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PB85-152387
Investigation of the Metabolism of
Chlorinated Hydrocarbons in Subhuman Species
Cincinnati Univ., Oil
Prepared for
Health Effects Research Lab.
Research Triangle Park, NC
Jan 85
EPA-600/1-85-001, January 1985
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EPA-600/1-85-001
January 1985
INVESTIGATION OF THE 'METABOLISM OF CHLORINATED
HYDROCARBON'S IN SUBHUMAN SPECIES
by
Carl C. Smith, Steven T. Cragg.
Orald'ne F. Wolfe, and Walter W. Wei<>el
Department of Environmental Health
University of Cincinnati
College of Medicine
Clnclnnntl, Ohio 45267
Grant *R-803963
Pro.Joet Officer
P.ohf;rt Mn£p,
Rptdomlolop.y Division
Hc.ilth Effects Rcac=»rch Laboratory
Cincinnati, Ohio 4526H
HEALTH KKFKCTS RKSEARCH LABORATORY
OKKICK OF RESEARCH AN'!) OEVKl.OPMENT
ir.S. ENVIRONMENTAL PROTECTION AfiENCY
RKSKARCH TRI ANCLE ('ARK , NORTH CAROLINA 27711
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TFCKNICAL REPORT DATA
(Plcaff ffttJ Instruct.et,t on the fctcrsc bcfof^ c
I. REPORT NO.
EPA-60Q/1-85-001
4 TITLE AN3 SUBTITLE
Investigation of the Metabolism of Chlorinated
Hydrocarbons in Subhuman Species
3. RECIPIENT'S ACCESSipi NO
PB85 152 38?/AS
S REPORT KATE
January 1985
ORGANIZATION CODS
L/H
Carl C. Saith, Steven T. Cragg, Ceraldine F.
and Walter W. Welgel
Wolfe
0 PERFORMING ORGANIZATION REPORT NO
1O PROGRAM ELEMENT NO.
CBMC1A
). PERFORMING ORGANIZATION NAME AND ADDRESS
University of Cincinnati
College of Medicine
Cincinnati, Ohio 45267
It COH l RACT/GKANT NO
R-803963
12. SPONSORING AGENCY NAME ANO ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina
13 TYPE Of REPORT ANO PERIOD COVERtp
?inal Project Report-lU/Vs/TS
14. SPONSORING AGENCY CODE
EPA-600/11
27711
1i SUPPLEMENTARY NOTES
P.O. H.P. Ringhand, CSSB, TMD, HERL (CI); original Project Officer Robert C.
Lingg.
16. ABSTRACT
The purpose of this research program was to provide metabolic data on four rather
conmon drinking water contandnants. The compounds were 1,2,4-trichlorobenzene (TCB),
tronodichloronethane (BDC). bis(2-chloroisopropyl) ether (BCIE) and bis (2-chloroethyl)
ether (BCZ£). The compounds, laJbeled with 14C, were administered orally and scraetirr.es
intravenously, to rats and rhesus monkeys. Studies on TCB included excretion and
tissue distribution in rats, phamacokinetic studies in mor.keys and subchronic studies
in monkeys. Daily oral doses of 174 ng/Xg were uniformly fatal to monkeys in less than'
one month. Activity of liver enzymes was increased. Pharwacokinetics and tissue dis- ,
tribution of BDC was examined in rats and monkeys. The compound was excreted primarily
via the lung either unchanged or as COj. BCIE in monkeys was also excreted via the
lung and was very toxic to the eyes and kidneys following multiple 30 mgAg oral doses.
Preliminary studies of BCEE in two rhesus monkeys produced the cane signs of peri-
orbital toxicity as BCIE when oral doses of 10 mgAg were administered.
WORDS ANO DOCUMENT ANALYSIS
DESCRIPTORS
b.lOENTIHlRS/OPlN INOtD THM5 C. COSATl Field/Croup
IB. DISTRIBUTION STATEMENT
Release to Public
«9. SECURITY CLASS ITIin
HncTnssf fi*
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N'OTTCK
The taaterlal has been funded wholly or In part by the t'nlted States
Environmental Protection Agency under Grant *R--B03963 to University of
Cincinnati. It has been subject to the Agency's review and it has been
approved for publication as an KPA document. Mention of trade r.iraes or
commercial products does not constitute endorsement or recotmeudatIon for
use.
11
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FOREWORD
The nany benefits of our nodorn, developing, inriustrl.il society are
accompanied by certain hazards. Careful assessment of the relative risk of
existinf, and new man-made environmental hazards is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance the
quality of our environment In order to promote the public health and welfare
and the productive capacity of our Nation's population.
The complexities of environmental problems originate in the deep Inter-
dependent relationships between the various physlca? and biological segments
of L-an's natural and social world. Solutions to these envlromental problems
require an Integrated progran of research and development using input from a
number of disciplines. The Hf.ilth Kffects Research Laboratory, Research
Triangle Park, NC and Cincinnati, 0!!, conducts a coordinated environmental
health research progran in toxlcoloey, epidemiology, and clinical studies
using hunan volunteer subjects. Wide ranges of pollutants known or suspected
to cause health problems are studied. The research focuses on air pollutants,
water pollutants, toxic substinoes, hazirdous wastes, pesticides, and non-
ion1;tlnc> radiation. The laboratory participates In the development and
revision of air and w.iter quality criteria and health assessment documents on
pollutants for which regulatory actions are being considered. Tjlr*»et support
to the regulatory function of the* Agency la provided in the for-a of expert
testimony and preparation of affidavits as well as expert advice to the
Adalnlst rtitor to assure the adequacy of environmental regulatory decisions
involving the protection of the health and welfare of all U.S. Inhabitants.
This report provides metabolic data. In rats and rhesus monkeys, on four
drinking wat<*r contaminants: I,!? ,4-t r Ich lorobenzene, hromodlchl oromethane,
bl sf3-rhloroIsopropyl) ether and bln(2-chloroethyI) ether. Pharmicoklnetlc
and metabolic data on the adsorption, distribution and elimination of the
parent compound and Its metabolites following oral and intravenous administra-
tion allows one to predict whether tho chemical or Its metabolites accumulate
In the Sody. Comparative phanacokI net: lc studies In subhuman species are
needed In thf» extrapolation of toxl rolojil r.il d.ita from anin.ili to nan.
F. Cordon Ilueter, Ph.D.
Director
Health Effects Research Laboratory
Hi
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ABSTRACT
The purpose of this research program was to provide metabolic data on
four rather common drinking water contaminants. The compounds were 1,2,4-
trichlorobenzene (TCB), bromoaichloromethane (BDC), bis(2-chloroisopropyl)
ether (BCIE) and bis(2-chloroethyl) ether (BCEE). The compounds, labeled
with ^4C, were administered orally and sometimes intravenously, to rats and
rhesus monkeys.
Studies on TCB included excretion and tissue distribution in rats fol-
lowing oral and intravenous doses. Pharmacokinetic parameters of TCB were
examined in rhasus monkeys. Subchronic studies on orally administered TCP
were conducted in monkeys. Daily oral doses of 25 mg/kg or less had no
observable effects on monkeys. Liver biopsies indicated further that such
doses had little effect on the drug metabolizing or mixed function oxidase
(KFO) activity of the liver. Using doses of 90 and 125 ng/kg one of four
monkeys on each of the dosage levels died but whether TCB toxicity was the
cause of death was uncertain. Daily doses of 174 mg/kg killed two monkeys
and would have been uniformly fatal if continued beyond one month. Recovery
from toxic effects was rapid and appeared to be complete. TCB in doses of
90 to 174 mg/kg produced significant increases in the levels of four MFO
enzymes in the primate liver studied in, vitro. The effect of TCB on the
in vivo metabolism of chlorguanide was alto investigated. The metabolic
fate of this compound can be used to estimate the level and type of hepatic
KFO activity. The results of this in_ vivo test of MFO activities correlated
well with the in vitro measurements of hepatic enzyme levels.
The pharmacokinetics and tissue distribution of BDC was examined in
rats and monkeys. UDC was excreted primarily via the lung as parent compound
or C02- The maximum amount recovered in the urine was 1* in rats and 6*
in monkeys. Monkeys excreted 0.5 to 1.5% in feces.
Pharmacokinetic studies cf BCIE following oral and intravenous adminis-
tration in rhesus monkeys showed that only about one-third of the adminis-
tered radio-activity was recovered in the urine and feces, therefore, one
of the primary routes of excretion munt have been via the lungs. BCIE was
very toxic to the eyes and kidneys whon multiple 30 mg/kg doses were admin-
istered. Preliminary studies of BCFE in two rhesus monkeys produced the same
signs of periorbital toxicity observed with BCIE; in monkeys BCEE was more
toxic than BCIE.
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CONTENTS
*'otice
Foreword
iv
Figures [[[ vii
Tables [[[ lx
Acknowledgment [[[ xiii
1. Introduction .................................................. 1
2 . Conclusions [[[ 2
3 . Recommendations ............................................... 3
4. In Vivo Studies on Trichlorobenzene ........................... *
I. Excretion and tissue distribution of 1 ,2 ,4-trichloro-
benzene (TCB) following single oral doses in rats ....... 4
II. Excretion and tissue distribution of 1 ,2 ,4-trichloro-
benzene (TCB) following single intravenous doses in rats. 5
Ill.Pharmacokinetic study of TCB- C in rhesus monkeys ...... 16
IV. First subchronic study of TCB in monkeys. Introduction
and preliminary findings ................................ 17
V. Second TCB subchronic study ..................... . ....... 26
5. In V] tro Studies on Trichlorcbenssne ................. ......... 42
I . In vitro enzyme assays .................................. 42
II. Extraction of TCB metabolites from rat urine ............ 53
III. Preliminary characterization of metabolites of TCB in
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6. Metabolisr. of Bromodichloromethane in Rats and Rhesus Monkeys .. 62
I. Introduction 62
II. Preliminary pharmacokir.etic studies on bromodichlorometh-
ane (BDC) in rats 62
Ill.Pharmacokinetics of BDC in rhesus monkeys 63
IV. Kutagenicity tests *>""
7. Studies on Bis(2-Chloroisopropyl)Ether 76
14
I. Pharmacokinetics of bis(2-chloroisopropyl)ether- C in
rhesus monkeys following single intravenous or oral doses. 76
II. Multiple dose study of bis(2-chloroisopropyl)ether in
monkeys .... 78
8. Phamacokinetic Study cf Bis (2-Giloroethyl) Ether in Two Rhesus
Monkeys 110
References 11 *>
Appendix t' 7
Metabolism of S-Haloethers in Rats and Monkeys. Smith, C.C.
and R.D. Lingg - Paper presented at the International
Symposium on the Analvsis of Hydrocarbons and Halogenated
Hydrocarbons in the Aquatic Environment, May 23-25, 1978,
Hamilton, Ontario 119
vi
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FIGURES
Kuaber Page
1 Cunulative Percent Excretion in Urine of 1,2,4-Trichloro-
benzene (as 14C) in vtfiesus Monkeys Following a Single
Oral or Intravenous Dose of 10 ing/kg . • 21
2 Effect of 30 Days TCB Pretreatment on Urinary Chlorguanide
Excretion Parameters (TCB Treated Monkeys Compared to
Vehicle Treated Controls) ., 39
3 Effect of 30 Days TCB Pretreatoent on Urinary Chlorguanide
Excretion Parameters (TCB treated "L's" compared to
control "A's", where A=post-treatnent values minus pre-
treatnent baselines) 40
4 Effect of 30 Days TCB Pretreatment on Urinary Chlorguanide
Excretion Parameters (Post-treatment values compared to
their O*..TI baselines by paired t test) 41
5 Aminopyrine U-Demethylase Activity (as nM/gm liver/rain)
Correlated With Eight Chlorguanide Urinary Excretion
Parameter a 48
Benzohetitfnir.e N-Demethylase Activity (as nM/gm liver/min)
Correlated With Eight Chlorguar.ide Urinary Excretion
Parar-etei a 49
Zoxazolamin« Hydroxylase Activity (AS nm/gra liver/min)
