Absorption, Distribution and Excretion
In a series of metabolism studies, [phenyl-U-14C]-AE 0172747 (/tembotrione/ Batch # Z 31053-4; radiochemical purity 99.5%) or [cyclohexyl-UL-14C]-AE 0172747 (/tembotrione/ Batch #s BECH 1517 or BECH 1523; radiochemical purity >98%) in PEG 200 was administered by oral gavage to groups of four Wistar rats/sex/dose at doses of 5 or 1000 mg/kg. The concentration time-courses of radioactivity in blood and plasma were calculated, the concentrations of radioactivity in tissues and excreta were determined, and metabolites were identified and quantified in the urine and feces. The test compound was absorbed rapidly, as radioactivity was detected in the blood and plasma of all animals at the first time point measured (30 min post-dosing) for both radiolabeled forms. Males had higher mean blood and plasma maximum concentrations (Cmax) than females. Also, males displayed higher AUC values than females in both blood and plasma at both doses. In both sexes, the AUC for both blood and plasma indicated a disproportionally higher mean systemic exposure at 1000 mg/kg than at 5 mg/kg (>200-fold) that was apparently due to a saturation of the initial elimination/biotransformation processes, resulting in a slower initial elimination phase. Other blood and plasma parameters were generally similar across doses and radiolabeled forms. In the 5 mg/kg animals dosed with either radiolabeled form, the liver and kidneys contained the highest mean levels of radioactivity. No other tissue exceeded 0.12% of the administered dose. In the 1000 mg/kg animals dosed with [phenyl-U-14C]-AE 0172747, the skin/fur and carcass contained the highest mean levels of radioactivity. No other tissue exceeded 0.06% of the administered dose. In the 5 mg/kg [phenyl-U-14C] males, the highest concentrations of radioactivity were detected in the, liver, kidneys, skin, and carcass. In the 5 mg/kg [phenyl-U-14C] females and [cyclohexyl- UL-14C] males and females, the highest concentrations of radioactivity were detected in the liver, kidneys, skin, and carcass. In the 1000 mg/kg [phenyl-U-14C] males and females, the highest concentrations of radioactivity were detected in the skin, liver, kidneys, stomach (and contents), and carcass and there was no evidence of bioaccumulation. Total recoveries ranged from 96.3-102.7% of the administered doses, with no differences observed between dose levels or position of the radiolabel. Substantial sex differences were observed in the routes of excretion. At 5 mg/kg, the majority of the radioactivity was recovered in the feces of the males, while in the females, the majority of the radioactivity was recovered in the urine. At this dose, the majority of the radioactivity in the urine was recovered during the first 6 h, while the majority of radioactivity in the feces was recovered during the first 24 h. Tissues and cage wash each accounted for <5.1%. Sex differences in the routes of excretion were also observed in the 1000 mg/kg group. In the males, approximately equal proportions of radioactivity were recovered in the feces and urine, while in the females, the majority of the radioactivity was recovered in the urine. At this dose, the majority of the radioactivity in the urine was recovered during the first 24 h, while the majority of radioactivity in the feces was recovered during the first 48 h. Tissues and cage wash each accounted for <10.1%. The test compound was extensively metabolized. The majority of radioactivity in urine and fecal extract samples was present as parent and up to eleven metabolites. Metabolic profiles were qualitatively similar for both radiolabeled forms; however, profiles for the high and low doses were dissimilar, and major differences were noted between sexes. The major route of metabolism was found to be hydroxylation (oxidative pathway) of the cyclohexyl ring of the molecule. In excreta, parent and identified compounds accounted for 68.1-93.2% of the administered