1,3-dichloro-2-propanol 
 ( dcp ) is a member of the broad chemical 
 class halohydrins . dcp is a semivolatile liquid used in high volume 
 as an intermediate in the production of epichlorohydrin ( ech ) , 1,3-dichloropropene , 
 and 1,2,3-trichloropropane . workers may be exposed 
 to 1,3-dcp via inhalation , dermal , or oral exposure during the manufacture 
 and use of these chemical agents . exposure to dcp may also occur from 
 ingestion of food containing hydrochloric acid - hydrolyzed vegetable 
 protein or from drinking water in which ech polyamine polyelectrolytes 
 are used as flocculants and coagulants for water purification . thermal degradation , metabolism , or hydrolysis of the flame retardant 
 tris(1,3-dichloropropyl ) phosphate ( fyrol fr-2 ) might also be a source 
 of consumer exposure to dcp . in subchronic drinking water or gavage studies 
 ( doses up to 100 
 mg / kg / day ) in male and female sprague  dawley rats , dcp caused 
 decreased body weights , increased liver and kidney weights , and histopathological 
 changes in the stomach , kidney , liver , and nasal tissue . in chronic drinking water studies ( doses up to 
 240 mg / l ) , dose - related increases in the combined incidences of hepatocellular 
 adenoma and carcinoma in the liver , squamous cell papilloma and carcinoma 
 in the tongue / oral cavity , follicular cell adenoma and carcinoma in 
 the thyroid , and the combined numbers of renal tubular adenoma and 
 carcinoma in the kidney ( males only ) have been reported in male and 
 female rats . in numerous bacterial and 
 mammalian test systems in vitro , dcp was genotoxic 
 in the presence and absence of metabolic activation . in humans , dcp is known to be moderately toxic via inhalation , 
 ingestion , and skin contact . acute hepatitis 
 was reported in 5 of 12 workers exposed to an unknown concentration , 
 likely via inhalation from the cleaning of a dcp manufacturing tank . in these individuals , dcp plasma levels at approximately 48 h after 
 exposure were 200 ng / ml . two of the five workers died from hepatic 
 failure , and autopsy showed hepatocellular necrosis in one of the 
 individuals . dcp is listed by the international agency for research 
 on cancer 
 ( iarc ) as being possibly carcinogenic to humans ( group 
 2b ) . california epa listed dcp on the 
 proposition 65 list of chemicals as known to cause cancer in humans . this assessment included consideration of the 
 prospective metabolism of dcp to carcinogenic metabolites , ech and 
 glycidol , and toxic metabolites , dichloroacetone ( dca ) and 3-monochloropropane-1,2-diol . ech is carcinogenic in experimental animals following exposure via 
 drinking water ( 375 to 1500 mg / l ) , gavage ( 2 and 10 mg / kg ) , and inhalation 
 ( 30 and 100 ppm ) . , there is no direct 
 evidence for the formation of ech or 
 dca following exposure to dcp in rodents or humans . indirect evidence 
 includes common metabolites observed in animals administered dcp and 
 ech . following oral administration of 50 mg dcp / kg daily for 5 days 
 in male sprague  dawley rats , -chlorolactate ( 5% 
 of the dose ) , n , n-bis(acetyl)-s , s-(1,3-bis(cysteinyl))propan-2-ol 
 ( 1% ) , and n - acetyl - s-(2,3-dihydroxypropyl)cysteine 
 were detected in urine . similar metabolites 
 were observed following oral administration of ech and hence ech was 
 proposed as an intermediate in the metabolism of dcp . in another study , following a single subcutaneous injection 
 of dcp at 68 mg / kg in male wistar rats , 1,2-propanediol ( 0.43% 
 of the dose ) and 3-chloro-1,2-propanediol ( 0.35% ) were detected in 
 the 24 h urine . on the basis of these 
 observations , two main metabolic pathways were proposed , as shown 
 in figure 1 , one via the formation of ech and 
 one via the formation of dca . several in vitro and in vivo studies reported the 
 ability of dcp to deplete gsh and to induce and/or be metabolized 
 by cyps . in wistar rats , diethyldithiocarbamate provided significant protection 
 against dcp hepatotoxicity and inhibited enzyme markers for cyp2e1 
 activity ; ech was proposed to be the toxic intermediate . in rat hepatocyte cultures , isoniazid increased 
 the rate and extent of gsh depletion as well as the toxicity , whereas 
 cyanamide did not , confirming the involvement of cyp2e1 in the metabolism 
 of dcp . pretreatment of cultures with 1-aminobenzotriazole ( a cyp 
 inhibitor ) prevented the toxicity of dcp , whereas pretreatment with 
 diethyl maleate or buthionine sulfoximine ( depletion of gsh or inhibition 
 of gsh synthesis ) increased its toxicity . the observation that pyridine in the microsomal incubations reduced 