Correlate-! With Eight Chlorguanida Urinary Excretion
Paraneteri: 50
Blood Concentrations of BDC (as C) in Female Rhesus Monkeys
Single Intravenous Dose of 10 ryj/kg 69
14
Blood Concentrations of BDC (as C) in Female Rhesus Monkeys
Single Oral Dose of 10 mgAg
10 Cumulative Urine Excretion of BDC in Kor>.eys. Single
Intravenoun Dose of 10 tug/kg .............................. 73
11 Cumulative Urine Excretion of BDC in Monkeys. Single
Oral Dose 3f 10 mgAg ..................................... 7*
vii
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Number Page
12 Blood Levels of BCIE in Monkeys. Single Intravenous
Dose of 30 rogAg .......................................... 8^
13 Blood Levels ^f BCIE in Monkeys. Single Oral Dose of
30
14 Cumulative Urinary Excretion of BCIE in Monkeys. Single
Intravenous Dose of 30 mgAg
15 Cumulative Urinary Excretion of BCIE in Monkeys. Single
Oral Dose of 30 mgAg
16 Urine Volumes in Rhesus Monkeys Before and After 3 Oral
Doses of BCIE 30 mgAg .................................... 98
14
17 Concentration of BCIE (as C) in Blood of Kiesus Monkeys
Oral Doses of 30 mgAg .................................... 1Q0
14
18 Concentration of BCIE (as C) in Plasma in Rhesus Monkeys
3 Oral Doses of 30 mgAg .................................. 102
14
19 Recovery of Urinary BCIE (as C) in mg in Rhesus Monkeys
3 Daily Oral Doses of 30 mgAg ............................
14 O
20 Cumulative Urinary Excretion of BCIE (as C) in 1 +
3 Oral Doses of 30 mgAg ..................................
14 o
21 Cumulative Urinary Excretion of BCIE (as C) in 96+
3 Oral Doses of 30 mgAg .................................. 107
22 Cumulative Urinary Excretion of BCIE (as 4C) in 98 ?
2 Oral Doses of 30 mgAg ..................................
viii
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TABLES
Page
14
Cur.ulative Excretion of 1,2,4-Trichlorobenzene (as C)
in Male Pats. Single Oral Dose of 10 ngAg ............... 7
ssue Distribution of 1 ,2 ,4-Trichlorobenzene (as C)
in Kale Rats. Single Oral Dcse of 10 mgAg ........ , ...... 8
14
ssue Distribution of 1,2,4-Trichlorobenzene (as C)
in Percent Dose. Single Oral Dose of 10 mgAg (Kale Rats) . 10
covery of 1,2,4-Trichlorobenz
Single Oral Dose of 10 mgAg
mulative Excretion of 1,2,4-
Kale Hats. Single Intravenous Dose of 10 mgAg
ssue Distribution of 1,2,4-Trichlorobenzene (as
Male Rats. Single Intravenous Dose of 10 mgAg
14
Recovery of 1,2,4-Trichlorobenzene (as C) in Male Rats.
14
Cumulative Excretion of 1,2,4-Trichlorobenzene (as C) in
14
Tissue Distribution of 1,2,4-Trichlorobenzene (as C) in
Percent Dose. Single Intravenous Dose of 10 mgAg
Recovery of 1,2,4-Trichlorobenzene (as C) in Male Rats.
Single Intravenous Dose of 10 ngAg
Blood and Plasma Concentration of 1,2,4-Trichlorobenzene
(as l^C) in Female Rhesus Monkeys. Single Intravenous
Dose of 10 mgAg and Single Oral Dose of 10 mgAg
14
10 Cumulative Excretion of 1 ,2 ,4-Trichlorobenzere (as C) in
female Rhesus Monkeys. Single Intravenous Dose of 10 mgAg *9
14
11 Cumulative Fxcretion of 1,2,4-Trichlorobenzene (as C) in
Female khesus Monkeys. Single Oral Dose of 10 mgAg ...... 2®
12 SKA 12/60 Results on Monkeys Before Treatment With TCB ...... 24
13 SKA 12/60 Results on Monkeys After Treatment With TCB
for Two Months ............................................ 7t>
14 Weights of Monkeys on Daily Treatment With TCB .............. 29
ix
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Number Page
15 Her.atocri.ts of Mcr.keys on Daily Treatment With TCB ........... 30
16 Liver Weight, Body Weight and Liver Weight/Body Weight
Ratios (as Percentages) ot TCB Treated Konkeys and Controls. 31
17 Significance Levels (P) Comparing Liver Weights and Liver
Weight /Body Weight Percentages Amon-g Control ana TCB
Treated Groups ............................................. 32
18 Effects of 30 Days TCB Treatment on Urinary Chlorguanide
Excretion Parameters ........ ........... . ................... 33
19 Effects of 60 Days TCB Treatment on Urinary Chlorguanide
Excretion Parameters ...................................... ^5
20 Effects of 90 Days TCB Treatment on Urinary Chlorguanide
Excretion Parameters ....................................... 37
21 Results of In Vitro Hepatic MFO Tests in Rhesus Monkeys
Treated With TCB (Nanoirole/Minute/Gram Liver ........... . ... 44
22 Significance Levels (P) Comparing In_ Vitro MFO Activity Anong
Control and TCB Treated Groups (Nar.omoles/Minute/Gran
Liver) ........................................... , . ........ 'lr>
^3 IlL yj-Jfcjg. Hepatic Enzyme Tests in Phesus Monkeys Treated With
TCB C.'anomole/Kinute/KG Kicrosomal Protein) ................ 46
24 Significance Levels (P) Which Compare In^ Vitro MFO Activity
of TCB Treated Monkeys to Controls (Nar.omoles/Minute/
KG Microsomal Protein) ..................................... 47
25 MFO Concentrations and Peak Locations from Control and TCB
Treated Monkeys ............................................ 51
26 Statistic-3 Comparing MFO Concentrations (as NM/G Liv«r)
Ariong Control and TCB Treated Groups ....................... '32
14
27 Extraction of C-Labeled Metabolites of Trichlorobenzene
in Hydrolyzed Rat Urir.e by Various Organic Solvents
Effect of Added (NHJ.SO. .................................. 54
42 4
28 Chromogenic Reactions ........................................ 55
29 Rf Values
30 CC Retention Times ........................................... 56
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Number
31 Recovery of Radict.-tivAty f r -re .Monkey Plasma in Percent ...... <;"
32 Treatment Protocol ........................................... 58
33 Toxicity (T) or Activity of TCB Metabolites .................. 60
34 Control Hutagenicity Test Data Using Ames Tester Strains
TA98 anJ TA100 ............................................. 61
* «
35 Tissue Distribution of Bromoditnlororw thane (as * C) in Kale
Hats. Single Intravenous Dose of 10 «ng/Xg/kg fl 1
14
43 Blood Concentrations of PCIE (as C) in Monkeys. Single
Oral Dose 30 r.v
14
44 Plasma Concentrations of BCIE (as C) in Monkeys. Single
Oral Dose of 30
14
45 Cumulative Excretion of HCIE (as C) in Monkeys. Single
Intravenous Dose of 30 nqA'J ST>
14
46 Cumulative Excretion of BCIE !*s C) in Monkeys.Single Oral
Dose of 30 KwAq :\~
14
47 Tissue Distribution of BCIE (as C) in Monkeys, Single
Intravenoua Doae of 30 wjA^ • • 89
xi
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Pacte
Tissue Distribution of BCIE (as "*O in Monkeys. Single
Intravenous Dose of 30 ngAg
ssue Distribution of BCIE (a
Cral Dose of 30 ngAg (wg/g)
14
49 Tissue Distribution of BCIE (as C; in .Monkeys. Single
14
SO Tissue Distribution of BCIE (as C) in Monkeys. Single
Oral Dose of 30 tngAg (* dose)
51 Ocular Effects of BCIE
14
52 Blood Concentrations of BCIE (as C) in Monkeys. Three
Consecutive Daily Oral Doses of 30 mg/kg ................. 9 9
14
53 Plasma Concentrations of BCIE (as C) in Monkeys. Three
Consecutive Daily Oral Doses of 30 rvg/kg
54 Recovery of BCIE in Monkey Urine. 30 ingAg Once Dally for
3 Days
14
55 Cumulative Recovery of BCIE (as C) in Monkey Urir.e Bused
on Single Dose (30 ngAg) or Additive Dose (30,60,90
3 Consecutive Oral Daily Doses of 30 mgAg
i «
Cumulative Recovery of BCIE (as "*O in Monkey Keces Eased
on Single Dose (30 ing/kg) or Additive Dose (30.60,90
3 Conaecutivo Daily Oral Doses of 30 rx/Ag
14
57 Cumulative Excretion of BCEE (as C) in Monkeys. Single
Oral Dose of 10 mq/kg
14
58 Urine Volumes and Feces Weights of C- BCEE- Treated Monkeys.
Single Oral Dose of 10 mg/kg .... .......................... 113
59 sy.A 12/60 Asisay un Khe!>u:J 1+ at 72 Hours Following Single
Oral 10 tng/kq/Dose of Bis U-Chloroc.thyl) Ether ..............
xii
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ACKN'OW LEDGEMENT
We wish to express cur thanks to Thomas Hatcher, Steve Batson and
James Buchanan for their dedicated assistance with the monkey experiments.
Dr. Robert G. Tardiff participated in the initial planning and provided
continuing support and guidance while the studies were in progress. The
active participation of Dr. Robert Lingg and fruitful discussions with other
members of the EPA staff are gratefully acknowledged. The editorial acumen
of Dot Reynolds and the typing skill of Mary Jo Loftus were important com-
ponents in the completion of the document.
xiii
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SECTION 1
INTRODUCTION
In the volume "Drinking Water and Health" the first summarized recom-
mendation for additional research lists "Studies of the physiological and
biochecical nechanistns by which the toxic substances in water produce theit
effects" (1). We have approached this objective by studying the metabolic
fate and pharmacokinetics of four of the common contaminants of chlorinated
drinkir.g water. The compounds chosen include trichlorobenzene, a bromine
analogue of chloroform and two bis-2-chloroalkyl ethers. We have used rats
and monkeys with special emphasis on the use of subhuman primates since
studies by the author and by many other investigators support the hypothesis
that in general the metabolic fate cf compounds in monkeys more closely
simulates the fate in man than do corresponding data obtained in ether
common laboratory animals such as dogs or rodents (C. C. Smith, in press).
Although there are some data on the metabolic fate of 1,2,4-trichloro-
ber.zer.e in rats and the effects of this compound on metabolism of foreign
ccnpounds (2,3) the data obtained in this study were designed to corroborate
and extend these previous studies.
In the case of the chloroform analogue and the two bischloroalkyl ethers,
very little previous data were available. Very recently Lir.gg et ad. , have
published data on the metabolic fate of bis(2-chloroethyl)cther and bis{2-
chloroisopropy1)ether after single oral doses in rats (4).
Wh^n these studies were undertaken, it was hoped that the data they
would provide using subhunan privates would be useful in judging the poten-
tial toxicity of these materials in ruin.
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SECTION 2
CONCLUSIONS
These studies on the metabolism of four halogen-containing water contam-
inants have shown certain characteristics and differences in metabolic
patterns and associated toxic effects. Trichlorobenzene (TCB) is probably
the least toxic compound when administered chronically but is probably the
only cocpound with the potential of inducing liver drug-metabolizing enzywes.
The compound has toxic effects at daily doses in excess of 125 F"j/Xg and is
lethal in about 25 days when the daily oral dose is 174 mgAg. The data on
effect of TCB on chlorguanide metabolism demonstrated in this study suggest
that measurement of the ratio of chlorguanide metabolites after singla oral
doses can be used as a method for estimating the rttia of P-450/P-448 enzyme
activities in vivo.
Bromodichlororaetharse appears to b« rapidly metabolized or exhaled but
the amount of labeled materials excreted in the urine appears to be greater
than occurs following chloroform administration. This is almost certainly
the result of the greater lability of the Br-C bond compared to the Cl-C bond.
The two bischloroethers appear to be exhaled either unchanged or ns CCH.
The occurrence of periocular irritation following single oral closes suggnsts
that significant anounts of the parent compounds or ac» ve metabolites are
being exhaled. Bis(2-chloroethyl)ether is probably t.i.~ghtly more toxic than
its isopropyl analogue, bis (2-chloroiscpropyl)e'"v;er.
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SECTION 3
RECOMMENDATIONS
The results on trichlorobenzene (TCB) obtained in this study correlating
the in vivo measurement of KFO activities and P-450/P-448 ratios using chlor-
guanide with in vitro measurements of typical MFC enzyme activities suggest
that this procedure merits further trial as a possible method for estimating
the state of hepatic drug metabolizing enzymes in vivo in human subjects.