dose, while unidentified metabolites accounted for 2.5-13.8% of the administered dose. The total administered dose accounted for in the excreta was 82.3-104.9%. Parent compound accounted for 1.9-59.9% of the total radioactivity eliminated, and was found in greatest quantity in the urine of the females (44.1-59.4%). Low dose males eliminated small amounts of parent (1.9-3.0%), while high dose males eliminated moderate amounts (33.8%). The metabolite found in the greatest quantity at both doses was 4-hydroxy-AE 0172747, with low dose males eliminating more than low dose females. High dose males and females eliminated approximately equal amounts. The only other metabolite found at >5% of the administered dose was 5-hydroxy-AE 0172747. Males excreted greater quantities than females.
Rat metabolism data indicate that tembotrione is well absorbed. More than 96.3% of the administered dose was recovered in urine and feces in 24 hours. Sex differences were observed in the routes of excretion. The primary routes of elimination were the urine in females and the urine and feces in males. At the low dose, males excreted up to 24.4% and 70.4%; females up to 79.1% and 20% of the administered dose in the urine and feces, respectively. At the high dose, females excreted up to 63.7% and 28.5%; males up to 44.2 % and 49.1% of the dose in the urine and feces, respectively. The highest mean levels of radioactivity were extracted from the liver (1.7-3.5%) and kidneys (0.14-0.26%) at the low dose. At the high dose, the mean levels of radioactivity were extracted from the skin/fur (0.22-0.33%) and carcass. The highest concentrations of radioactivity were found in the skin followed by the liver, kidneys, stomach (and contents) and carcass. Males had higher mean blood plasma maximum concentrations (Cmax) and AUC values than females. In both sexes, the area under the AUC for both blood and plasma indicated a disproportionally higher mean systemic exposure at 1000 mg/kg than at 5 mg/kg (>200-fold) that was apparently due to a saturation of the initial elimination/biotransformation processes, resulting in a slower initial elimination phase.
In an in vivo dermal penetration study, [phenyl- UL-14C]-AE 0172747 (/tembotrione/ >98% radiochemical purity; batch # BECH 0857) in a suspension concentrate formulation containing 420 g/L AE 0172747 and 210 g/L Isoxadifen-ethyl was applied to four male Wistar (Rj:WI[IOPS HAN]) rats/group on 2 x 6 sq cm skin areas at dose levels of 0, 6.6, 66, or 660 ug/sq cm. Exposure times were 0.5, 1, 2, 4, 10, and 24 hr for each dose. At the end of each exposure period, the skin was swabbed, and urine, feces, treated skin, cardiac blood, kidneys, liver, brain, spleen, and residual carcass were collected and analyzed for radioactivity. Recovery of the applied dose was 90.8-98.7% of the administered dose. The distribution profile of radioactivity was qualitatively similar between the dose groups. The majority of the administered dose was recovered from the skin swabs, accounting for 76-93% of the administered doses. A total of 76-94% of the applied doses was not absorbed. A general trend of increasing dermal absorption with increasing time was observed, and the amount of radioactivity found in the treated skin generally increased with decreasing dose level. Estimates of dermal absorption were based on the sum of the treated skin + the total directly absorbed (urine + feces + cage wash + carcass + brain + spleen + liver + kidneys + blood + non-treated skin + surrounding skin). Dermal absorption was 8.3-14.9% (low), 4.8-12.8% (intermediate), and 1.7- 4.8% (high) of the applied doses. The amount of dermal absorption was not proportional to dose. All treatments (dose levels applied) were for exposure periods for up to 24 hr. The most conservative value for risk assessment is a dermal-absorption of 15% observed at the low dose (6.6 ug/sq cm) at 4 hr after application. This value should be considered to protect commercial applicators.