 gsh depletion but did not completely eliminate depletion suggests 
 the existence of a second pathway , which was thought to be dca as 
 a metabolite of dcp via the action of alcohol dehydrogenase or cyps . proposed metabolic pathway of dcp in rodents . adapted 
 with permission 
 from ref ( 20 ) . the genotoxic and carcinogenic 
 potency of dcp is thought to arise 
 mainly due to the metabolism of dcp to ech and/or dca , although , as 
 pointed out earlier , there is no direct evidence in the literature 
 for the formation of these metabolites . this is likely due to the 
 reactive nature of these electrophilic metabolites that are postulated 
 to be short - lived in vivo . dcp was nominated to the 
 national toxicology program ( ntp ) by the national institute of environmental 
 health sciences for toxicological characterization , including metabolism 
 and disposition , reproductive toxicity , and carcinogenicity studies . unambiguous identification of ech and/or dca from the metabolism of 
 dcp is critical for deciding whether further toxicity and carcinogenicity 
 studies of dcp are needed . therefore , the purpose of the current work 
 is ( 1 ) to develop a sensitive analytical method to detect ech and 
 dca in selected biological matrices , ( 2 ) to investigate the half - lives 
 of ech and dca in selected biological matrices in vitro to determine the feasibility of detecting these reactive metabolites in vivo , and ( 3 ) to investigate the formation of ech and 
 dca in adult male and female harlan sprague  dawley rats following 
 oral exposure to dcp . the dose was set at 50 mg / kg based on the rat 
 ld50 values as well as the doses used in toxicity studies . dcp was obtained from acros 
 organics ( geel , belgium ) with a specified purity of 99.5% . ech was 
 obtained from aldrich chemical co. ( st . louis , mo ) with a vendor purity 
 of 99.9% . prior to use in studies , the identity of dcp and ech was 
 confirmed by a combination of gc - ms and h and c nmr spectroscopy . purity , estimated by a combination of gc - fid 
 and gc - ms , was 99.4 and 99.9% , respectively , for dcp and ech . ( h5)()-epichlorohydrin 
 ( ech - d5 ) ( 98.7 atom % d ; 98.8% chemical 
 purity ) and ( h5)1,3-dichloro-2-propanol ( dcp - d5 ) ( 98.8 atom % d ; 98.1% chemical purity ) was 
 from c / d / n isotopes ( pointe - claire , quebec , canada ) . ethyl acetate and 
 methyl tert - butyl ether were obtained from aldrich 
 chemical co. ( st . ten microliters of ech 
 in acetonitrile was added to 90 l of rat blood , rat plasma , 
 rat liver homogenate ( 250 mg / ml ) , or mouse blood at 37 c or 
 on ice to give a final ech concentration of 1 or 80 m . samples 
 were mixed and incubated for 5 , 15 , 30 , and 60 min . at the end of 
 the reaction , 100 l of ethyl acetate containing an internal 
 standard mixture ( 1 m ech - d5 ) was 
 added , and the samples were vortex mixed and centrifuged . the supernatant 
 was transferred into a microcentrifuge tube containing 10 mg of 3a 
 molecular sieves , vortex mixed , and centrifuged . standards were prepared containing 
 0.1 , 0.25 , 0.5 , 1.0 , and 2.5 m ech , prepared by adding 10 l 
 of ech in acetonitrile and 90 l of internal standard in ethyl 
 acetate . samples were analyzed by the gc - ms method developed ( but not validated ) 
 below . a method involving 
 the extraction of blood with ethyl acetate and drying the ethyl acetate 
 extract with 3a molecular sieves followed by gas chromatography mass 
 spectrometry ( gc - ms ) in positive ion chemical ionization mode was 
 developed and validated to quantify dcp and ech as described below . the recovery of dca from all matrices evaluated was extremely low , 
 likely due to its high reactivity , and hence was not evaluated any 
 further . solvent calibration curves for dcp and ech were prepared 
 in ethyl acetate over the range 0.01 , 0.025 , 0.05 , 0.1 , 0.2 , 0.5 , 
 and 1 m . matrix calibration curves were prepared using 90 l 
 of rat blood , plasma , or liver homogenate prepared in water ( 250 mg / ml ) 
 in a microcentrifuge tube , to which 10 l of a spiking solution 
 of analytes in acetonitrile was added to give final concentrations 
 of 0.01 , 0.025 , 0.05 , 0.1 , 0.2 , 0.5 , and 1 m in matrix . calibration 
 curves were also prepared in water similar to that for the matrix 
 calibration curves except that 90 l of water was used in place 
 of biological matrix . following the addition of 100 l of ethyl 
 acetate containing the internal standard mixture ( 0.25 m dcp - d5 and ech - d5 ) , samples 
 were vortex - mixed and centrifuged for 5 min . the ethyl acetate layer 
 was dried in a second microfuge tube containing 10 mg of 3a molecular 