Although an attempt was made to test the effect of a typical P-448 inducer
(3-methylcholanthrene) the experiment provided inconclusive data and should
b« repeated. The data on hepatic enzyme induction in monkeys produced by
TCB suggests that an attempt be nade to see if TCB induces the came pattern
of response in humans.
The data on bromodichloromethane (BDC) and on the two chloroethers are
interesting and suggest the need for additional studies. Specific goals of
these studies should be to treasure the chronic toxicity of BDC in rodents
and subhunan primates and to define the nature of the urinary metabolites.
More extensive metabolic and chronic toxicity studies of the two chloro-
alkylethers studied in this report are necessary to decide whether the renal
and periorbital toxicity of these compounds is due to the parent compound or
to a more active metabolite. There is a suggestion in thes.e limited studies
that these chloroethers may affect the menstrual cycle in monkeys and this
jx>ssible sidfl effect requires better definition. Since these chloroalkyl
ethers are unstable, future studies should include special precautions to
insure that the results obtained are due to defined compounds and not to the
effects of undefined decomposition or degradation products.
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SECTION 4
IN VIVO STUDIES CN TRICKLOFCBENZENE
I. EXCRETION AND TISSUE DISTRIBUTION OF 1 ,2 ,4-TRICH:X>RCBENZENE (TCB)
FOLLOWING SINGLE OFAL DOSES IN RATS
The purposes of this study were to determine the excretion and tissue
distribution of TCB and metabolites in rats following single oral doses and
to compare these results with those obtained when the compound was adminis-
tered intravenously.
Materials and Methods
Seven groups of four young (300-350 g) Charles River male albino rats
were housed in stainless steol metabolism cages permitting separation of
urine and feces. One group of four was killed at i, 6, 12, 24,48, 72, and 96
hours post-tredtnent.
Treatment solution contained 2.0 mg '0.45 uCi -4C) 1,2,4-TCB per ml of
water :ethanol :Driulr.hor** (8:1 :l ,v/v/v) . Each rat was given this solution by
oral gavage in a dose of 10
Urine and feces werw collected at 24 hour intervals or at necropsy.
Aliquots of urine were prepared ii-mediatoly after collection for liquid
scintillation counting (LSC) and the remainder of each sample stored plet« by 2-1 hours
*f:muiphor-EL-62CH"- w*3 obtained from the GAF Corporation and consists of a
polyoxyethylated vegetable oil soluble in water.
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and was COL etc by 48 hours. One can estimate the T*s of TCB in rats follow-
ing oral do-,-s to range from four to six hours. This fast or a phase is
followed by i much longer slow 6 phase.
Data on tissue distribution of TCB and metabolites (as *4C) are -present-
ed in Tdble 2. The highest tissue concentrations were observed at three
hours. By 24 hours most tissues except the GI tract contained less than
3 u g/g as C. In percent dose, the tissues, other than the GI tract, whicii
accounted for most of the TCB-derived 14C were blood, liver, and kidney
(Table 3). At 24 hours there was very little ^4C in the stomach, about 3.5%
in the rest of the GI tract, and only 1% in all other tissues. The snail
amounts in the stomach compared to the persistence of C in the rest of the
GI tract suggested tha«- the compound and its metabolites were not actively
secreted by the stomach, but nay have been actively secreted via the bile.
Some radioactivity persisted in the fat, kidney, and liver for at least 96
hours after single oral doses.
Data on excretion, tissue distribution, and total recovery are sunmar-
ized in Table 4. Essentially all the administered ^-4C was accounted for in
the urine, feces, and analyzed tissues after 12 hours. The C not accounted
for during the early periods after treatment was probably present in the fat
and other pools, presently unidentified, which provided temporary depots
until the levels in blood, liver, lung, etc., began to fall due to metabol-
ism and urinary excretion of metabolites. Probably less than 2% of the ^ C
administered persists in the rat beyond 48 hours.
II. EXCRETION AND TISSUE DISTRIBUTION OF 1,2,4-TRICHLOROBENZENE (TCB)
FOLLOWING SINGLE INTRAVENOUS DOSES IN RATS
The purposes of this study were to (1) determine the excretion and
tissue distribution of TCB in rats following single intravenous doses and
(2) compare these data to the results obtained following single oral doses.
Materials and Methods
Seven groups of four young (300-350 g) Charles Pdver male albino rats
were used. The rats were housed in stainless steel metabolism cages per-
mitting separation of urine and fccas. One group of four rats was killed
at 3,6.12,24,48,72 and 96 hours post-treatrxsnt.
The treatment solution was prepared by adding 193 vl (280.7 mg) of un-
labeled 1,2.4-TCB and 150 ul (218.1 nq, R4 uCi) of 14C-labeled 1,2,J-TCB to
10 ml of a 1:1 (v/v) nixturo of I'mulphor® and absolute alcohol; this solution
was diluted to SO ml with distilled HTO. The final treatment solution con-
tained io rrvy 1,2,4-TCB (1.68 uCi 14C) per ml.
One femoral vein of each rat was exposed under light other anesthesia
and injected with & dose of 10 r.«j TCB (labeled as above) por kg body weight.
Urine, fecca, and tissues wore handled and assayed for C activity
using tho sane proctsduroa described previously.
-------
Pesultb
""he results of these studies expressed as ug TCB/gran tissue or percent
dose (as ^4C) are surwvarized in Tables 5 to 8. As shown in Table 5, excre-
tion of ^4C was pnnarily via Vhe urir.e (83-86%) and about 12% appeared in
the feces. Excretion of 14C was 90% complete by 24 hours and virtually 100%
by 48 hours.
Data on tissue distribution of TCB and metabolites are presented in
Tables 6 and 7. The data in Table 6 indicate that maximum tissue levels of
^4c occurred in sone tissues at six hours (blood ar.d plasma, liver and kid-
ney) while in the other tissues the peak occurred at three hours or the three
and six hour values were equal. Concentrations were higher in fat than in
any other tissue analyzed. After 24 hours concentrations were higher than
1 wg/g in fat, kidney, and liver. Levels of ^C were roost persistent in the
fat but at 96 hours were still appreciable in kidney and liver.
The data in Table 7 point out the inportance of the blood con-.partaent,
liver, and kidney in the pharaacokinetics of TCB in the rat. Since the drug
was administered intravenous, the prominent radioactivity in the snail in-
testine, cecum and large intestine demonstrate the biliary excretion of TCB
and/or trtetabolites and the probability of enterohepatic circulation (see GI
tract data in Table 8).
In sunsnar/, after intravenous doses of ^C-labeled XCB, 85 to 90% of
the ^*-C appears in the urine with only about 10 to 15% appearing in the
feces. There was early accumulation of radioactivity (assumed to be parent
compound) in the fat and l^C activity persisted in this tis3ue longer than
in any other. Only about 2% of the dose was present in the body at 24 hours
and much less at later periods.
Discussion
If we compare these data after intravenous injection with the previous
data following equivalent oral dosos, we note that the overall metabolic
patterns arc quite similar following the two routes. The intravenous data
denwnstrate 1) that the compound undergoes active enterohepatic circulation,
and 2) that the tissue levels are higher after intravenous injection. This
is particularly evident when we compare the tissue levels (as 1<5C in ug/g)
at six hours in the fat, liver, spleen, kidney, lung, heart, brain, mu»cl«,
and Rkin. Nevertheless, the overall excretion patterns in urine and feces
are essentially the same.
-------
14
TABLE 1. CUMULATIVE EXCRETION' OF 1,2,4 - TRICHLOROBENZEHE (AS C) IN MALE RATS
SINGLE ORAL DOSE OF 10 MG/KG
Cu-ulative Excretion in Per Cent Dose
Collection Tericxl
Hours
0-3
0-6
0-12
0-24
0-43
0-72
0-96
Urine
12.
37.
79.
84.
87.
87.
8S.
5
8
7
5
2
3
1
i 5
.4
i 20.0
i 7
i 6
i 5
i 5
± 5
.Q
.8
.4
.0
.4
(3)
(•J)
(•1)
(16)
(12)
(8)
(-5)
Feces
0.4 (1)
0.2 (2)
2.1 i 1.1 (4)
11.2 i 0.8 (4)
14.3 ± 0.4 (4)
14.7 1 0.6
14.9 i 0.7
Urine and Feces
12.9
38.0
81.8
9f>.7
101.5
102.0
100.0
-------
TABLE 2. TISSUE DISTRIBUTION OF 1,2,4 - TRICHLOROBENZENE (AS 14C) IN MALE RATS
SINGLE ORAL DOSE OF 10 KG/KG
Tissue
Blood
riasr-a
Liver
$i>lcen
Kidney
Uir.5
Heart
Testes
Brain
rat
ug/g at Various Hoars Post-Treatment
3
5.7a
1.0-11.3
10.4
1.6-20.9
8.1
1.9-15.7
0.92
0.11-2,0
31.7
7.1-64.8
3.0
0.57-6.2
2.1
0.45-3.4
1.1
0.19-2.3
0.52
0.13-0.76
17.2
4.6-24.4
6
2.7
2.5-4.0
5.1
3.9-6.8
4.6
4.1-5.2
O.J3
0.27-0.69
22.4
17.0-25.4
1.6
1.3-2.0
1.1
0.65-1.6
0.67
0.50-0.82
0.27
C. 23-0. 29
13.8
6.8-17.1
12
1.1
0.65-1.5
1.7
0.94-2.4
2.1
1.5-2.9
0.29
0.14-0.51
6.2
4.1-7.4
0.71
0.48-0.96
0.59
0.40-0.96
0.33
0.24-0.49
0.15
0.10-0.24
4.3
2.7-6.6
24
0.42
0.2C-0 08
0.62
0.32-0. 52
1.1
0.76-1.5
0.19
0.11-0.28
2.4
1.7-3.4
0.45
0.27-0.61
0.33
0.12-0.52
0.18
0.06-0.33
0.12
0.06-0.17
2.8
2.0-3.8
48
0.09
0.02-0.20
0.15
0.06-0.24
0.59
0.45-0.79
0.11
0.06-0.19
0.06
0.43-1.3
0.21
0.15-0.33
0.18
0.09-0.25
0.08
0.03-0.17
0.06
<0. 01-0. 12
1.5
C. 87-2. 6
72
0.03
<0. 01-0. 04
0.05
0.03-0.07
0.38
0.27-0.44
0.03
<0. 01-0. 07
0.57
0.37-0.88
0.08
0.03-0.12
0.05
0.02-0.13
0.07
<0. 01-0. 11
0.05
<0. 01-0. 07
0.80
0.57-0.99
96
<0.02
<0.01-0.u5
0.03
0.02-0.06
0.32
0.22-0.44
0.02
<0. 01-0. 04
0.29
0.18-0.37
0.04
<0. 01-0. 08
0.03
<0. 01-0. 07
0.03
<0. 01-0. 05
0.03
<0. 01-0. 06
0.62
0.33-0.90
(continued)
-------
TABLE 2 (continued)
Tissu*
Muscle
SXin
pg/q at Various Hours Post-Treatment
3
0.74
0.09-1.5
2.78
0.64-4.6
6
0.49
0.32-0.8:
1.24b
l.CX-1.34
12
0.17
0.06-0.31
0.63
0.34-1.4
24
0.09
0.01-0.14
0.43
0.22-0.93
48
0.05
0.02-0.09
0.31
0.15-0.44
72
O.Ob
<0. 01-0. 07
0.26
0.15-0.34
96
0.03
<0. 01-0. 05
0.15
0.09-0.26
Average of 4 ar.ir.als ar.d ran<]«.
b
Average of 3 anir-als and range.
