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I-MENTHOL : ABSORPTION, EXCRETION AND BIOTRANSFORMATION IN RATS, MICE, SYRIAN HAMSTERS AND GUINEA PIGS

CONFIDLNTIAL 7D/BATS/239 I-MENTHOL : ABSORPTION, EXCRETION AND 8IOlRANSFORMATION IN RATS, MICE, SYRIAN HAMSTERS AND GUINEA RIGS To: British-American Tobacco Company Limited, Group Research & Development Centre, Regent's Park Road, Southampton. SO9 IPE From: J.W. Daniel, g. RycrofP, Life Science Research, Stock, Essex. CM4 9PE 7 Dctober 1976 5708J -870

L|FE SC|ENCE RESEARC~ I-MENTHOL : ABSORPTION, EXCRETION --AND BIOTRANSFORMATION IN RATS, MICE~ SYRIAN HAMSTERS AND GUINEA PIGS LSR Report No : 76/BAT8/23g We, the undersigned, hereby declare that the report following constitutes a true and faithful account of the procedures adopted, and the results obtained, in the performance of this study. J.W. Daniel, Ph.D., D.Sc., F,I.Biol., C.Chem., R.R.I.C. (Director, Metabolic Studies) D. Rycroft, C.Chem., M.R.].C. (Metabolic Chemist)

CONTENTS I. SUMMARY ..................... 2. INgRODUCTION .................. 3, MATERIALS ..................... 4, METHODS ..................... 5, RESULTS ..................... 6. DISCUSSION .................. 7. COMMENT ..................... TABLES I, The excretion of radioactivity in the urine and faeces ef male rats given I3H]-i-menthol by intraperitoneal injection ...... -- ............... 2. The excretion of radioactivity in the urine and faeces of male mice given [3HJ }-menthol by intraperitoneal injection ..................... S. The excretion of radioactivity in the urine and faeces of male Syrian hamsters given 13H] ~menthol by intraperitoneal injection ............... 4. The excretion of radioactivity in the urine ant faeces of male guinea pigs given 13B]-~-menthol by intraperitoneal injection ............... g. The excretion of radioacLivity iu the t;~ine and i~eces of male rats after the oral administration of [eH]-l-menthol (225 mg/kg) ............ 6, IHe excretion of radioactivity in the urine and faeces of male rats after the oral administration of {~H]-~-menthol (675 mg/kg) ............ 7, Cumulative excretion of radioactivity in the bile of male rats given a single oral dose of ~3H] l menthol .., D, The excretion of radioactivity in the u~ine and faeces of male rats after the oral administration of bile obtained from rats dosed with I~(]-~-mentho! (225 mg/kg) .., Pa~ l 3 4 g 9 12 14 15 16 17 18 19 20 21 22 (i) o: s2.8 z

C 0 N T E N T S - continued TABLES - continued 9A Concentration of radioactivity in the blood of rats, mice, hamsters and guinea pigs at intervals foilowing a single intraperitoneal dose of [SH]-l-menthol ......... gB Concentration of radioactivity in the blood of rats after a single intraperitoneal dose of [SH]-!-menthol ...... IDA Concentration of l-menthol in the blood of rats, mice, hamsters and guinea pigs following a single intraberitoneal dose of [SH]-1-menthol ......... 10B Concentration of l-menthol in the blood of rats following a singl~ intraperitaneal dose of f~H]-l-menthol ,,, FIGURES I. Thin ]ayer histogram mf the distribution of radioactivity from rat urine, after intraperitoneal administration of 13HI-l-menthol, before and after hydrolysis with B-glu~uronidase .................. Thin layer histogram of the distribution of radioactivity from mouse urine, after intraperitoneal administration of L~il]-1-menthol, bmfcre and after hydro]ysis with B-glucuronidase .................. 3. Thin layer histogram of the distribution of radioactivity from Syrian hamstmr urine, after in~raperitoneal administration of l~H]-l-menthol, before and after hydrolysis with B-glucu~onidase ............ 4. Thin layer histogram cf the distribution of radioactivity from guinea pig urine, after intraperitoneai administration of {~H] l-menthol, before and after lydrolysis Iiilh ~ glucuronidase ............ 5. Thin layer histogram ef the distribution of radioactivity from rat urine, after intraperitoneal administration of [SHl-l-mentbol, before and after hydrolysis with ~-glucuronidase .................. 6. Thin layer histogram of the distribution of radioactivity frOllIInouse urine, after intraperitoneal administration of [~H] l-menthol, before and after hydrolysis with #-glucuronidase .................. Page 23 24 25 26 27 2B 29 C 31 32 (ii}