 sieves . after vortex mixing , the sample was centrifuged , and the ethyl 
 acetate layer was transferred to a 200 l silanized insert in 
 a 1.5 ml amber gc vial . the method was validated in blood using solvent and matrix standard 
 curves prepared as above . to evaluate the extracted sample storage 
 stability , six sets of quality control ( qc ) rat blood samples , each 
 with four replicates , were prepared at 0.025 m ( low qc ) , 0.1 
 m ( medium qc ) , and 0.5 m ( high qc ) and were processed 
 as described above . another set was stored in an autosampler tray for 48 h at room temperature . four sets were stored at 20 c for 1 week , 3 months , 
 or 6 months prior to analysis . one set of samples was subjected to 
 three freeze  thaw cycles during a 1 week storage period . study 
 samples were prepared similar to that for the matrix calibration curve 
 samples as described above without the addition of the spiking solution 
 containing dcp and ech standards . all samples were analyzed 
 on an agilent 6890 gc equipped with a 
 programmable temperature vaporization inlet coupled to an agilent 
 5973 msd ( agilent technologies , santa clara , ca ) operated in positive 
 chemical ionization mode with selected ion monitoring , with the exception 
 of samples from female rats administered 25 mg / kg ech by gavage and 
 male and female rats administered 1 mg / kg ech by intravenous injection 
 ( see below ) . one microliter was injected via pulsed splitless injections 
 onto a db-5 ms ui column ( 30 m  0.25 mm i.d  0.25 m , 
 thickness ) ( agilent technologies , santa clara , ca ) . the injector was 
 programmed from 75 c ( 0.1 min ) to 200 c ( 0.5 min ) at 500 
 c / min with a pulse pressure of 50 psi at the time of injection . the gc oven 
 temperature was maintained at an initial temperature of 30 c 
 ( 1 min ) and then ramped at 5 c / min to 50 c . late eluting 
 compounds were removed by increasing the oven temperature to 200 c 
 at 80 c / min . dcp , dcp - d5 , ech , and ech - d5 were eluted at 6.65 , 6.60 , 4.02 , and 3.99 min , respectively . mass 
 spectrometer parameters were as follows : ion source temperature , 110 
 c ; quadrupole temperature , 106 c ; and transfer line temperature , 
 150 c . samples from female rats administered 25 mg / kg ech by 
 gavage and male and female rats administered 1 mg / kg ech by intravenous 
 injection and corresponding calibration curves ( with extension of 
 the calibration curve to 8 points , including 0.005 m ) were 
 analyzed on an agilent 7890a gc coupled to an agilent 7000 triple 
 quadrupole mass spectrometer ( agilent technologies , santa clara , ca ) . 
 all gc and mass spectrometer conditions were the same as those given 
 above except for the following : the injector temperature was constant 
 at 150 c , the ion source temperature was 150 c , and the 
 total run time was ca . 8 min with dcp , dcp - d5 , ech , and ech - d5 eluting at 4.10 , 
 4.06 , 3.15 , and 3.12 min , respectively . in all cases , ions monitored 
 were m / z 111 ( dcp ) , 116 ( dcp - d5 ) , 93 ( ech ) , and 98 ( ech - d5 ) . calibrations for both dcp and ech were performed using 
 seven - point standard curves prepared in rat blood . a 1/x weighted linear regression equation was calculated for dcp and ech , 
 relating the response ratios of the analyte / internal standard to its 
 concentration in matrix standard . all studies 
 were conducted at rti international 
 ( rtp , nc ) and were approved by the rti institutional animal care and 
 use committee . animals were housed in a facility that is fully accredited 
 by the association for assessment and accreditation of laboratory 
 animal care international . animal procedures were in accordance with 
 the guide for the care and use of laboratory animals . male and female harlan sprague  dawley rats ( males , 
 235277 g ; females , 184232 g ; 810 weeks old 
 at dosing ) with implanted jugular vein cannulae were obtained from 
 harlan laboratories ( indianapolis , in ) . the animals were quarantined 
 for up to 6 days before they were used in a study . animals were provided 
 certified ntp 2000 wafer diet ( zeigler brothers , gardners , pa ) and 
 city tap water ( durham , nc ) ad libitum . during the 
 quarantine and study periods , room temperature was maintained at 22 
  2 c and relative humidity was maintained within 35 to 
 65% , with a 12 h light / dark cycle . oral 
 dose formulations of dcp were prepared at 10 mg / ml in deionized water 
 on the morning of use and stored on wet ice . dose formulations were 
 diluted 1:20 with acetonitrile and quantified by gc with a flame ionization 
 detection against a calibration curve from 11000 g / ml . 