-------
TABLE 3. TISSW DISTRIBUTION 0V 1,2,4 - TRICHLOROBEN7,ENE (AS 14C) IN F'ER CENT DOSE
SINGLE ORAL DOSE OF 10 HG/KG
KALE KATS
TJ.ssue
Blood
Liver
Kidney
Lur.9
Stonv.ch
Srwill
tr.tcstine
Cccua
Large
Intestine
Other
Tissues
Per Cent Dose
3
4.6
0.82-9.3
2.8
0.79-4.8
2.3
0 56-4.2
0.13
0.02-0.29
6.2
0.3-9.9
13.4
5.2-21.5
1.2
0.06-2.4
0.23
0.06-0.33
0.23
O.C6-0.44
6
2.6
2.2-3.2
1.5
1.4-1.6
1.9
1.4-2.4
0.07
0.00-0.08
0.50
0.05-1.4
8.0
6.0-9.3
3.2
1.5-5.6
1.6
0.23-2.9
0.12
0.09-0.16
12
0.94
0.59-1.3
0.76
0.60-0.95
C.48
0.32-0.62
0.03
0.02-0.04
0.07
0.05-0.09
4.1
2.2-5.8
3.1
2.4-3.7
4.7
2.6-6.3
0.07
0.04-0.10
At Various Hours rest-Treatment
24
0.36
0.15-0.57
0.39
0.28-0.49
0.19
0.11-0.26
0.02
0.01-0.03
0.05
0.02-0.08
1.1
0.46-1.6
1.1
0.5-2.7
1.3
0.5-3.9
0.04
0.01-0.06
49
0.09
0.02-0.19
0.25
0.16-0.37
0.07
0.03-0.11
0.01
<0. 01-0. 02
0.02
0.01-0.03
0.17
0.12-0.24
0.20
0.14-0.29
0.23
0.05-0.47
0.02
0.01-0.04
72
0.02
<0. 01-0. 04
0.15
0.11-0.19
0.05
0.03-0.07
<0.01
0.01
0.01-0.02
0.07
0.04-0.00
0.05
0.04-0.06
0.05
0.03-0.10
0.01
<0. 01-0. 01
96
0.02
<0.01-0
0.12
0.09-0
0.02
0.01-0
<0.01
0.01
0.01-0
0.06
0.0-J-O
0.05
0.03-0
0.07
0.02-0
-------
TABLE 4. FiCCVERV OF 1,2,4 - TKICHLOPOatNZENE (A3 "O IN MALE RATS
SI:K.;LK CK,V~ rxtE CF 10 MVK^
lit' JilU
Faces
G-I Tract
Tissues
Total
Recovery
3*
12.!*
0.4
2i.O
10.2
44.1
Per Cent
6
17.0
0.2
13.3
e.i
57.4
Douc
12
7-J.7
"> 1
12.1
2.3
96.2
at Various Hours
24
U0.4
11.2
3.6
1.0
102.2
Post- Treatment
4B
Ud.4
14.3
0.6
0.4
103.7
72
Ui,.*
14.7
0.2
0.3
105.1
'JO
o ^ . y
14.9
0.2
0.2
100.3
4 rats per tir.c period.
-------
14
SIN'.IX i:,.r/.VL.';^-;s I/XL a 10 KG/KG
Car.uiativi- fcxcrotion in For Cent of Dose
y.car. ar.d Standard Deviation
Collection Period
Hcurs Urine l-'ecoa
C-3 €.0 ± !.!> !4)a <0.1 (1)
O-o 31.1 1 4.2 (4) 0.1 (2)
0-12 50.0 1 11.2 (4) 0.1 (4)
0-24 77. C i C.e (16) 7.0 * 3.6 (1C)
0-4B 83.2 i C.e (12) 11.5 i 3.2 (12)
0-72 80.0 i 5.C (8) 11.0 ± 2.6 (8)
0-96 83.2 i 4.7 (4)b 12.4 i 1.9 (4)
Urino anJ l-'t-coy
fc.O i 1.5 (4)
31.1 i 4.2 (4)
57.0 t 9.1 (4)
B4.6 i 5.U (10)
94.7 « 4.4 (12)
97.1 t 3.7 (d)
95.5 i 3.3 (4)
•
Sj.rvix.-r of aniT^l".
-------
TASLE 0. TISSUE DISTRIBUTION OF i.2,4 - TRICIiLCKCHLNIENE (AS 14C) IN MALE PATS
srsju: iriTfvY.T.'iC'JS DOSC or 10 KG/KG
— __. .... 1 . ._.
Tissue
yg/g at Various Hours Post
3
biv^a 3.70
3.19-3.94
Plasr-a
Liver
Spleen
Kidney
Lur.g
Heart
Tcstcs
train
tat
Muscle
am
3
3.43
5
5.10
.79
-4.55
.47
-5.93
O.fci
0.63-0. *:>
10
15.2
2 . GO
.0
-i7.5
-3.05
2 . 04
1.08-2.27
0
rl U f
J * ;; v
1.13
51
3-:. 7
0.9--
3
1.30
.9S
-1.01
."*..•
-l.t-,7
.6
-04.7
-",3
.24
-4.62
0
4.40-5.74
7.02
6.17-3.08
S.74
5.10-6.03
0.82
0.72-0. Si
32.2
22.8-30.7
2.17
1.95-2.32
1.60-1.00
O.b9
0 . 02
O.S3-S.7;
41. >
29.0-47.1
1.20
0. 97-1.40
1.05
1.30-1.91
12
2 . •
0.10
0.05-0.
0.40-1.
0.11
C. 09-0.
0 . 1 >j
0.12-0.
17
O'J
52
20
95
50
35
19
10
4 J
u-
- J
-------
TABLE 7. TISSUE DISTJ-H^TION OF 1,2,4 - TRlCHLOKGbLNZKHi: (AS C) IN PER CENT LOLE
sitiGLE ir;TfAVE:;ous DCGL OK 10 MG/KG
""* " ~ " TT---T- --, . -u. __ __ i i - ill ™-._L_ j^r L . — - . . T — - i r. - — __-jiMi-mr J _. _ _. .i~ _. TV— -_ .. T--± . ... . . -T-: JL -_.. _ — -. - __ ,.- -_ •_, i i ii ,..- — —
Tissue
Blcod
Livur
Ridr.cy
LUT/J
Stcwch
Sr^ll
Ir.*.estine
Cccun
Large
Ir.tcstir.c
Other Tissues
3
2
1
0
0
8
C
0
1
3
3.39
.07-3.61
2.30
.10-2.72
1.17
.07-1.26
0.10
.O-0. 13
0.57
.40-1.00
9.41
.73-10.1
0.35
.25-0. CO
0.34
.19-0.53
1.93
.53-2.26
Per Ce
t>
4.43
4.12-4.93
2.20
2.01-2.40
2.23
1.77-2.86
0.03
0.08-0.10
0.34
0.17-0.49
15.8
13.4-18.5
3.55
2.27-4.42
0.79
0.5f>-1.22
1.59
1.16-1. 68
fit
1.
0.
0.
C.
0.
3.
1.
2.
0.
Lose at
12
2.16
59-2.65
1.11
95-1.39
1.03
75-1.64
0.0&
04-0.09
0.34
17-0.4%
4.31
5C-5.24
4.83
97-6.90
4.38
90-6. 34
0.81
76-0.33
Varioun Hours
24
0.64
0.47-0.79 0
0.49
0.39-0.48 0
0.23
0.19-0.28 0
0.02
0.02-0.03 0
0.09
0.05-0.13 <0
1.26
1.01-1.56 0
1.80
0.92-2. 12 0
2.06
0.60-3.16 0
0.30
0.18-0.48 0
Post -Treatment
4b
0.21
.15-0.31
0.27
.19-0.33
0.10
.04-0.13
0.02
.02-0.03
0.03
.01-0.07
0.21
.05-0.37
0.25
.18-0.35
0.29
.07-0.79
0.14
.10-0.18
0
0
0
<0
<0
0
0
C
0
72
0.13
.09-0.19
0.22
.18-0.27
C.04
.03-0.00
0.01
.01-0.01
0.02
.01-0.04
O.C9
.03-0.13
0.06
.02-0.11
0.15
.04-0.52
0,07
.05-0.09
96
0.09
C.G4-0.
0.20
0.18-0.
0.04
0.03- 0.
<0.01
(
-------
3. KrCCVXKY O.r 1,2,4 - TKICHLCF.Cl'ENr4:NU (AS 4C) IN KAI^ RATS*
S:::-.I.E XNTKAYE:;::US DOSE OK lo MG/KG
j
urine 6.0
^ Fecss <0.1
en
G-I Tract 10.7
Tissues 8.3
Total Recovery 25.6
Per Cent
-------
III. PHAKXACCKINETIC STtDY CF TC3-14C IN RHESUS MONKEYS
14
TC3- C has b«en administered in sir.gle 10 mg/kg doses to 4 rhesus non-
keys; 2 received the corpo-ond intravenous and 2 orally. The purpose of this
study was to collect range-finding data en the pharcacokinetics of TCB in
rhesus oonkeys. dsta to be used in designing the subchronic studies to follow.
Materials ar.d Methods
The treatrent solution was prepared by dissolving 220 u 1 (320 wj) of
l^.S-trichlorober.zene-^C (0.56 uCi/nl ) and 124 u 1 (180 ng) of unlabeled
TCB (Eastman Kodak Lot »1641) in 10 nl of EnulphosO.ethanol (1:1, v/v) and
Diluting this to a total volume of 50 rsl with distilled HjO. This solution
contained 10 tag TCB sr>d 2.14 u Ci **C per nl.
Mult fervale rhesus monkeys were tranquilired with ketamine HC1 (4.5
rig/kg) and placed in restraining chairs three days prior to the administra-
tion of the coryound. There was no apparent reaction to either intravenous
or oral treatments with the compound. Blood samples were taken before treat-
ment and at 1.2.4,6,8,12,24,48,72 ar.d 96 hours post- treatment, frir.e sarplts
were collected at 6,12,24,48,72 and 96 hours post-treatment; feres were col-
lected at 24 hour intervals for seven days.
Hentatocrit was determined on each blood sar.ple, and whole blood, plasma,
urine, ar.d feces samples were analyzed for total ^C content employing the
standard procedures used in this laboratory.
on tho blood and plasma levels of intravenous and orally treated
animals are contained in Table 9. When TC8-"C was Administered intravenous
there was A aranatic drop in * C content of whole blood 'row an <-stirmted
rote tine level of about 112 u g/tnl to a level of 2.0-2.9 ug/ml one hour
; r.ext
four days to levels of 0.2 ug/sl. The plasma concentrations of TCB-^C wcr«
higher th.in the r«"Sf>«ctive whole blcxxi concentrations rnnyim) fron 3.1 to
4.9 ug/nl at orm hour and declining biphasically to 0.24 ug/ml at 9b hours.
Wiesus nonkey Ik). 3 which received th« cowpound orally, exhibited a
p«.ik blood level of 3 ug/ml (4.6 ug/ml plasna) at one hour. Khesua ronkey
No. 4, also orally treated, had a peak blood lev?»l of 2.3 ug/sl (3.6 u ij/nl
;>l.t:i--i) at twn hoars j>O3t-tr<"Atswnt . P-jrir.>-> the rorvsinlnq y#nols ot
the intravenous .'.nrl or«\lly treated .«ninvi)a.
Urinary oxcreiion following intravenous administration accounted for
About Jy* of the do»« in four day*; 2.'i was excreted during the first 34 hours
(r.ne Table 10). In the jnonkeys receiving TCB orally, urinary excretion
accounted for a significantly greater onount of the cor4X>und; 36 to 40* WAS
recovered durin'i th« firat 24 hours and 56 to 73* of the dos« in fovt days
CUbio Ii).
16
-------
Figure 1 shows the draruitic difference between the urinary excretion of
7CB following single oral cr intravenous doses. This difference in urin-
ary excretion between the 'ani~als receiving this compound intravenous and
orally nay be due to the fact that the parent, TCB, is very lipid soluble
ar
-------
9.
AND PLASMA CC.VCLNTKATICN ci* 1,2,4 - TRictiLOKQi>r.N::uJE (AS c) IK mavLE KHLCUS MONKXYS
TIRO Post \
Hours
1 45
2 44
4 43
6 42
e 41
12 41
24 45
4& 37
72 35
96 34
Sir.-jie Jritraver.c
No. 1
Blood riaw.,i
2.9 4.9
2.0 4.5
1.6 3.0
1.3 2.3
1.2 2.1
0.8 1.4
0.43 0.7fc
0.35 0.54
0.23 0.36
0.18 0.24
us Done of 10
NO. 2
%
iict Bloc•') 1.5 2.0
:;s 30 1,4 2.2 41 l.i 1.6
0.75 32 0.94 1.4 39 0.07 1.0
0.53 32 0.54 0.79 37 0.43 O.vO
0.33 32 0.29 0.30 37 0.27 O.Jii
32 0.19 0.25 3d 0.22 0.30
31 0.10 0.19 39 0.17 0.21
Phesus ronkey Slo. 2 dtvclcpvd a detached scat-pad and was returned to C&-JQ at 50 hours post
trcatrcr.t.