C 0 N T E N T S ~ continued FIGURES - continued 7. Thin layer histogram of the distribution of radioactivity from Syrian hamster urine, after intraperitoneal administration of [3H]-Irmenthol, before and after hydrolysis with ~-glucu~enidase ............ 8. Thin layer histogram of the distribution of radioactivity from guinea pig urine, after intraperitoneal administration of [3H]-l-mentho], before and after hydrolysis with B-gluou~onidase ............ 9. Cumulative excretion of radioactivity i9 the bile of rats after a single oral dose of [3H]-~-menthol ...... lO, Thin layer histogram of the distribution of radioactivity in rat bile after oral admlnistra,ion of [3HI-l-menthol, before and after hydrolysis with 5-glucuronida~e ...... II. Thin layer histogram of the distribution of radioactivity in rat bile after oral administration of [3HI ]-menthm], before and after hydrolysis with ~-glucuronida~e ...... 12, The concentration of radioactivity and l-menthol, ug/ml of blood, in rats after intraperitoneal administration of [~H]-i-menthol .................. 13. The concentration of radioactivity and i-menthol, ~g/ml of blood, in mice after intraperitoneal administratlon of [Sill-l-menthol .................. 14. The concentration of radioactivity and l-menthol, ~g/ml of blood, in Syrian hamsters after intrapeTitoneal administration of [3H]-~-menthol .................. 15. ]he concentration of !~di3aetivity an(] I menthol, l~g/ml st blood, in guirea pigs ~fter intra~eritoneal administration of [3H1 l-menthol .................. APPENDIX I. Estimation of l-menthol in blood ............ 33 34 35 36 37 38 39 40 41 42 (iii) 57031 8 4

I. SUMMARY ],I Rats, mice, Syrian hamsters and guinea pigs given a solution of tritiated l-menthol (200 mg/kg) in polyethylene glycol 200 by intraperi~neal injection excreted all average of 65.5, 86.0, 88,3 and 85,4 percent of the radioactivify in the urine and 14,0, 2.1, i.4 and 8.8 percent in the faeces in four days. Mice, hamsters and guinea pigs excreted 70 80 percent of the radio- activity within 24 hours, whereas only 45 percent was excreted by rats in the same period. ].2 Thin-layer chromatographic examination of the 24-hour urines indicated the presence of menthyi glucuronide as a major component of the urine of rat, hamster and guinea pig, whereas the major component(s) in mouse urine had chromatographic characteristics similar to that of l-menthol. No evidence was obtained for the presence of a sulphate conjugate in any species. Maximum average blood levels of radioactivity> equivalent to 37, 172, 158 and log ~g l-menthol/ml, were obtained within 2 hours in all four species Tollowing the administYation of tritiated l-menthol (200 mg/kg) by intraperitorleal injection. Blood levels of radioactivity in mice, hamsters and guinea pigs renl~ined constant for the duration of the experiment (5 ho~rs), whereas in rats the levels declined with a hal# life of approximately 1,5 hours, 1.4 Gas chromatographic analysis of the individual blood samples indicated maximum average levels of l-menthol of 4, 17, 17 and 38 pg/ml in rats, mice, hamsters andguinea-pigs respectively, within 20 minutes of dosing, Because of variability between individual dnimals it ilrowd impossible to derive a precise value for the half-life of l menthol hl blood. It wo~id appear however that the concentY~tion declines at a comparable ~ate in ell four species. 1.5 Rats given an oral dose of tritiated 1 menthol at 225 and 675 mg/kg excreted an average of 83.3 andgO percent of tile radio- activity in the urine and the remainder in the faeces within four days. i.6 Approximately 52 percent of the radioactivity was excreted within 24 hours in the bile when tritiated I menthol (225 mg/kg) was administmred by garage to rats in which the bile duct had been cannulated prior to dosing. 1