 oral and intravenous dose formulations of ech were prepared in deionized 
 water at 0.2 and 5 mg / ml and in sterile isotonic saline at 0.5 mg / ml , 
 respectively , on the morning of use and stored on wet ice to prevent 
 any hydrolysis of ech . ech dose formulations were analyzed by gc - ms 
 following extraction with chloroform and quantified against a calibration 
 curve from 0.04 to 1000 g / ml . dcp dose formulations were also 
 analyzed for the presence of ech using the same procedure as that 
 used for the analysis of ech dose formulation . male or female 
 rats ( 3 rats per sex per group ) were administered a single oral dose 
 of dcp at 50 mg / kg or ech at 1 and 25 mg / kg . oral doses were administered 
 via intragastric gavage using a syringe equipped with a ball - tipped 
 16-gauge stainless steel gavage needle in a dose volume of 5 ml / kg . male or female rats ( 3 rats per sex per group ) were administered a 
 single intravenous dose of ech at 1 mg / kg . doses were administered 
 via the tail vein using a syringe equipped with a 23-guage needle 
 in a dose volume of 2 ml / kg . after dosing , blood was collected 
 into a heparinized syringe via indwelling jugular cannulae from 3 
 animals per each time point . a blood sample was also collected from 
 each animal prior to dosing , and the cannulae were refilled with heparinized 
 isotonic saline . blood collection time points were as follows : predose , 5 , 15 , and 
 30 min and 1 , 2 , 4 , and 6 h ( oral , 50 mg / kg dcp by gavage ) ; predose , 
 5 , 10 , 15 , 30 , 60 , and 90 min ( 25 mg / kg ech by gavage ) ; predose , 5 , 
 10 , 15 , 30 , 45 , 60 , and 90 min ( 1 mg / kg ech male rats by intravenous ) ; 
 and predose , 5 , 10 , 15 , 20 , 25 , 30 , and 45 min ( 1 mg / kg ech female 
 rats by intravenous ) . following blood collection immediately upon collection , blood 
 was weighed in a 1.5 ml plastic centrifuge tube , an equal volume ( based 
 on the weight of blood in the tube and the density of ethyl acetate ) 
 of internal standard solution ( 0.25 m each of dcp - d5 and ech - d5 in ethyl acetate ) 
 was added to the tube , and the sample was vortex - mixed . tubes were immediately 
 placed on wet ice after internal standard addition and before subsequent 
 extraction steps . the samples were centrifuged , and the ethyl acetate 
 was removed , dried with 3a molecular sieves , and analyzed by gc - ms 
 as described above for dcp and ech . for each analyte , a triplicate 
 standard curve and qc toxicokinetic analysis was conducted 
 using winnonlin software , version 5.2 ( pharsight corporation ) . individual animal data was modeled , with input 
 for the dose of dcp or ech administered to calculate the dose - normalized 
 area under the curve in blood for dcp or ech . parameters estimated 
 were as follows : t1/2 , half - life of elimination ; cmax , maximum concentration ; cmax / d , dose - adjusted maximum concentration ; tmax , time at which the maximum concentration 
 was achieved ; auc(0t ) , area under 
 the blood concentration vs time curve to last time point ; auc(0t)/d , dose - adjusted 
 area under the blood concentration vs time curve to last time point ; 
 auc(0 ) , area under the blood concentration 
 vs time curve to infinity ; auc(0 ) , dose - adjusted 
 area under the blood concentration vs time curve to infinity ; and vz / f , volume of distribution 
 adjusted to bioavailability of test chemical . various approaches 
 including solid - phase microextraction and solvent extraction methods 
 coupled with detection by electron capture or mass spectrometry with 
 electron ionization , negative ion chemical ionization , or positive 
 ion chemical ionization were investigated to detect and quantify ech 
 and dca , the postulated reactive metabolites of dcp , in biological 
 matrices . solvent extraction followed by drying over molecular sieves 
 and analysis by gc - ms in the positive ion chemical ionization mode 
 was found to be the most promising method and was selected as the 
 final method . plasma , blood , and liver homogenate were evaluated 
 to determine the most suitable matrix to detect these reactive metabolites in vivo . in addition , samples were processed on ice to prevent 
 hydrolysis and/or reaction of ech and dca in biological matrices . 