-------
14
TABLE 10. CL-MULATIVE EXCRETION CF 1,2,4 - TBICHLORCEEI.'ZEI.T (AS C)
I!J FEMALE P-KESUS MONKEYS
SINGLE INTFAYE^GL'S DOSE CF 10 MG/KG
.'to. 1
HOMTS
0-24
24-48
48-72
72-96
96-120
liO-144
144-163
'Jrir.e Feces
Peritd Total Period Total
a b
22 22 -IS
5.4 27.4 <0.01
6.2 33.6 0.1 0.1
4.7 33.3 1.7 1.3
1.1 2.9
0.5 3.4
0.4 3. Q
orir.e and Feces
Period Total
22 22
5.4 27.4
6.3 33.7
6.4 40.1
1.1 41.2
0.5 41.7
0.4 42.1
!.'o. 2
Hours
0-24
24-48
48-72
72-96
96-120
120-144
144-108
Urine Feces
Period Total I'criod Total
21. B 21. a r;s
6.3 23. C KS
1.3 29.3 rJS
- c - 0.4 0.4
0.9 1.3
0.2 1.5
0.3 1.8
Urin«» jrxJ Feces
Period Total
21.8 21. H
6.3 28.0
1.3 29.3
0.4 29.7
0.9 30.6
0.2 30. b
0.3 31.1
Approximately 752 of total sanple lost.
SS- no snnplc.
Khcuua roonkev No. 2 developed a detached seat-pud and w.is returned
to cnpo at bb hra post treatment.
-------
TABU; '11. CUMULATIVE EXCRETION CF 1.2,4 - '."
IN FI-^-M-E RHESUS MCNYXY
SIJJGLE CPAL DOSE CP 10 HC/KG
(AS
Jto. 3
Hours
0-24
24-48
43-72
72-9o
96-120
120-144
144-1C8
Urine
Period Total
39.8 J9.8
25.6 65.4
4.5 69.9
3.2 73.1
a
-
-
Feces
Period
<0.1
0.9
2.0
0.8
0.1
0.2
0.1
Total
-
0.9
2.9
3.7
3.B
4.0
4.1
Urine ar.d
Period
39.8
26.5
6.5
4.0
0.1
0.2
0.1
Feces
Total
39.8
66.3
72.8
76.6
76.9
77.1
77.2
No. 4
Hcurn
0-24
24-48
48-72
72-96
96-120
120-144
144-KB
Urine
Period Total
3G.7 36.7
10.1 46.8
5.7 52.5
4.2 56.7
_a
-
-
Fc
I'wriod
0.1
0.1
<0.1
0.5
O.B
G.6
0.6
cos
Total
0.1
0 2
0.2
0.7
1.5
2.1
2.7
Urine and
Period
36.0
10.2
5.7
4.7
0.8
0.6
0.6
Feces
Total
36.8
47.0
52.7
57.4
58.2
W. a
S'>.4
Aninal returned to cac,e—no further urin«. collections evade.
20
-------
ioo
so
UJ
I/)
O
u.
O
i. 60
ce
UJ
Q.
UJ
cc
CJ
X
UJ
20
0
FIGURE 1
CUMULATIVE PERCENT EXCRETION IN URINE OF 1,2,'t - TRICHLOROBEHZEilE (AS WC)
IN RHESUS nONKEYS FOLLOWING A SINGLE ORAL OR INTRAVENOUS DOSE OF 10 MG/KG
O ORAL
A INTRAVENOUS
'13
TIME IN HOURS
72
_J
96
-------
When urine and feces collections were required, the animals were placed
in individual metabolism cages specially fabricated for primate iretabolisa
studies. During these periods, Mnne was collected in iced containers and
the portion not analyzed iimediately was stored at -20°C.
«
V^or.V.eys were gavaged with TC3 each mcrr.i:;q, seven days per weeh for
three nor.ths. Treatments occurred at the same tire each irsorr.ir.g ar.d re-
quired no triore than 45 minutes. Each week when the monXeys were weighed, a
blood sanple was drawn for TC2 netabolite analyses. Cr.ce ncnthly, addition-
al blood was taXen for SMA ll'/60 clinical chenistry analyses. Chlorguanide
(CG) netatolite profile tests were performed at 30-day intervals after ini-
tiation of TCB treatment.
Liver biopsies for enzyire analyses were obtained at the end of the
three ncnth treatment period through the generous cooperation of the staff
veternariar.s in Dr. Steele Mattingly's Department of Laboratory Anical Medi-
cine, University of Cincinnati, College of Kedicine. The aniir.als all
recovered without sequelae in five days fron this procedure.
B. Chemicals. 1.2,4-trichlorobenzene (TCB), was obtained frcn Aldrich
Chemical Co., and most of the chenicals used in the in vitro KFO assays
were obtai.-sd from Fisher. Sigtsa or Aldrich. One graa of benzphetanine
hydrochloride i»as procured directly from the UpJohn Co., Kalamazoo, Michigan.
Chlorguanide hydrochloride (CG-HC1) was obtained fron the DuFont Chen-
ical Co. This compound was recrystallized to a cvelting point range of lose
than 1°C and an extinction coefficient at 258 nn of 712 in inethanol. Kadic-
labeled CXJ'HCl was purchased from Kew England Nuclear, Inc. with ^4C at
position 1 or 2 of the phenyl rir.g. This material had a very narrow neltinq
point range and showed r.o radiolabeled contamination when chrotnatound
metabolized primarily by this aystcm. Ideally, th« co:npouiicl selected for
22
-------
this purpose should be netabolized to discrete products by each of the two
najor types of Mi'O, P-450 and P-448. In this way, the urinary concentrations
of each metabolic prodvct can be used to cstinate the relative activities of
P-450 and P-443 in the liver. Furtherr»ore, the ccrpound should normally be
excreted in part unchanged so that or.e could raeasure the relative activity of
each nvonoxygenase cor-ponent present by cotrparing '•IFO-specific metabolites to
parent coapour.d.
One compound neeting these criteria is the intirnalarial. chlorguanide
(CG). It is oxidized by P-448 to the active antinalarial metabolite, chlor-
guanide tnazir.e (CGT), and by P-450 to the inactive product, p-chlorophenyl
biguanide (PBG) (5,6). Parent CG is also excreted (6). The metabolic path-
way is shown below:
tCCT)
To determine its metabolic profile in urine we have developed cclori-
netric and microbiological rwthods for measuring CG, CGT, PBG (after conver-
sion to CGT) and total CG tnet.«Jx>lites. These procedures have been used in
estimating CG and its principal metabolites CGT and PBG in blood, plasna,
aj>d urine ( 6 ). More recently, when radiolab«led CG became available, we
developed a method for separating CG and its metabolites using TLC (S.T.
Cragg, Doctoral Thesis, University of Cincinnati, 1980) &nd thia latter pro-
cedure was the one enployed principally in the studies reported here. Essen-
tially the cvcithod is based on th« extraction and separation of CG, cr,T and
PbG from a two day urine pool foJlowing CXJ dosing. An appropriate amount of
the extract is spotted on a TLC plato and after development, the radioacti-
vity in spots corresponding to the lor«tion of tho 3 CG rvetabolic products
is determined by LSC. The oothod has been shown to b« reproducible and as
will b« (lcff>onstrated fiubsequently appears to be predictive of the relative
activities of the "P-448" and "P-45G" components of the ?. Thus, if th«
"P-450" component is induced we would «xp«ct n»re PBG in th« urine and
correspondingly less CG and/or CGT. A "P-448" inducer like 3-HC should in-
crease CGT formation and excretion at the expense of CG and/or PBG while an
induc«r like the PCB Aroclor 1254 should increase the forrvation of both CGT
and PBG with a concurrent reduction in urinury CC.
Results
Tho monkeys, which wore treated with doaos of TCB of 1,5, arid 25 wgAl
for 3 nonths, showed neither weight loss, changes in clinical chemistry an-
alyses (SMA 12/60) Tnbles 12 and 11, nor aiijnificar.t changes in their urin-
ary CG rwt.sboilt« profiles. Furthermore, thnro wore no significant differ-
ences in the T*> values for labeled TCB (aa ^C) when a single intrav«nou«
douo w,-« adr-.ir.i atered to t!*e control and 23 mg/Xg (TCB) groups at the end of
3 raontt.H tre«t.T«nt,. for the aak« of brevity these dat* »ro not included
but cor t-o r-id« available to anyone upon request.
-------
TABLE 12. SKA 12/60 KS:SUL7S ON MONKEYS IU.FCKE TREATMENT Will! TCB
Dose Monkey
03 /x<| ;:o .
0
1
5
25
125
Basclir.o
Values
S.X.
573
433
Jcu
405
543
223
264
i04
634
505
237
39
343
110
503
276
012
C-14
C-18
253
Sugar
rwOO
304
250
257
249
166
290
196
223
237
171
225
170
245
175
271.2
+24.6
P
4.7
3.6
2.8
2.1
3.2
2.4
4.3
2.8
5.6
4.3
3.4
4.1
4.4
3.4
5.1
3.0
3.3
2.6
2.8
3.3
3.56
+0.21
Alk.!'.
mU/rol
183
81
252
195
75
252
>350
159
189
120
62
223
105
118
83
134
225
91
274
154
166.3
+ 17.5
Crt
9.4
10.2
9.5
9.5
9.3
8.1
9.9
10.3
11.0
10.4
9.7
9.9
9,1
9.1
9.3
9.2
10.7
10.1
10.9
10.0
9.78
+ 0.16
Direct
Bili-
rubin
ing %
0.1
0.1
0.1
0.1
0.1
0.2
0.1
0.0
0.0
0.1
0.1
0.1
0.2
0.1
0.2
0.1
0.0
0.0
0.0
0.0
0.12
+ 0.01
Total
Bili-
rubin
my %
0.2
0.3
0.3
0.3
0.3
0.5
0.2
0.1
0.1
0.3
0.3
0.1
0.4
0.3
0.3
0.2
0.1
0.2
0.2
0.2
0.26
+0.02
Tro-
tcin
g %
8.0
7.7
7.4
7.2
e.r
C.4
8.1
6.6
8.5
8.2
7.9
8.1
8..)
'J.I
8.4
7.5
7.6
\J • m.
8.0
0.3
8.01
+0.13
Alb
g %
3.6
3.8
3.9
3.7
3.5
2.6
2.7
4.3
3.8
3.9
4.2
3.5
3.6
3.9
4.0
3.3
4.3
4.0
4.t
4.2
3.77
+ 0.11
Hor.fceys 573-276 assayed 12/17/76.
«:rJK-ys CI2-253 assayed 6/24/7?.
-------
7A3I£ 13. SKA 12/60 KLSCLTS ON MONXfcYS AFTER TREATMENT WITi! TCB FOR TWO MONTHS
Dcso
0_
1
5
2rj
125
baseline
Values
Mcr.kcy
573
463
366
495
543
223
264
C04
634
505
267
39
343
110
500
276
C-12
C-14
C-18
m t
77
81
85
82
80
65
1U
95
84
85
78
90
63
97
88
55
' 85
77
73
67.3
Uric
acid
ag *
0.3
0.2
0.2
0.2
0.3
0.2
0.2
0.3
0.2
0.2
0.2
0.2
0.3
0.1
0.3
0.1
1.1
1.0
1.0
0.69
Choi
ISO
151
175
162
175
205
150
150
125
100
198
160
188
200
200
205
142
145
169
173.9
SOOT
rVYnl
31
24
25
19
29
20
20
4:
17
19
23
25
17
20
16
20
9
5
16
30.4
LDH
r.U/nl
295
226
255
215
255
249
210
335
232
360
255
335
223
189
245
210
155
123
195
288.0
Tig %
3.6
3.1
2.5
2.3
4.2
3.8
3.7
4.4
3.3
2.2
2.4
2.5
4.2
3.2
4.9
4.3
4.7
3.5
2.9
3.70
Alk.P.
ir.U/ml
255
137
206
217
96
227
324
170
185
178
95
144
131
117
140
188
247
159
167
161.3
Ca
Iftf] \
8.8
9.*
9.4
9.2
9.4
9.5
9. J
10.0
9.9
10.4
10, 3
9.6
9.0
9.8
10.3
9.8
9.6
9.7
10.6
9.62
Direct
Bili-
rubin
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
. o.o
0.0
0.0
0.1
0.0
0.0
0.12
Total
BJli-
rubln
ir.cj %
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.3
0.1
0.1
0.1
0.1
0.2
0.2
0.2
0.1
0.1
0.0
0.0
0.28
Pro-
tein
8.0
7.9
7.6
7.6
Q.9
6.1
B . (•
8.9
7.8
8.1
8.4
8.4
8.4
9.0
9.2
9.0
7.8
8.5
8.6
7.99
Alb
-------
V. SECCJ.'O TC3 SCBCHROMC STJDY
Afrer the first TCB stuiy h«d t-een in progress about one nonth it be-
case cbvicas that nor.e of t_r.e ar.isals vere shoving tcxic symptoms and that
cr.e of the irrrortant objectives of the study, ar. estimation of the chronic
caxxeun tolerated dose of 1CB, was not likely to be accomplished. Therefore,
we added another geometrically-spaced dose level, 125 »g/Xg, which we ex-
pected to be toxic. After one zienth it again becane clear that although two
nc-rjceys frors the 125 ng/kg group showed teaporary weight loss and one aninal
died an 1C- days, the three retiring anir-als appeared healthy, began to gain
wight, and showed no signs cr syr$>toeis of toxicity specifically related to
TCB.
