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 .
 the observed toxicity and carcinogenicity 
 of 1,3-dichloro-2-propanol 
 ( dcp ) in rodents is thought to be due to the formation of reactive 
 metabolites , epichlorohydrin ( ech ) and dichloroacetone ( dca ) . 
 however , 
 there is no direct evidence for the formation of these metabolites 
 from exposure to dcp in rodents due to the challenges of measuring 
 these reactive intermediates directly in vivo . 
 the 
 objective of this work was to investigate the metabolism of dcp to 
 ech and dca in vivo by first developing a sensitive 
 analytical method in a suitable biological matrix and analyzing samples 
 from rats administered dcp . 
 dca reacted rapidly in vitro in rat blood , plasma , and liver homogenate , precluding its detection . 
 because ech rapidly disappeared in liver homogenate , but was relatively 
 long - lived in plasma and blood in vitro , blood was 
 selected for analysis of this metabolite . following a single oral 
 dose of 50 mg / kg dcp in male or female harlan sprague  
 dawley 
 rats , ech was detected in blood with a maximum concentration reached 
 at 13.7 min . 
 ech was cleared rapidly with a half - life of ca . 
 
 33 and 48 min in males and females , respectively . following a single 
 oral dose of 25 mg / kg ech in male and female rats , 
 the elimination 
 half - life of ech was ca . 
 34 and 20 min , respectively ; the oral bioavailability 
 of ech was low ( males , 5.2% ; females , 2.1% ) , suggesting extensive 
 first pass metabolism of ech following oral administration . 
 the area 
 under the concentration vs time curve for ech following oral administration 
 of dcp and intravenous administration of ech was used to estimate 
 the percent of the dcp dose converted to ech in rats . on the basis 
 of this analysis 
 , we concluded that in male and female rats following 
 oral administration of 50 mg / kg dcp , 1.26% or 1.78% 
 of the administered dose was metabolized to ech , respectively . 
Introduction
Materials and Methods
Results
Discussion
Conclusions