 the recovery of dca from all biological matrices investigated was 
 very poor . the clearance of ech from liver homogenate , blood , and 
 plasma was evaluated at 37 c and on ice at ech concentrations 
 of 1 or 80 m . ech was relatively stable in blood and plasma , 
 but it rapidly cleared in liver homogenate and was not detectable 
 at any of the time points measured . the half - lives estimated for ech 
 in blood and plasma are shown in table 1 . in 
 rat , ech cleared more rapidly in blood compared to that in plasma , 
 and at 37 c , the half - life in rat plasma was between 50 and 
 53 min , compared with between 20 and 25 min in rat blood . decreasing the temperature resulted in a substantially longer half - life in both 
 blood and plasma ( table 1 ) . at 37 c , 
 ech was cleared in mouse blood more slowly than in rat blood , with 
 a half - life between 43 and 45 min . the initial concentration of ech 
 had no effect on half - life at 37 c ; however , on ice , the estimated 
 values were longer at higher concentration in both rat blood and plasma 
 ( table 1 ) . therefore , the final method was 
 optimized for the analysis of dcp and ech in blood . with sample stability being a concern , the additional manipulation of samples to generate 
 plasma was avoided by choosing blood as the matrix of choice for in vivo studies . half - life 
 was determined from semi 
 log plots of ech concentration vs time curve using the equations y = a e , and half - life = 0.693/k . to investigate the effect of the 
 sample matrix on extraction efficiency , 
 standard curves were prepared for dcp and ech in ethyl acetate , water , 
 and blood . the accuracy of response of dcp in blood or water was similar 
 and was 94102% of the solvent standard . similar results were 
 observed for ech , with a response between 91 and 112% over the concentration 
 range examined . the data indicates efficient extraction of both dcp 
 and ech with ethyl acetate from blood and water and an absence of 
 a matrix effect . the analytical method was validated to quantitate 
 dcp and ech in blood over the concentration range from 0.005 to 1 
 m ; the method was linear ( r > 
 0.997 ) , accurate ( relative error  15% ) , and precise ( relative 
 standard deviation  15% ) for the quantitation of both dcp 
 and ech over the concentration range of 0.005 to 1 m . stability 
 of analytes in blood extracts was verified using qc samples prepared 
 at low , mid , and high concentrations for up to 48 h at room temperature 
 ( 105 , 98.3 , and 102% ) , 1 week frozen ( 102 , 103 , and 106% ) , and after 
 three freeze  thaw cycles ( 109 , 96.1 , and 102% ) . for dcp , dilution 
 into the concentration range of method was verified for samples above 
 the highest standard . because two analyses needed to be conducted 
 on each sample , with ech in the range and dcp above the range , the 
 dilution was verified for dcp only . additional standards of dcp in 
 ethyl acetate were prepared in the range of 2400 m 
 and were diluted with 0.25 m dcp - d5 in ethyl acetate to be in the range of 0.10.6 m . dcp dose formulations were analyzed for the presence of ech prior 
 to administration in animals . ech was detected in the 10 mg / ml dcp 
 dose formulation by gc with flame ionization detection , with a peak 
 area corresponding to approximately 0.1% of that of dcp . the peak 
 area ratio did not appear to change when dcp was sampled immediately 
 after being dissolved in water or after 24 h at room temperature . the concentration of ech in the dcp dose formulations was determined 
 by gc - ms . the dcp and ech concentrations in the dcp formulation administered 
 in male rats were 9.54 and 0.000424 mg / ml , respectively , and in female 
 rats , 10.13 and 0.00041 mg / ml , respectively . these data indicate that 
 animals received negligible amounts of ech via administration of dcp . the doses of dcp administered in male and female rats 
 were 49.8  1.6 mg / kg ( 0.386  0.014 mmol / kg ) and 50.7  
 1.6 mg / kg ( 0.393  0.014 the estimated 
 doses of ech administered in male and female rats via the dcp formulation 
 were 2.23  0.08 g / kg ( 0.0239  0.0009 mol / kg ) 
 and 1.81  0.07 g / kg ( 0.0197  0.0007 the dose of dcp administered in male rats was approximately 
 22  500-fold higher than ech on a mass basis and 16  140-fold 
 higher on a molar basis . similarly , the dose of dcp administered in 
 female rats was approximately 27  830-fold higher than ech on 
 a mass basis and 19  963-fold higher on a molar basis . ech was detected in blood following administration of dcp in both 
 male and female rats starting from about 5 min through 120 min . ech 
 concentrations fell within the calibration range in blood of animals 
 following administration of dcp ; however , for dcp , the concentration 
 for the majority of samples was substantially above the calibration 
 range . therefore , analyses were conducted with two runs per sample : 