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Metabolism / Metabolites
The parent molecule and 11 metabolites were identified & isolated from urine and feces /of the rat/. Metabolic profiles were qualitatively similar for both radiolabeled forms; however, profiles for the high and low doses were not the same and differences were noted between sexes. Females excreted the greatest quantity of the parent molecule in urine (44.1-59.4%). While low and high dose males eliminated 1.9-3.0% and 33.8%, respectively, in the urine. The metabolites found in the greatest quantities were 4-hydroxy-tembotrione and 5-hydroxy-tembotrione. Other identified metabolites found at <5% were the 4,5-dihydroxy, benzylic alcohol, dihydroxybezophenone, 4-hydroxy-benzylic alcohol, and ketohydroxy-hexanoic acid ([cyclohexyl-UL-14C] only). Males excreted greater quantities of both major metabolites than females; except, at the high dose where 4-hydroxy-tembotrione was eliminated in approximately equal amounts in both sexes. The primary step in the metabolism of tembotrione is the hydroxylation (oxidative pathway) of the cyclohexyl ring of the molecule.
In a series of metabolism studies (MRIDs 46695726, 46695727, 46695728, and 46695729), [phenyl-U-14C]-AE 0172747 (Batch # Z 31053-4; radiochemical purity 99.5%) or [cyclohexyl-UL-14C]-AE 0172747 (Batch #s BECH 1517 or BECH 1523; radiochemical purity >98%) in PEG 200 was administered by oral gavage to groups of four Wistar rats/sex/dose at doses of 5 or 1000 mg/kg. The concentration time-courses of radioactivity in blood and plasma were calculated, the concentrations of radioactivity in tissues and excreta were determined, and metabolites were identified and quantified in the urine and feces. The test compound was absorbed rapidly, as radioactivity was detected in the blood and plasma of all animals at the first time point measured (30 min post-dosing) for both radiolabeled forms. Males had higher mean blood and plasma maximum concentrations (Cmax) than females. Also, males displayed higher AUC values than females in both blood and plasma at both doses. In both sexes, the AUC for both blood and plasma indicated a disproportionally higher mean systemic exposure at 1000 mg/kg than at 5 mg/kg (>200-fold) that was apparently due to a saturation of the initial elimination/biotransformation processes, resulting in a slower initial elimination phase. Other blood and plasma parameters were generally similar across doses and radiolabeled forms. In the 5 mg/kg animals dosed with either radiolabeled form, the liver and kidneys contained the highest mean levels of radioactivity. No other tissue exceeded 0.12% of the administered dose. In the 1000 mg/kg animals dosed with [phenyl-U-14C]-AE 0172747, the skin/fur and carcass contained the highest mean levels of radioactivity. No other tissue exceeded 0.06% of the administered dose. In the 5 mg/kg [phenyl-U-14C] males, the highest concentrations of radioactivity were detected in the, liver, kidneys, skin, and carcass. In the 5 mg/kg [phenyl-U-14C] females and [cyclohexyl- UL-14C] males and females, the highest concentrations of radioactivity were detected in the liver, kidneys, skin, and carcass. In the 1000 mg/kg [phenyl-U-14C] males and females, the highest concentrations of radioactivity were detected in the skin, liver, kidneys, stomach (and contents), and carcass and there was no evidence of bioaccumulation. Total recoveries ranged from 96.3-102.7% of the administered doses, with no differences observed between dose levels or position of the radiolabel. Substantial sex differences were observed in the routes of excretion. At 5 mg/kg, the majority of the radioactivity was recovered in the feces of the males, while in the females, the majority of the radioactivity was recovered in the urine. At this dose, the majority of the radioactivity in the urine was recovered during the first 6 h, while the majority of radioactivity in the feces was recovered during the first 24 h. Tissues and cage wash each accounted for <5.1%. Sex differences in the routes of excretion were also observed in the 1000 mg/kg group. In the males, approximately equal proportions of radioactivity were recovered in the feces and urine, while in the females, the majority of the radioactivity was recovered in the urine. At this dose, the majority of the radioactivity in the urine was recovered during the first 24 h, while the majority of radioactivity in the feces was recovered during the first 48 h. Tissues and cage wash each accounted for <10.1%. The test compound was extensively metabolized. The majority of radioactivity in urine and fecal extract samples was present as parent and up to eleven metabolites. Metabolic profiles were qualitatively similar for both radiolabeled forms; however, profiles for the high and low doses were dissimilar, and major differences were noted between sexes. The major route of metabolism was found to be hydroxylation (oxidative pathway) of the cyclohexyl ring of the molecule. In excreta, parent and identified compounds accounted for 68.1-93.2% of the administered dose, while unidentified metabolites accounted for 2.5-13.8% of the administered dose. The total administered dose accounted for in the excreta was 82.3-104.9%. Parent compound accounted for 1.9-59.9% of the total radioactivity eliminated, and was found in greatest quantity in the urine of the females (44.1-59.4%). Low dose males eliminated small amounts of parent (1.9-3.0%), while high dose males eliminated moderate amounts (33.8%). The metabolite found in the greatest quantity at both doses was 4-hydroxy-AE 0172747, with low dose males eliminating more than low dose females. High dose males and females eliminated approximately equal amounts. The only other metabolite found at >5% of the administered dose was 5-hydroxy-AE 0172747. Males excreted greater quantities than females.
Rat metabolism data indicate that tembotrione is well absorbed. More than 96% of the administered dose was recovered in urine and feces in 24 hours. Minor sex differences were observed in the routes of excretion. The primary routes of elimination were the urine in females and the urine and feces in males. The highest concentrations of radioactivity were found in the skin followed by the liver, kidneys, stomach (and contents) and carcass. Males had higher mean blood, plasma maximum concentrations (Cmax) and area under the concentration-time curves (AUC) values than females. The primary step in the metabolism of tembotrione is the hydroxylation (oxidative pathway) of the cyclohexyl ring of the molecule.