We had demcnstrated to our satisfaction that the 1,5 and 25 xq/Y« cages and th« urinary profile* of chlorguanii'.e (CO)
netalxjlite* w«ro rwavutcd. Alto at ccnthly interval* blood 5*.v.-.pl«u w«re
collected for SMA 12/fcO clinical chemistry analysas. At the end ot 12 w**ks ,
or at death. th« .ini.-sal* v«re nccropsied and liver slices uore froren in
liquid tJj tor ^~45C r*latcd ensyc* assays.
t^r.eral Tcxxcity of 7rn
Th« ronkcys rec«ivir;<7 these higher dos«K of TCB exhibited definite
;icf«-rclat«d evidence cf toxicity. 7 ho or.e an in A! on th« 90 sna/kg rto»e
thit dl«id in tho ?th wok (47 d) lest {).'; kq. However, the lack o£ .!rofil« (T^blc 14). ""!'.<• wt»i:r«4tcst
to TCB .it 4-t.
The ct-.ar.fjosi in reut>e!.R* to the Iwj r/;A<; done hav» b«en r«ntloncJ abov« .
Main vm note that tt.« ts,ixjp-y» t*»fonii« to TCh Arp«ar» to coac alfr 4-6
.reatiwM. A riot which ih* anir^ld either IJOC.VT* stabl* or i
2 6
-------
The 174 rag/kg dose was definitely lethal ar.d would hav« killed all four
ffenkeys in this group had we not discc-r.tir.ued treat-sent a:ter day 25. At
this tine the two surviving ncr-Aeys shewed s/Eptons of extrer* intoxication
including weight loss, weakness, trerors, ar.d increased susceptibility to
pulmonary infection.
pjecovery fron the visibly toxic effects of TCB was rapid &ft\.: treat-
ment was discontinued. Although one additional rer-key difed after treat r«r.t
was discontinued, this anicjl (*343) appeared to be recovering but apparent-
ly succuc-Jyed to acute gastric dilatation, probably the result of overeating
due to careless overfeeding.
The weight changes in the nor.keys treated with large doses of TCB are
shown in Table 14. The controls showed ho significant weight changes during
the 10 weeic period sr.own in the table. For the 90 ar.d 125 =?gAg groups the
responses were quite variable. Tvo nonkeys on tho 90 rag/kg lost 1 and 2 kg,
respectively. A third raonkey did not charge weight significantly. The
fourth aninal died but lost weight prinarily in the last week of treatment.
This death we believe tr-ay not be due to TCB. In the case of the 125 eg/kg
dose the monkey that died (»I53) lost 0.9 kg in two weeks and died on day 16.
The other two aor.keys (C18 ar.d C14) showed e^tly large weight losses tC.»
ar.d 0.9 kg) but then becaae stable ar.d at the end they, and C12, were gaining
weight.
It would appear likely that if the ants-*! responds to TCB by developing
hepatic Ki'O induction, then the liver will b* able to better hindle the
ccer.-ound ar.d from this i«oir.t on the increased snetacdisn will reduce th«
effective dose.
Change* in henvatocrit -at* *hovp in Table IS .ir,i in general on« se«» th«
sane trends observed with th« ch«»:.'jes in weight. TCB tr«>*tn«nt tends to c..^us«
a decline in tho packed r«-J cell volu»e. Xe *.jv» not nad« ar-.y .utterq^t to
pxawine th« unJerlyir.q cause* of this ph«nor*r>on.
The changes in li%'er to tc-Jy w«i jht ratios (ai *) are shov^ in Table*
10 and 1?. >or '.*•.<• (»3 rxi/kg dose, t!-.<* oniy highly significant change WAS
seen in r«r.kcy «11-,) wt.ich died a!tcr 47 days of trpatfwr.t . The j>ercent.v3es
for the other three rxinkey* wwre scarcely atxjve the ncrw-al ranae. All the
-cr.kcys r<»cpivinn 1.'5 And 1^4 P«J.>l
jfter rtijriniatrat lor. of I'.'V f.;r r.r.o month 4i«? '>j'ict«>cl graphically in TtT-ire
2. "'!>* graphs in
-------
ratio.
Ccnparison of the data after one no nth TCB treatment (Table 18 ar.d
ii-ijre 2) with the findings after two and three norths (Figures 3 and 4)
suqqests that administration of TCB lei to an induction of MFO activity in
the liver and that this ir.cre.ise appedred to be restricted to the P-450 class
cf KTO's. We found a decrease in CG excretion, an increase in FBG, and a
decrease or no change in CGT excretion as well as changes in certain ratios,
especially CGT/FBG.
At the termination of TCB administration the nonkeys were necropsied,
•.he li-.-ers vere weiqhed, ar.d slices of liver were stored in liquid N2 for
ill V3-trc assay of MFC activity using several substrates. The results of
t.r«ese in vitro studies are described later.
-------
TABIZ 14. WEIGHTS CF KCNKEYS ON DAILY TRl^TyXNT WITH TCB
Do'.te JVr.key
r»j/Xt| No.
0 573
433
366
4>S
DO 6 34
505
110
SOO
1^5 cia
cu
C12
253
174 267
33
343
276
iMt ial
K'J '
7.5
6.0
5,5
5.6
7.8
6.4
5.9
6.3
5.1
6.3
5.0
5.3
6.2
6.3
6.4
6.3
1
7.4
5.9
5.5
5.5
7.0
(j.'j
5.9
6.3
4.7
5.8
5.1
5.1
5.8
5.9
5.4
5.8
•>
* . 4
5.9
5.5
5.6
6.4
6.3
5.8
6.4
4.3
5.4
5.1
4.4
5.5
5.2
5.2
5.4
3
7.C
6.2
5.6
5.6
5.6
6.1
5.7
6.4
4.3
5.6
5.1
Died
5.0
4.5
5.4
5.0
Time in Weeks
456
Woifjht in Kg
7.6
6.3
5.7
5.7
5.3
5.8
5.6
6.3
4.3
5.b
5.2
Diod
4.2*
4.1J*
Died
7.5
6.2
5.6
5.6
5.6
5.6
5.5
6.5
4.5
6.1
5.2
4.3
5.2
7.6
6.2
5.6
5.7
5.2
5.7
5.2
6.5
4.6
6.6
5.3
4.8
Died*
7
7.6
6.4
5.6
5.9
5.1
5.5
Died
6.3
4.9
6.6
5.2
5.2
8
7.6
6.4
5.5
5.8
5.4
5.5
6.4
5.3
6.7
5.1
5.6
9
7.0
6.4
5.5
5.8
5.6
5.5
6.3
5.3
6.7
5.4
5.6
10
7.6
6.5
5.6
5.7
5.8
5.4
6.1
5.3
6.7
5.5
5.0
cJiscor.tinucd on day 25.
Iij«d of a; j'-trer.t overeat ir,
-------
TABU: 15.
u:s cr K,:;KJ.YS
DAILY TI-XAIMI.NT WITH TCH
Do:;« .Her. key
,) 573
4d3
3'- 6
435
90 634
5:5
no
5C3
i::» cis
C14
C12
253
174 :;7
32
343
276
Initial
Hct
45
43
47
45
' 47
41
31
46
44
43
4ti
41
46
41
44
1
_
-
-
-
46
46
42
31
42
41
39
40
42
46
46
44
2
49
52
51
39
45
44
42
33
45
41
3b
(Dit;i
40
46
43
44
3
47
52
49
39
44
••••S
42
34
40
.36
37
day If,)
39
43«
39*
4:
4
47
50
47
40
40
44
42
35
36
39
40
(Died
40
41
(Die,!
Time in
5
49
53
51
42
37
42
43
35
36
37
40
day 28)
38
40
day 24)
Wcfka
6
47
51
50
41
40
41
43
35
39
34
37
-
39
(Died
-
7
47
49
48
43
34
39
(Oied
35
39
34
J9
-
41
day 35
-
8
48
54
48
42
37
40
day 47)
35
40
40
38
-
43
of over«:at
-
9
48
52
49
42
39
40
-
35
39
3!;-
3B
.
44
in-;)
-
10
49
52
49
43
41
41
-
35
40
J7
39
-
45
-
-
.~ , . _
disccnr.ir.ucl or, day 25.
-------
10. UVtR WEIGHT, fcCOY «UGHT AND LIVJ:K WtlCHT/UODY WEIGHTS RATIOS (AS ri:r0.135
Initial
B&Jy Wt
7.50
6. CO
5.50
5-fiO
7. BO
0.40
5.90
6.30
5.10
6.30
5.00
5.30
f>.20
6.40
6.30
6.30
Sacrifice
IV- '.Jy wr
7.40
6.50
5.35
5.80
6.10
5.40
4.70
5.95
5.10
5 90
5.85
4.40
4.70
5.20
6.10
4.50
I.iver Wt x 100
Init Ko.jy Wt
1.93
2.51
2.39
2.37
2.20
2.40
3.73
2.37
3.04
3.40
3.63
4.04
3.53
3.25
2.02
>2.1
Uvf>r Wt x 100
Z,\<: IV.Jy Wt.
1.90
2.32
2.45
2.28
2.81
2.05
4.08
2.50
3.04
3.70
3.10
4.06
4.66
4.00
2.70
>3.0
Mor.keys wf.ich ditj during trcat,ner.t.
Thi* subject hj-1 t«en withdraw lto>a trc.itr»»r.t for over one month prior tc-sacrifice.
»2
Not all of this subject'* liver was recovered.
-------
: 17. SlC'.r.'Ir ICA.NCE I.KVI.LS (D COMt'AKINC. LIVt:K WEIGHTS AND UVi:R WT./UODY WT.
CONTKOL A.ND TCB TKLATKD GROUPS*
I.iver Wt . , „„
.1: inon
"Overall" F
I.ivor
p « 0
Wts.
.0141
I n 1 1 1
-------
TABU: 13. i:m:crs CF 30 DAYS ics TKI:/OU,::T 0:1 VKJNAKY CHIX>KGUANIDE KXCRI;TION
TVB Dc:u» K-.-.key
r»;.-*=7/J.iy No.
0 573
483
366
4?5
Mean
SE.M
S3 634
505
110
500
Ke.in
SEM
125C C13
C14
C12
.'x an
StM
174d 343
39
Mean
i
B*
10.1
17.8
17.7
17.6
15.8
11.90
12.0
14.5
11.5
n.i
12.0
10.92
15.4
14.6
15.2
15.1
10.24
10.7
14.1
12.4
crcent C
Treated
13.9
22.0
17.0
18.3
17.8
11.C3
12.2
15.7
9.C-6
13.2
12.3
11.19
9. BO
12.6
10.5
11.0
+ 0.64
10.6
11.0
10.8
»
3.8
4.2
-0.7
0.7
2.0
11.2
0.2
1.2
-1.5
3.1
0.7
+ 1.0
-5.6
-2.0
-4.7
-4.1
11.1
-0.1
-3.1
-1.6
I
B
37.5
39.7
36.6
40.8
38.7
10.97
36.9
39.6
41.6
38.6
39.2
I0-98
59.0
40.5
43.4
41.0
11.29
30.8
33.6
38.7
'ercent. TDG
Treated D
38.4
39.5
37.5
41.9
39.1
10.97
47.2
46.2
46.0
44.4
«!5.9
10.58
54.1
44.1
50.0
49.4
12 . 90
47.3
49.0
40.2
0.9
-1.2
0.9
1.1
0.4
10.5
10.3
6.6
4.4
5.8
6.8
11.3
15.1
3.6
6.6
8.4
O.4
8.5
10.4
9.5
rercent CX'T
H Trc.'itejd D
52.4
42.5
45.7
41.5
45.5
12.46
51.1
45.9
48.9
47.3
4h . 3
— *
45-. 7
44.8
41.4
44.0
11.31
50.4
47.3
48.8
47.8
39.4
45.4
39.8
43.1
+ 2.08
40.6
38.0
44. .2
42.4
41. J
11.32
36.1
43.3
39.5
39.6
12.08
42.2
39.9
41.0
-4.6
-3.3
-0.3
-1.7
-2.5
+ 0.94
-10.5
-7.9
-4.7
-4.9
-7.9
11.4
-9.6
-1.5
-1.9
-4.3
O.6
-0.2
-7.4
-7.8
(continued)
-------
7ABIX 18 (continued)
TCS Scsc Mor-key
rvj/Xq 'r!,iv Jk>.
