Va 1470 to horses. Unpublished report No. 23405 from Research and Development Department, Chemagro Corporation. Submitted to WHO by Bayer AG, Leverkusen, Germany. Nelson, D.L., White, R.G. Bauman, E.K., & Allen, A.D. (1968b). The effect of large injected doses of BAY Va 1470 to dogs. Unpublished report No. 23679 from Research and Development Department, Chemagro Corporation. Submitted to WHO by Bayer AG, Leverkusen, Germany. Parton, J.W., Probst, G.S., & Garriott, M.L. (1988). The in vivo effects of 2,6-xylidine on induction of micronuclei in mouse bone marrow cells. Mutat. Res., 206, 281-283. Parton, J.W., Beyers, J.E., Garriott, M.L., & Tamura, R.N. (1990). The evaluation of multiple dosing protocol for the mouse bone-marrow micronucleus assay using benzidine and 2,6-xylidine. Mutat. Res., 234, 165-168. Poklis, A., MacKell, M.A., & Case, M.E.S. (1985). Xylazine in human tissues and fluids in a case of fatal drug abuse. J. Anal. Toxicol., 9, 234-236. Ponder, S.W. & Clark, W.G. (1980). Prolonged depression of thermoregulation after xylazine administration to cats. J. Vet. Pharmacol. Ther., 3, 203-207. Pütter, J. & Sagner, G. (1973). Chemical studies to detect residues of xylazine hydrochloride. Vet. Med. Rev., 2, 145-159. Reynolds, S.M. (1994). Xylazine hydrochloride: teratology study in the rat. Unpublished report No. 94/BAG240/0268 from Pharmaco LSR. Submitted to WHO by Bayer AG, Leverkusen, Germany (GLP statement and report summary, only). Robertson, S.A., Carter, S.W., Donovan, M., & Steele, C. (1990). Effects of intravenous xylazine hydrochloride on blood glucose, plasma insulin and rectal temperature in neonatal foals. Equine Vet. J., 22(1), 43-47. Ruckebusch, Y. & Toutain, P.L. (1984). Specific antagonism of xylazine effects on reticulo-rumen motor function in cattle. Vet. Med. Rev., 1, 1-12. Rudd, C.J., Mitchell, A.D., & Spalding, J. (1983). L5178Y Mouse lymphoma cell mutagenesis assay of coded chemicals incorporating analyses of the colony size distributions (Abstract No. Cd-19). Environ. Mutagen., 5(3), 419. Samanta, A., Roffe, C., & Woods, K.L. (1990). Accidental self administration of xylazine in a veterinary nurse. Postgrad. Med. J., 66, 244-245. Sagner, G. (1967). Bay Va 1470, anaesthetic, analgesic and sedative for veterinary medicine. Unpublished report No. 1470 from Bayer AG Institute of Pharmacology. Submitted to WHO by Bayer AG, Leverkusen, Germany. Sagner, G., Hoffmeister, F., & Kroneberg, G. (1969). Pharmacological principles of a new preparation for analgesia, sedation and relaxation in veterinary medicine (Bay Ve 1470). Vet. Med. Rev., 3, 226-228. Short, C.R., King, C., Sistrunk, P.W., & Kerr, K.M. (1983). Subacute toxicity of several ring-substituted dialkylanilines in the rat. Fundam. Appl. Toxicol. 3, 285-292. Short, C.R., Joseph, M., & Hardy, M.L. (1989). Covalent binding of [14C]-2,6-dimethylaniline to DNA of rat liver and ethmoid turbinate. J. Toxicol. Environ. Health, 27, 85-89. Singh, J., Peshin, P.K., Singh, A.P., & Nigam, J.M. (1983). Haemo-dynamic, acid base and blood gas alterations after xylazine administration in calves. Indian J. Vet. Surg., 4, 10-15. Skarda, R.T., Jean, G., & Muir, W.W. (1990). Influence of tolazoline on caudal epidural administration of xylazine in cattle. Am. J. Vet. Res., 51(4), 556-560. Spoerke, D.G., Hall, A.H., Grimes, M.J., Honea, B.N., & Rumack, B.H. (1986). Human overdose with veterinary tranquilizer xylazine. Am. J. Emerg. Med., 4, 222-224. Swift, B.L. (1977). A technique for the surgical removal of the spleen in calves. Vet. Med.