Interactions
In a subchronic toxicity study, two groups of 10 male and 10 female Wistar rats (Groups 1 and 3) were fed basal diet while two groups of 10 male and 10 female Wistar rats (Groups 2 and 4) were fed diets supplemented with 20,000 ppm (2%) L-tyrosine (Lot/batch No. 078H06822 and 123K0376; purity >99%) for 28 days. (Tyrosine supplementation was approximately three to five times the normal dietary intake.) Rats in Groups 3 and 4 received 10 ug/kg bw/day 2-(2-nitro-4-trifluoromethyl-benzoyl)-1,3- cyclohexanedione (NTBC), an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, daily by gavage. The study was done to determine the effects of increased plasma tyrosine to the eye, kidney, liver, pancreas, and thyroid of rats. One Group 3 female rat died during the study, but its death was unrelated to treatment. No treatment-related effects were noted on body weight, body weight gain, or food consumption. Nine of ten male and 3/10 female rats in Group 4 (2% tyrosine + 10 ug/kg bw/day NTBC) developed white areas on the eye between Days 23-26 on one or more occasions. Following opthalmoscopic examination prior to sacrifice, 9/10 male rats in Group 4 had developed corneal edema and all male and 3/10 female rats had developed 'snow flake' corneal opacities. In addition, three Group 4 male rats had developed congestive iritis. None of the male and female rats in Group 2 (2% tyrosine) or Group 3 (10 ug/kg bw/day NTBC) developed ocular abnormalities. The average plasma tyrosine concentration of Group 3 and Group 4 male and female rats was markedly increased 18-23 fold on the day of sacrifice, while plasma tyrosine was unaffected by treatment in Group 2 rats. Although the liver to body weight ratio of male and female rats in Group 4 was statistically increased, no histological correlates were found. No other treatment-related effects were noted on organ weight. Microscopic treatment-related effects were found in the pancreas, thyroid, and eyes of Group 4 rats. The incidences of focal/multifocal acinar atrophy/ fibrosis and/or acinar degeneration/apoptosis, as well as the incidence of focal/multifocal or diffuse inflammation were increased in the pancreas of Group 4 male and female rats. In the thyroid, an increased incidence of colloid alteration was found in male, but not female rats of Group 4 rats. In the eye, the incidence of unilateral and bilateral keratitis was markedly increased in male rats while minimal keratitis was found in 1/10 Group 4 female rats. No treatment-related effects were noted in male or female Group 2 and Group 3 rats.
In a subchronic toxicity study, two groups of five male and five female Wistar rats (Groups 1 and 3) were fed basal diet while two groups of five male and five female Wistar rats (Groups 2 and 4) were fed diets supplemented with 20,000 ppm (2%) L-tyrosine (Lot No. 114K0375, purity 98.9%) for 28 days. (The tyrosine supplementation was approximately three to five times the normal dietary intake.) Rats in Groups 3 and 4 received 10 ug/kg bw/day 2-(2-nitro-4-trifluoromethyl-benzoyl)-1,3-cyclohexanedione (NTBC), an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, daily by gavage. The study was done to determine the effects of increased plasma tyrosine concentration to the eye, kidney, liver, pancreas, and thyroid of rats No toxicologically significant effects on body weight or food intake were noted. All male and 1/5 female rats in Group 4 (2% dietary tyrosine + 10 ug/kg bw/day NTBC by gavage) developed white areas on the eye beginning on Day 24 through the end of the study. In addition, the eyes of 4/5 Group 4 male rats were half-closed beginning on Day 22 through the remainder of the study. The average plasma tyrosine concentration of Group 4 male and female rats increased with time from approximately three to five fold on Day 2 to a 24-fold increase in males and 18-fold increase in females by Day 21. Treatment with 10 ug/kg bw/day NTBC alone had little effect on plasma tyrosine in male and female rats until Day 29/30 when it was increased 3-fold and 5.8- fold in males and females, respectively. After an overnight fast, plasma tyrosine was increased in NTBC-treated rats 18-fold in males and 27-fold in females. Treatment with 2% dietary tyrosine alone induced a < 5-fold increase of plasma tyrosine in male and female rats that decreased with fasting. There were no effects on the absolute or relative liver, brain, kidney, or thyroid weights of tyrosine-, NTBC, or tyrosine/NTBC-treated rats. Macroscopically, minimal to slight bilateral ocular opacity was observed in all male and 1/5 female rats treated with tyrosine/NTBC and microscopically, treatment-related effects were found in the eye, pancreas, and thyroid. Bilateral keratitis was observed in the eyes of all males and one female and diffuse interstitial mixed cell inflammation was noted in the pancreas of two males and one female rat treated with tyrosine/ NTBC. The pancreatic changes were associated with an increased incidence of focal/multifocal acinar degeneration and apoptosis. Minimal to slight thyroid colloid alteration was noted in 3/5 Group 4 male rats. No treatment-related effects, to the eye, pancreas, or thyroid, were noted in rats treated only with tyrosine or NTBC. This study demonstrated a prolonged threshold tyrosine concentration exists in rats, above which macroscopic and/or microscopic effects occur to the eye, pancreas, and thyroid. These effects occurred when rats were fed diets containing three to five times the normal dietary intake of tyrosine while one of the tyrosine catabolizing enzymes was inhibited.