 first , with analysis for ech followed by dilution of the samples into 
 the range for quantitation of dcp . blood concentration vs time profiles 
 in male and female rats are shown in figure 2a and b for dcp and ech , respectively . for dcp , the parameters were similar between male and female 
 rats , with an elimination half - life between 42 and 44 min . the apparent 
 volume of distribution exceeded the reported aqueous body water volume 
 in rats of 0.688 ml / kg , suggesting extensive 
 distribution of dcp into the peripheral compartment . the maximum ech 
 concentration , cmax , was detected at 10 
 and 13.7 min following administration of dcp in males and females , 
 respectively . ech was eliminated in male rats ( half - life , 33.2 min ) 
 slightly faster than that in female rats ( half - life , 47.9 ) . as seen 
 with dcp , other toxicokinetic parameters estimated for ech were very 
 similar between males and females . for both males and females , the 
 apparent volume of distribution was lower than the reported aqueous 
 body water volume in rats , suggesting limited distribution of ech 
 into the peripheral compartment . blood ( a ) dcp and ( b ) ech concentration 
 vs time profiles following 
 a single oral administration of 50 mg / kg dcp in male and female harlan 
 sprague  dawley rats . initially , a group of male rats was administered 
 a single dose of 1 mg / kg by gavage , but no ech was detectable in blood 
 at time points ranging from 5 min to 6 h. a higher dose of ech was 
 then used . the oral doses of ech administered to male and female rats 
 were 26.2  0.3 mg / kg ( 0.283  0.003 mmol / kg ) and 25.3  
 1.1 mg / kg ech was detected 
 up to 1590 min following administration in male and female 
 rats . blood concentration vs time profiles in male and female rats 
 are shown in figure 3 , and toxicokinetic parameters 
 obtained from noncompartmental analysis are given in table 3 . a sex difference in toxicokinetics was observed 
 following administration of ech in male and female rats . cmax and auc(0 ) were about 3-fold 
 lower in females compared with that in males , suggesting that females 
 received less internal dose of ech than males following oral administration 
 of ech . ech was eliminated in females ( half - life , 20.1 min ) faster 
 than in males ( half - life , 33.8 min ) . blood concentration vs time profiles following 
 a single oral administration 
 of 25 mg / kg ech in male and female harlan sprague  dawley rats . the intravenous doses of ech administered in male 
 and female rats 
 were 1.02  0.02 mg / kg ( 0.0111  0.0002 mmol / kg ) and 1.03 
  0.03 mg / kg ( 0.0111  0.0003 blood 
 concentrations of ech fell rapidly in male rats following administration 
 and were below the limit of quantitation ( loq ) of the analytical method 
 in some animals at 30 min and in all rats at 45 min . therefore , in 
 female rats , blood was collected only up to 45 min following administration . 
 in general , concentrations start falling below the lowest standard 
 of 0.005 m between 10 and 25 min in females . as a result , for 
 one animal , the concentration after 10 min was determined by interpolation 
 below the loq in order to have sufficient data points for toxicokinetic 
 analysis . blood concentration vs time profiles in male and female 
 rats are shown in figure 4 , and toxicokinetic 
 parameters obtained from noncompartmental analysis are given in table 4 . in general , toxicokinetic parameters between males 
 and females were similar except that , as observed following oral administration , 
 ech was eliminated in females ( half - life , 3.1 min ) faster than in 
 males ( half - life , 6.51 min ) . blood concentration vs time profiles following 
 a single intravenous 
 administration of 1 mg / kg ech in male and female harlan sprague  dawley 
 rats . area under the ech concentration 
 vs time curve following oral and intravenous administration of ech , 
 after adjusting for the dose , was used to estimate oral bioavailability 
 of ech . for male rats , auc(0)/dose following 
 oral and intravenous administration was 42.9 and 827 m min 
 kg / mmol , respectively . therefore , the oral bioavailability was estimated 
 to be 5.2% ( ( 42.9/827 )  100 ) . similarly for female rats , auc(0)/dose following oral and intravenous administration 
 was 14.4 and 676 m min kg / mmol , respectively . therefore , the 
 oral bioavailability estimated for females was 2.1% ( ( 14.4/676 )  
 100 ) . although ech and dca were proposed to be 
 reactive species , to the 
 best of our knowledge , there is no direct evidence for the formation 
 of these metabolites from exposure to dcp . the work described here 
 is the first report of the formation of ech in rodents following exposure 
 to dcp . in this study , the feasibility of detecting dca and 
 ech in dcp - administered 
 animals was investigated by developing a sensitive analytical method 
 to detect these metabolites and by identifying a suitable matrix . 