0 573
433
366
405
Mean
SLM
9O 634
505
110
500
MC3U1
SLM
125C CH
C14
C12
Mean
sr.M
174J 343
39
Ko an
B*
5,17
2.38
2.59
2.35
3.12
+0.69
4.25
3.16
4.26
4.0.8
4.0-3
+0.33.
2.93
3.07
2.72
2.92
+ 0.10
4.72
3.35
4.04
CGT/CG
Tre-itp 1
3.44
1.78
2.68
2. 18
2.52
t_0.36
3.35
2.45
4.52
2.38
3.43
+ 0.42
3.68
3.42
3.77
3.62
+ 0.10
4.10
3.61
3.86
jjb
-1.73
-C.60
0.10
-0.17
-o.to
+_0.40
-0.90
-0.71
0.26
-1.30
-0.66
I0-33
0.70
0.35
1 . 05
0.70
^0.20
-0.62
0.26
0.18
B
3.71
2.22
2.07
2.31
2.58
+_0.38
3.07
2.73
3.46
4.22
3.37
+ 0.32
2.54
2.77
2.86
2.72
+ 0.10
3.64
2.74
3.19
PBG/CC.
Treated
2.7C.
1.75
2.21
2.29
2.25
+^0.21
4.88
2.96
4.70
3.49
4.01
+_0.46
5.52
3.48
4.77
4.59
+^0.60
4.39
4.44
4.52
D
-C.-55
-0.47
0.14
-0.02
-0.33
±°-25
1.81
0.23
1.24
-0.73
0.64
+0.56
2.98
0.71
1.91
1.87
+0.66
0.95
1.70
1.33
n
1.40
1.07
1.25
1.02
1.19
+0.09
1.38
1.16
1.23
1.11
1.22
±° • OG
1.17
1.11
0.953
I.Ob
I0-06
1.30
1.22
1.26
Tro.itvd
1.24
1.02
1.21
0.95f>
1.11
±°-07
0.863
O.R24
0.901
0.959
0.904
_+0.030
0.668
0.982
0. 790
0.812
+0.090
0.890
O.H14
O.HSO
v
-0.lf.-0
-0.050
-0.040
-0 .004
-0.0t!.0
+0.030
-0.517
-0. 3?0
-0.269
-C.I SI
-0. 320
+0.080
-0.502
-0.126
-0. 163
-0.264
+C.120
-0.410
-0.406
-0.408
8 m Biselir.c (pret-rcatafnt values).
t
D (i>clt
-------
7A3l£ 19. ErFECTS OK CO DAYS TCB TRLATKUJT ON URINARY CHLORGUANIDi: EXCKKTION FAKAMtTLJvS
TO> rose Hcr.Xf/
0 573
493
366
4)5
Moan
sun
90 634
505
5: j
.He, in
SEM
125° C18
CU
CJ2
MCTica!.o^
47.2
40.2
46.4
43.1
44.2
+ 1.61
41.4
41.8
43.H
42. 1
+_0.74
36.8
39.4
40.6
39. G
+ 0.53
.T
D
-5.2
-2.3
0.7
l.f.
-1.3
I1-5
-9.7
-4.1
-3.5
-3.8
+ 2 . 0
-6 . )
-5.4
-0.6
-4.4
+ 1.8
14.1 11.;* -2,9 38.6 41.3e 2.7 4/.3 47.5* 0.2
(continued)'
-------
TABU; U (continue i)
7Cfa Ccs« !Vjr.>.cy
ru-'Vi M.V.- S'o.
0 573
4t!3
366
415
Xoar.
StM
90 634
505
5:0
.Ho tin
5J-M
125C C18
C14
C12
He if.
St."
174d 39°
n
5
T
*k
2
T
3
+ 0
4
3
4
4
+ 1
1
3
2
2
1°
3
a
.17
.3a
.58
. 35
.12
.69
.25
.16
.63
.03
.45
.98
.07
.72
.92
.10
.35
c^r/cx;
Tf <-• 3t f !
3.32
2.01
3.18
2.72
2. el
+ 0. 30
2 . 99
2 . 99
3.36
3.11
+ 0.12
3.30
1.99
3.20
2.93
*_0.42
4.26C
,J
-1
-0
0
0
-0
1°
-1
-0
-1
-0
+ 0
0
-1
0
-0
1°
0
L>
.155
.37
.60
.37
.31
.55
.26
.17
.32
.92
.37
.32
.08
.48
.09
.50
.91
U
3.71
2.22
2.07
2.31
2.58-
±0.38
3.07
2.73
4.22
3.34
K>.45
2.54
2.77
2.86
i.72
^0.10
2.74
TBG/CG
2,71
1.98
2.67
2.58
2.49
+ 0.17
3.24
3.16
3.30
3.23
+ 0.04
4.21
2.07
3.67
3.32
+_0.64
3.71C
D
-1.00
-0.24
0.60
0.27
-0.09
+_0.35
0.17
0.43
-0.02
-0.11
+ 0.41
1.67
-0.70
0.81
0.59
+ 0.69
0.97
n
1.40
1.07
1.25
1.02
1.19
+,0.09
1.38
1.16
1.11
1.22
+ O.OB
1.17
1.11
0.953
1.08
+0.06
1.22
CGT/i Ji;:;
1.22
1.01
1.20
1.06
1.12
+0.050
0.924
0.946
1.02
0.900
+ O.CV-)
0.784
0,965
0.871
0.870
+_0.050
i.M-
r •,
-0.1HO
-0.060
-0.050
0.040
-o.oto
+O.C50
-0.456
-0.214
-0.090
-0.250
+0.110
-o. :<«(>
-0.145
-0.082
-0.204
+0.090
-0.080
B « iUsiel:r.o (|.: etre,it merit valucn) .
D (Delta! • 30 djy trcJtc 5'V.iluot. r.ir.us bJi.clir.CK.
^
Cr,c ncrj.cy died t«forc tt.c first CC, teat.
d
A thlrJ Wir.kcy frcsi this group cUed t* first And second CG tcstSi
^2
Kcr.k«y J9 h.^j teen withdrawr. frctr. TCB treatncnt for over 1 month prior to this CG test.
-------
7A3IX 20. in~l.CT3 OF 90 DAYS TCB TREATMENT ON URINARY CHLORC-UAWIDE EXCRETION PARAMETERS
.
TCB I»s«
r^-'V-T/J^
125°
MorJwy
Ko.
C18
C14
C12
Mean
SE.M
Icrcent OG
B°
15.4
14.6
15.2
15.1
*0.24
Tre.U*-;!
6.41
18.4
12.7
12.5
*3 46
Db
-8.99
3.80
-2.50
-2.56
+ 3.t9
Percent PBG
n
39.0
40.5
43.4
41.0
+ 1.29
Treated
55.6
43.8
47.0
48.8
+ 3.52
D
16.6
3.3
3.6
7.8
+4.4
Percent CX.T
B
45.7
44.8
41.4
44.0
+1.31
Treated
37.9
37.7
40.3
38.6
+ 0.84
D
-7.8
-7.1
-1.1
-5.3
+2.1
(continued)
-------
7A3U: 20 (continued)
TCii COk
2-
3.
t
4 .
2.
+0.
9->
07
72
32
10
CC.
Tr
5
2
3
3
-l
T/CG
.92
.05
.18
.72
.15
D*>
2.
-1.
0.
0.
I1-
94
02
46
79
16
&
2.54
2.77
2 . H6
2.72
±C.10
ruc/cr.
Treated
6
2
3
4
+_!
.6fl
.30
.71
.92
.92
D
6.
-0.
0.
2.
-*'
14
39
85
20
00
b
1.17
1.11
0.953
1.08
+ 0.06
a.
Trc
0.
0.
0.
0.
i°-
T/I i.u;
ated
662
Hf>0
BS9
600
060
D
-0.4(39
-0.250
-0.0')4
-0.283
+0.110
B » Bds*;li.-:e (pretteatncr.c v
D (C
-------
FIGURE 2
£->"£Cf Of 30 DAYSTC8 PRETPEfMLf, ON URINARY CHLDRGUAN/DE EXCRETION PARAMETERS
( !C8 tree.'** -f<,- ±crr\porcd lo vehicle healed con'.rois)
% CG
W" _
40
30
»P« UUD
»*p
-------
FI'uFI 3
EFFECT CF 20 WyS TCO WE TREATMENT ON UPlf^PY CHLORGUAMDE EXCRETION PARAMETERS
( 7C3 frccfetf *&V compared fo confro/ "A'iT *^C'C A* pos'-'reofmerrf vo/ues minus pre»feofmcnf bose/tnci J
A % CG
T
*p« 005
OOi
A%PBG
to
.[p ?>
93
174 <•
A(CGT/CG)
-I
1
o—d11-
O 90 Kb 174 ported
TCB
WOup*
A (PBG/CG)
rce
A % CGT
0 90 125 J74
pooled
TCB
T
-4
-6
-eu
j
A(CGTXPBG)
o 90 i;
pooled
,74 JS
90 125 174 pootd
^
-Ol
•0.2
-03
L.J*
-------
F1GUKE H
EFFECf Of 20 W*-S TC8 PPCTi'CATMCHT OH UK'iARY OlORGUAMDE EXCKET/CW W?AMET£RS
fo ?httf own
%PBG
3v
4Ci
^
?f -A
!•» 1
t
*
^
tl
J
£> SKJ
„,. CGT/CG
40-
20
*—
IOJ-
;
ir*,
'
o
T
rf
1
^
s
i
i
«.3~
*»p« 00!
*»*p« OCOi
40
30
20
•«•
• f
II
I
]
10
._
r*1*
- ^rt
h-
L
-J'
*
^
*
in
-i
*
f
f-
*
*
#
AT
-
-
""
1
t
*J
.T.
*
T
1]
*
^
fj
*J
*
J1
*
*
*
1^5 1/4 t'i"**j O *X) '25 iM p<-oied 0 9O I«S 1/4 pnoird
!tfi tea ir.u
CGT/PBG
.0 PBG/CG T I0
t»0p j Iv)
rf
ph
I T
T » 40^ rH
i
r
*
._
30
20
10
!
^
i
!
I
^i
rf
*
p
F
j
*
1
1
m*
rf
*
* 40
30
20
10
-
-
-
-
t
ll
ft
I]
*
J]
T
Tl
T
Tl*
^
^
h*
*
*
»"i iJ4 f«;«a U 90 u;i >M pro'»i3 0 SO U'i i?4 poo td
TCB
-------
SECTIC-M >
iE" C,S TRICHLCRCBENZEKE
I. IN VITRO ENZYME ASSAYS
Preparation of Mi crosc .?»..•> 1 Susp-ensior.s
Liver Sir-tie* were taken directly frors liquid nitrogen ar.d placed ir. a
large be.3>.er .->•;•. taming XCl-Tns buffer (0.15 M ar.d . ^'j n . respectively, at
; h ~.~> -.-plea ar.d buffer were continually nixed inside larqe tweakers
vh _ .ere placed in a large volun* of water to help thaw the sarnies within
.; to 30 rr.ir.utes. This process had to be performed rapidly ar.d consistently
since thawing tine will affect nixed function oxidase (KFO) stability. All
subsequent procedures (until incubation) were carried out at 0-5°C.