-Small Anim. Clin., January, 77-79. Symonds, W.H. (1976). The effect of xylazine on hepatic glucose production and blood flow rate in the lactating dairy cow. Vet. Rec., 99, 234-236. Takase, I., Terada, H., & Fujii, T. (1976) Xylazine residues in organs and tissues of calves and milk of cows. Unpublished report No. 76/ 8278a from the Department of Veterinary Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology. Submitted to WHO by Bayer AG, Leverkusen, Germany. Tettenborn, D. (1969). BAY Va 1470. Subchronic toxicity for dogs during application as feed additive. Unpublished report No. 26692 (1731) from Bayer AG, Institute of Toxicology. Submitted to WHO by Bayer AG, Leverkusen, Germany. Tettenborn, D. & Hobik, H.P. (1968a). BAY Va 1470. Chronic toxicity to rats from oral administration. Unpublished report No. 23414 (956) from Bayer AG, Institute of Pharmacology and Toxicology. Submitted to WHO by Bayer AG, Leverkusen, Germany. Tettenborn, D. & Hobik, H.P. (1968b). BAY Va 1470. Subchronic toxicity to dogs from oral administration. Unpublished report No. 23415 (958) from Bayer AG, Institute of Pharmacology and Toxicology. Submitted to WHO by Bayer AG, Leverkusen, Germany. Thurmon, J.C., Neff-Davis, C., Davis, L.E., Stoker, R.A., Benson, G.J., & Lock, T.F. (1982). Xylazine hydrochloride-induced hyperglycemia and hypoinsulinemia in thoroughbred horses. J. Vet. Pharmacol. Ther., 5, 241-245. Thurmon, J.C., Steffey, E.P., Zinkl, J.G., Waliner, M., & Howland, D. (1984). Xylazine causes transient dose-related hyperglycemia and increased urine volume in mares. Am. J. Vet. Res., 45(2), 224-227. Trossmann, G. & Hobik, H.P. (1970). BAY Va 1470. Histopathological changes in organs of rats after feeding for 32 weeks. Unpublished report No. 28576 (956) from Bayer AG, Institute of Pathology and Histology. Submitted to WHO by Bayer AG, Leverkusen, Germany. US National Toxicology Program (1990). Toxicology and Carcinogenesis Studies of 2,6-Xylidine (2,6-Dimethylaniline) (CAS No. 87-62-7) in Charles River CD Rats (Feed Studies). US National Toxicology Program, Research Triangle Park, NC, USA (NTP Technical Report No. 278; NIH Publication No. 90-2534). Vernot, E.H., Macewen, J.D., Haun, C.C., & Kinkead, E.R. (1977). Acute toxicity and skin corrosion data for some organic and inorganic compounds and aqueous solutions. Toxicol. Appl. Pharmacol., 42, 417-423. Wagner, A.E., Muir, W.W., & Hinchcliff, K.W. (1991). Cardio-vascular effects of xylazine and detomidine in horses. Am J. Vet. Res., 52(5),:651-657. Wasak, A. (1983). Haemetological and electrocardiographical changes in dogs after xylazine. Med. Weter., 39, 235-237. Weiss, L.D., Generalovich, T., Heller, M.B., Paris, P.M., Stewart, R.D., Kaplan, R.M., & Thompson, D.R. (1987). Methemoglobin levels following intravenous lidocaine administration. Ann. Emerg. Med., 16(3), 323-325. Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., & Mortelmans, K. (1988). Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals. Environ. Mol. Mutagen., 11(12), 1-158.
See Also: Toxicological Abbreviations XYLAZINE (JECFA Evaluation)