 the high reactivity of dca in blood , plasma , and liver homogenate in vitro precluded its investigation ; dca was cleared in 
 these matrices with a half - life  1 min . ech rapidly disappeared 
 in the liver homogenate . in the presence and absence of diethyl maleate 
 ( gsh depleter ) or cyclohexene oxide ( epoxide hydrolase inhibitor ) , 
 we confirmed in vitro that 80% of the loss of ech 
 in liver homogenate was due to epoxide hydrolase activity and 18% 
 was due to the reaction of ech with gsh ( data not shown ) . although the estimated half - life of ech in plasma was longer than 
 that in blood , the time necessary to prepare plasma from blood from 
 animals would result in loss of ech . therefore , blood was selected 
 as the preferred matrix for detecting ech following administration 
 of dcp or ech in rats . processing samples on ice slowed the hydrolysis 
 of ech on blood and plasma in situ and hence was 
 incorporated into the final method . an analytical method was developed 
 and validated for successful quantitation of ech in blood with a limit 
 of quantitation of 0.01 m , which was subsequently extended 
 to 0.005 m . ech was detected and quantified in blood 
 following oral administration 
 of dcp in male and female rats . to the best of our knowledge , trace 
 levels of ech were detected in dcp dose formulations corresponding 
 to 0.0239 and 0.0197 mol ech / kg administered in males and females , 
 respectively , suggesting that animals received negligible amounts 
 of ech compared to dcp via administration of dcp . the initial dose 
 of 1 mg ( 10.8 moles ) ech / kg used for gavage administration 
 in male rats produced no detectable ech in blood , indicating that 
 the extremely low levels of ech in the dose of dcp administered would 
 not have caused the ech in blood from dcp - dosed rats . these data along 
 with the low bioavailability of ech in male ( 5.2% ) and female rats 
 ( 2.1% ) following oral administration provide evidence for the formation 
 of ech following oral administration of dcp in rats . ech was 
 detected in blood from rats following a single oral dose 
 of 25 mg / kg ( half - life of 33.8 and 20.1 min in males and females , 
 respectively ) or a single intravenous dose of 1 mg / kg ( half - life of 
 6.51 and 3.14 min in males and females , respectively ) . in a study 
 by rossi et al . , following a single oral dose of 50 mg / kg ech in male 
 cd1 swiss albino mice , levels of ech were below the limit of quantitation 
 ( 50 ng / ml ) of the analytical method used ; however , ech was detected 
 following an oral dose of 200 mg / kg . the 
 peak blood ech concentration reported at 5 min following administration 
 was 0.49  0.13 g / ml . the estimated half - life in mice 
 ( 5.5 min ) was much shorter than that observed in male rats ( 33.8 min ) 
 following oral administration of ech in our study , indicating a clear 
 species difference in the disposition of ech following oral administration . following administration of dcp in male and female rats , the maximum 
 ech concentration was achieved 13.7 min . there was no clear 
 sex difference in the disposition of ech in rats except that the half - life 
 in males ( 33.2 min ) was slightly shorter than that of female rats 
 ( 47.9 min ) . this may be partially explained by the reported higher 
 microsomal epoxide hydrolase activity in males compared to that in 
 females . in a pilot study , ech was also 
 detected and quantified in male b6c3f1/n mice following oral administration 
 of 25 mg / kg dcp ( data not shown ) ; ech was rapidly eliminated in blood 
 with a half - life of 10.1 min in male mice compared to a half - life 
 of 33.2 in rats . in addition , cmax and 
 auc were about 10-fold and 40-fold lower in male mice compared 
 with those in male rats , showing a clear species difference in the 
 disposition of ech following administration of dcp . the percentage 
 of the dose of dcp converted to ech in rats following 
 oral administration of dcp in rats was estimated in several ways . an initial evaluation used auc(0 ) values 
 for ech following oral administration of ech and following oral administration 
 of dcp . in male rats , for ech , the dose - adjusted auc(0 ) was 42.9 m min kg / mmol following oral administration of ech , 
 and auc(0 ) was 4.02 m min following 
 oral administration of dcp ( tables 2 and 3 ) . therefore , the dose of ech formed in dcp - treated 
 ( 0.386 mmol / kg dcp ) male rats was estimated to be 0.094 mmol / kg ( 4.02 