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Non-Human Toxicity Values
LC50 Rat inhalation > 5.03 mg/L/4 hr
LD50 Rat dermal >2,000 mg/kg
LD50 Rat oral >2,000 mg/kg

Tembotrione is an aromatic ketone that is 2-benzoylcyclohexane-1,3-dione in which the phenyl group is substituted at positions 2, 3, and 4 by chlorine, (2,2,2-trifluoroethoxy)methyl, and methylsulfonyl groups, respectively. It is a post-emergence herbicide used (particularly in conjunction with the herbicide safener cyprosulfamide) for the control of a wide range of broad-leaved and grassy weeds in corn and other crops. It has a role as a herbicide, an agrochemical, an EC 1.13.11.27 (4-hydroxyphenylpyruvate dioxygenase) inhibitor and a carotenoid biosynthesis inhibitor. It is a sulfone, a cyclic ketone, an aromatic ketone, a member of monochlorobenzenes, an organofluorine compound, an ether and a beta-triketone.

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Mechanism of Action
Tembotrione is a broad-spectrum early and mid-postemergence herbicide that belongs to the triketone class of herbicides. It acts by inhibiting 4-hydroxyphenylpyruvate dioxygenase (HPPD), which leads to chlorophyll destruction by photooxidation and causes bleaching of emerging foliar tissue. In mammals, HPPD is a key enzyme in the catabolism of tyrosine. It catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) to homogentisate. Inhibition of HPPD leads to a reconversion of HPP to tyrosine and a consequent increase in blood tyrosine concentrations (tyrosinemia).

C17H16O6F3SCL

440.81854

440.031

335104-84-2

11556911

Beige powder

1.458g/cm3

612.86ºC at 760 mmHg

123 °C
MP: 117 °C

324.446ºC

1.519

4.024

0

9

6

28

714

0

ClC1C(COCC(F)(F)F)=C(S(C)(=O)=O)C=CC=1C(C1C(=O)CCCC1=O)=O

IUQAXCIUEPFPSF-UHFFFAOYSA-N

InChI=1S/C17H16ClF3O6S/c1-28(25,26)13-6-5-9(15(18)10(13)7-27-8-17(19,20)21)16(24)14-11(22)3-2-4-12(14)23/h5-6,14H,2-4,7-8H2,1H3

2-[2-chloro-4-methylsulfonyl-3-(2,2,2-trifluoroethoxymethyl)benzoyl]cyclohexane-1,3-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)