1.7 Chromatographic analysis of the 24-hour samples of urine and bile obtained following the oral administration of tritiated l-menthol to rats indicated the presence of two major radio- ~ctive components, one of which was identified as mentbyl glucuronide. 1.8 Rats given a single oral dose of bile from rats treated with tritiated l-menthol excreted 38 percent of the radioactivity in the urine and 25 percent in the faeces within four days. Those data were interpreted to indicate the presence of an entero- hepatic circulation of !-menthol in rats. I STO3X2,3 6 2

2. INTRODUCTION Comparatively little is known of the metabolic disposition of I menthol in laboratory animals. Previous studies in these labora- tories have shown that the material is rapidly removed from the blood when administered intravenously to dogs, and the low circulating levels of 1 menthol after oral adminlstration to rats is consistent with eithe~ poor intestinal absorption or rapid metabolism (LSR Report NO. 75/BAT6/~78). The toxicity of both I- and dl-menthol when administered orally to rats is low (LSR R~port NOT 75/BAT4/217). No attempt has been made, however, to assess the sub-acute or chronic toxicity of either compound by systemic application. The selection of the most appropriate species of laboratory animal for such studies is somewhat empirical because of the absence of a~y comparative metabolic data. This report describes the results of experiments to examine the pharamcokinetics and biotr~nsformation of l-menthol when administered by irltraperiLoncal injection to rats, mice, Syrian hamsters and guinea pigs, and the absorption and elimination of l-menthol after oral administration to rats. Tritium labelled l-m~ntbol was used in all these experiments. AS previous toxicity studies in rats had failed to demonstrate any related differecces in response, the present investigations were performed using only mal~ animals. / • •°..i , 570 12877"

3, MATERIALS 3.1 Chemicals ]-Mentho] was provided by British-American Tobacco Co., on IO April 1974. L-Menth~ne was obtained from Eastman - Kodak Ltd.; bovine B-glucuronidase (2,500 Fishman units/ml) from William R, Warner, Eastlelgh. Bants.; a crude extract of Helix pomatia containing both sulphatase (5,000 units/ml) and 6-glucuronidase activity and ] : 4-saccharolactone from Sigma Chemicals Ltd.; Ana]ar grade reagents from Fisons Scientific Apparatus Ltd., ~o0glT~orough; Gas-chrom Q from Field Instrument co. Ltd., Richmond and Carbowax 20 M from Chromatog~apby Services Company~ Birkenhead and thin-layer plates of Silica 9e] GFzs4 from Merck. Darmstadt. West Germany. Polyethylene glycol 200 (PEG 200) was obtained from Hopkins and WiT]Jams Ltd.. Romford. Analysis of the material by British-American Tobacco indicated the followhlg values: Ash - Nil. Density - 1,0794 g/ml at 29nC. Impurities Ethylene glycol - 1.1% w/w. Diethylene glycol - 6.6% w/w, 3.2 Preparation of [g - 3HI _l-menthol L-Men%hone (22.4 mgs) in methanol C] ml} was stirred at room temperature for 4 hours with sodium boro[gH] hydride (7 mgs). Carrier l-menthol (].l g) together v~irh glacial acetic acid (50 ~I) were added and the solutioT~ concentrated to dryness. Methanol (I ml) was added and the process repeated to remove Bll traces of labile tritium. The product was purified by preparative thin layer ebrcn!atogra~hy on si)ica go! irl rl-Ke×ane ethyl ~cetate {9:1, v/v) and the I menzhol eluted w~Th hexane-ethyl acetate (50% v/v). The product {246 mgs) had a specific radieectivity of 155 m~i/F,T~l and a purity greater" than 97 Dercent. 3.3 Animals Male CD-rats (160 !80 g bodyweight} and CD l mice (25 - 30 g) were obtained from Charles River U.K. Ltd., M~rgate; male Syrian hamsters {50 - 60 g) from Coombehurst Breeding Establishment%, Basingstoke, =nd ma]e guinea pigs (~80 ~ 300 g) from A. Tuck and So~1 Ltd., ~ayleigh. / 4