 m min/42.9 m min kg / mmol ) ; this is equivalent to ca . 24% ( 0.094/0.386  100 ) conversion of dcp to ech in male rats . similarly , in female rats , for ech , dose - adjusted auc(0 ) was 14.4 m min kg / mmol following oral administration of ech , 
 and auc(0 ) was 4.67 m min following 
 oral administration of dcp ( tables 2 and 3 ) . 50 mg / kg 
 ( or 0.393 mmol / kg ) dcp - treated female rats was estimated to be 0.324 
 mmol / kg ( 4.67 m min/14.4 m min kg / mmol ) ; this is equivalent 
 to ca . 82% ( 0.324/0.393  100 ) conversion of dcp to ech in female 
 rats . these estimates may be higher than actual values since ech may 
 be hydrolyzed in the gut following oral administration of ech , leading 
 to lower auc(0 ) following oral administration 
 of ech . this is supported by the estimated low oral bioavailability 
 of 5.1 and 2.1% in males and females , respectively . an alternative 
 approach was then used that avoids the issue of absorption , using 
 the auc(0 ) following intravenous administration 
 of ech to estimate the percent dcp converted to ech . following intravenous 
 administration , the dose - adjusted auc(0 ) values were 827 and 676 m min kg / mmol in males and females , 
 respectively ( table 4 ) . on the basis of this , 
 the dose of ech in dcp - treated rats can be calculated as 0.00486 mmol / kg 
 ( 4.02 m min/827 m min kg / mmol ) and 0.00696 mmol / kg ( 4.67 
 m min/676 m min kg / mmol ) , which is equivalent to 1.26% 
 ( 0.00486 mmol ech/0.386 mmol dcp  100 ) and 1.78% ( 0.006961 mmol 
 ech/0.393 mmol dcp  100 ) of dcp converted to ech in male and 
 female rats , respectively . these values may be slightly underestimated 
 because ech , once formed in the liver from the metabolism of dcp , 
 can undergo hydrolysis and gsh reaction , thereby decreasing the amount 
 entering systemic circulation . therefore , we concluded that the percent 
 dose of dcp converted to ech in male and female rats following a single 
 oral exposure to dcp was 1.26% and 1.78% , respectively . the investigation into the formation of dca from the metabolism 
 of dcp proved to be unsuccessful due to its rapid disappearance . indirect 
 evidence for the formation of dca could be provided by the analysis 
 of further metabolites such as glutathione conjugates and protein 
 adducts . however , the major conjugation products of dcp and ech separately 
 administered to male rats were the same : n - acetyl - s-(2,3-dihydroxypropyl)cysteine and n , n-bis - acetyl - s , s-(bis - cysteinyl)-2-hydroxypropane . in 
 addition , n - acetyl - s-(3-chloro-2-hydroxypropyl)cysteine , 
 reported to be a metabolite of ech ( gingell et al . ) , is also expected to be a metabolite of dcp . this was 
 confirmed by investigation of the reaction of gsh with dcp or ech 
 ( unpublished observations ) . the formation of adducts from reaction 
 of ech with globin has been described , and several adducts were identified , 
 including 2,3-dihydroxypropylvaline ( which 
 is not specific for ech but can be formed by reaction with glycidol ) 
 and chlorohydroxypropylvaline , which , 
 although formed by ech , could also be formed by reaction of 3-chloro-1,2-propanediol . therefore , glutathione conjugates , mercapturic 
 acids , or protein adducts could not be used to distinguish dcp and 
 ech . the biological conversion of dcp to ech has been demonstrated 
 in 
 bacteria and is catalyzed by haloalcohol dehalogenases in gram - positive 
 and -negative bacteria . production of 
 ech from dcp has been reported in a biochemical reactor containing escherichia coli expressing haloalcohol dehalogenase 
 and epoxide hydrolase . similarities between the active sites of the 
 bacterial haloalcohol dehalogenase and mammalian epoxide hydrolases 
 have been drawn . the mechanism by which this conversion 
 occurs and its location within the body should be investigated further 
 to understand the toxicity and carcinogenicity of dcp . it has been suggested that the observed toxicity and carcinogenicity 
 of dcp in rodents is due to the formation of a reactive metabolite , 
 epichlorohydrin ( ech ) . to date , there is no direct evidence for the 
 formation of ech following exposure of rodents to dcp . the work described 
 here in reports the first unambiguous detection of ech following oral 
 administration of dcp in rodents . following a single oral dose of 
 50 mg / kg dcp , 1.26% and 1.78% of the administered 
 dose toxicokinetics 
 of ech in male and female rats were similar in males and females , 
 eliminating ech with a half - life of 33.2 and 47.9 min , respectively .