After sar-ples v>ere thawed, blotted dry and carefully weighed, 3 Rls of
ice cold KCl-Tris buffer (as above) were added per qraa of liver. A "Tis-
suizer" (TeXrvar Inc.) was used to blend buffer and liver and the resulting
hoooqenate was spun dcvn at 10, COO x g. for 20 sunutes in & refriqerated
Scrvail KC-2B centrifuge. "his process was recited on the supernatant
whereujon 10 r-.la of the second sup«rn\tant were placed in ultraeentr xf uqe
tube* and spun at ICC, 000 x q. for t>0 mnutes in a refrigerated tfeck.»an
Spinco .vodel L^-t>'iB preparative ult racentri f uae . Su; r in v:tri- Kf'O tests
,>Md .v.ad to b» u«c>5 within ars. Ir, th« carbon ronoxi^'.e rirJing
studies for the >;-jant i t.ttxon and oh.tr.icTsrif atlcn of KfO, phcs;:h.ito buffer
v.ia substxtatud for fCl-'Iris buffer. I rotom concentration* of tnese cicro-
suspensions wwre rt-asurpd by the* rethcxl of Lowry *t al. ( "> } as
by Killer *_t jj_. ( a ).
In Vitro HtX> Testa
KFO cn2yn.it tc activity (P-44C and P-4^C) wa» quantitateJ t_n_ vt^ro usinq
fcur mubstrAtes wMc-h wi»re ir.cvX.ttr«?rar,st icnn *r,.i «?ss«>n-
t»al cr-f actcr 3 . Asitnot-yr »(•.« »r.,.i i^n-'-hrt _>-sr.r f.'--:irr«?thy lat lot. r.itcs were
a'>9.iyc«i by p«?.»sur ir. . T-- xazol,rnSr.» hydrorj'lat xon activit,'
waa i!et*imr.«"d fcy r^.r.it or in ; d i»,ij p«*r .inrj? r,f parent (11!. Th« (reduction
<,{ p-anini^phifiol v^s -j-iAtit it .itcJ in • Ivn ,^-iline hydroxyl.^ais reaction *ft#r
tr.« r«tthc«.J« of C -.latino «-?. -•> 1 . £1.^.15). '• t P 1 : slnary »tu fc^ro con "uctod
with pr>r-k*y livor ricro K<.?W a to initurv lir.Bj»rity of tho roactlcn r.>t«>» of
4li fc-ur sul^ttat«!» ovr>r ?. lie in.v.b^f.lon j«tlods. Thus, klfi«sti«:« v-nre B.itur-
at^j arul V.I o v.-i* niable t^,rr^^'7^.f1ut thn it-.', ufr* tic-ton. ^«'^ction r.it»» *«pr«
tFportcd *-st?cr as r.M -,-r R-jimf r nitt i-r>r .vot t«»<5 {-or an liver t-«r tainu»« or an
f.M. i«r r- ; mf ir>s>r4Ml prn'oin p«r rihijtc, Aba^n t inns w«r« c1«t«r»inei1 on *
i pr* tn-f ir.r.r '>'?*> nt w.»« «l?.o
•„*•:.. I «,o <(«.•«*-,« s t. at« KK'' by i-.irl^ n fw>rfOKt-ie Mn;!lr;
-------
In Table 21 we report t.".«* results of the i_n v x t r o assays using four
substrates, -isir.opyr i.-.e (Ai>). t-er.rpheta:Mre (BZ) , zcxazci Arune (~X) ar.d ani-
line (AJL) . The first two substrates are prir.&rily N-deaethylated while the
last two are rrinarily hy-ircxylated. Inspection cf the data in Table 21
shows t-hat t.-.ere were sivjnifica.it increases in activity against all four
substrates. for Af- the increases following 90 and 125 ng/kg TCB doses
averaged J02 and 2sfc%; for bZ. 331 ar.d 302%; for ZX. 356 ar.d 310%; ar.d for
AL, 3£4 ar.d 234%. Although the averages for the scnkeys on the 174 &g/k-}
dose were elevated, the data are ccrplicatcd fcy overwhelming tcxicity of the
cossycund at this dcse level requiring cessation of treacner.t. Thus, th*
jxjssxble ir. •. :r itirg or potentiating effects of acute weight, loss ar.d reduced
fcod intake %-:.ot be evaluated frees the data ir. this jrirt of the experirsnt.
If or.* ar. a; >'?es the data using a cne-way analysis of variance (ANG\fA)
af.d elir.ir.ates hi^n dos« r»or.Jcey« *39 and #343 froa the analysis ve obtain
the data ir. Table 22. The "overall" F coepanng TCB-treated groups to
vehicle-treated controls has a f of 0.003 to 0.01. Evaluation of th<» con-
trasts indicated in the table shov that th« ?0 Bq/Vg and 125 svq/kg doses are
accotrpanied by significant increases in activity against all four substrates.
The 174 ng/Tc(j dose also produces increases in all the associated enzym
activities but the induction it. net as great probably b«caus« the dos« also
produces both hepatic ar.d general toxicity.
Sir.ilar results were obtained when the data are expressed on tne
of r.sx>l/Pir./p<| ra c ro SCJEW 1 srotein. T?» data ar« not quite as consistent
Sdjta not included for brevity) .ma oii« ext/lanation of this nay b« the var-
iability we ct»«rv«d with th«s Lowry ricro»<«'-al protein procedure.
Tata on the ht-j-.atic >U O concent rat jor.s pr*i*f-.t in «?*--^i contiol and
treated liver, based or. CO bin ling stu«Jt«s«, are ahov» In T.tM« *.}. lh« ilat*
arm {resented A* rjnols/grA* of liver ar.d r.nol* KJ'C/rsq S5lcro»f!c.al protein and
the corr««jx>r.-l»r,<7 i-eak location of th« KF<"> raxiem in rjs *re also listed.
Ct.p c.in sen that tl* fcak location* for th« TCB-tr»»«t«d ar.Jrala w«i« indik-
tir.guish,xble Jri-a th« contruls but th« activity in njRola wjs always ircr«*zed
(«xco[)t for tr.c toxic 1?4 rKj/Tcfi anirula) and this was nost {>rc««incnt in ty.e
gr liver figures (Table 22).
v« h.tv» r.»d« s<>n* correlations (as *catt«r
t>,« v.trio.js ehlorcxrujr. id* urir,,»ty «xcr»tion i ararwtcrs aurh at %IHG. O.
etc., an.! rf*» various )n virr,; «»tim,»r«n of ni'O activity usir,-; t*ic c!af*
obtained with .irir.c; yr irv , i«>r.ri •hetar.xt.e- , Kv^xaycl.vaine and .tr.ilin*.
of t?-oa« ccr rci A? .if n» a.» jTetontod in iigui^n S to 7. 7^0 fi'jurcs w«r»
rui;tf«;! i-;ir.>j t.v«s voiwrs ctt ASr.eti for nil treated ar.isjil's on which w«
in vivo c» **c r«>t lor. r.»rjl |jitt<>rn8 vfir.'i nil thrro Bjb»tr^t«»s j rr>»f>r. tr J. With--
th«i» in A !-ot»ti%'«? cvrro lotion i«^WT>«n urir-ary »1! M*>.-', CtiT v,*lu
-------
r^:,,.,,,,.,^ uv,,,
TCI* less K:r.fc«y
r-jAj/dv/ flo.
0 D7)
4BJ
366
49i
93 634
'03
no
-^
125 CIS
c:4
C12
253
174 ;r»7
39*
343
276
r.r ir.cj/yr ir.c
r<-rr t hj-l »:;c
76.6
53.2
55.5
54.4
199
165
192
168
192
125
173
195
142
73. a
76.6
64.4
txrr. r; ^t.x.T.lr:«
155
120
131
123
-62
376
5J5
363
435
2C2
393
508
375
158
liiO
256
! Zox.izolarr.ir.e
llyiiroxyloro
32.4
6.90
19.8
19.0
67.2
60.3
96.4
53.4
65.2
49.4
55.4
76.1
74.1
17.8
19.3
29.2
Anilii.e
14.2
11.4
11.7
12.0
31.0
35.0
61.8
50.5
35.0
19.9
31.4
59.4
42.4
12.1
-
40.9
This &• r.xoy h,v.1 \+<>n witKiravri fror, treatncnt. for over 1 ronth prior to r,.icr i! ice.
-------
SIG.'iU :CA.';CT l£Vs.i-S {!') CGKTAKINC; £N VITRO MKO ACTIVITY AM'.'NG CONTKOL
AND TCB TK.A"LD cro'.:*:i*
(SW:<:f,.; L-^S/KINCTE/CRAM MVCH)
,11.-.-. .1 - . -.
Corp a r i so n
•overall" F
Cor, irol vs. i 'co lei
Do sc • PC s- ; or. s c
Curvilir.carity
Cor.'; re In vs. 'JO's
Cc.n-rois vs. 12S'»
Cor.trols va. 174's
Arir.oj yr ir.c
p " O.CC03
0 C " 0 ^
0.0079
0.135S
0.0001
O.C002
0.0:>5S
licr.rj;het.ur,ir.e
0.0008
0 0"02
0.0872
O.CC45
O.OC02
0.0005
0.0184
Zoxazolanino
MydroxylaKc
O.OOUO
n on??
0.2 560
0.9274
0.0018
0.0054
0.0514
An! lir.c
Hyflroxy In'.v
0.0130
o on? \
0.7047
0.7C57
o. oojrjon capable of contrast
-------
TA3U. ;:i. IN VI7K) HKTATIC
7C3 :,06C Kcr.ir.cy
0 573
493
3CO
4«
93 634
505
110
500
125 CIS
C14
C12
2S3
174 287
3'>*
343
276
Ar ir.oj •/*»«"•«
!>•:•«• '..hyl.v P
2.18
1.87
1.61
1.39
4.02
J.69
4.81
4.17
3.77
2.92
3.75
4.6'J
3.18
2.C2
2.89
1.75
thrift hylii' e
4.42
4.21
3.80
3.14
') . 3 .1
8.18
13.4
9.50
8.53
6.14
6.54
12.2
8.40
4.32
6.79
6.99
PI'OTUIN)
Zox.izolamino
Hy.Jroxyl xae
0.92
0.24
0.57
0.48
1.36
1.31
i.42
1.32
1.28
1.16
1.20
1.82
1.66
0.49
0.73
o.eo
Anil ir.c
0.36
0.40
0.34
0.26
0.64
0.76
1.56
1.08
0.69
0.49
0.68
1.16
O.H7
0.33
-
0.77
his ssorJtcy had been vi*hrir
-------
4. sir.rjirzcANct u;vx:^ ID WHICH COMI-AKT. IN VITKO wo ACTIVITY CK rcu
TH-AIt-D .«.. •:;>•!.Vi 71; CONTROLS
Aair.-.; y: ir.c lx-n/j>het anino
** '~» **•*«• _ ,*• '• ~L \ ,\ r •-, t ^
..-•erJii 1 J' ^ • >• — •* u.U'Oj
7CB Trc.itcJ Crou;.a 0.0007 0.0014
&-se-;-*:,;-or;^c O.:'0'}5 0.1742
C.rvil ir.pdrity 0.2633 0.9714
Control:* vu. O0'» O.CC-02 0.0010
Cor. « re, is* vs. 125's 0.0008 O.OC42
Ccr.troU v*. 174's 0.2106 0.0437
ii
-------
V"!
:.I.iL ..-..:_. L.
:EL'7L_ :'!T.1-: cr.:.,
E
»
o
a
E
r * 0.772
p < 0.001
jC
f
0
E
a
r =-0.625
p< 0.02
% RGB
A CGT
x
**
I
Q
a
E
r --0.493
p< 0.1
0
E
0
o
r s-0.673
p < 0.01
% CGT
4(CGT/PBG)
0
E
0
O
a
E
r s-0.724
p < 0.005
o
E
0
o
a
E
CGT/PBG
r * 0.736
p< 0.002
APBG - &CGT
0
E
0
a
a
E
r * 0 696
p< 0.005
0
E
0
o
ex
E
r "-0.635
p<002
PGB
PBG "ACGT
-------
'i.'.L .'i-JL £11.'; LACE. AC II. 'I TV (.',„ ;,.'/:.",
C^
.
..l C.'.i_. .-^,.».i.L L.. .i .1. , . ;
£
•*
0
E
O
00
r = 0.741
p< 0.002
PBG
I
o
O
e
CD
r =-0.582
p< 0.05
CGT
E
0
O
c
o
0
r =-O.435
P N.S.
% CGT
£.
•*
0
E
e
CO
r =-0.646
p<0.01
AtCGT/PBG)
0
E
0
CO
r =-0.681
p < 0.01
CGT/P8G
0
0
O
N
0
00
r * 0.686
p < 0.005
A PBG ACGT
0
O
0
00
r « 0 651
p < 0 01
A PBG
0
0
O
r =-0 686
p<0.05
A PBG "&CGT
-------
C.~£L'.7L; ..'ITH El-lil
- .•...< '...vi
x
o
rsi
662
x
O
% PBG
rs-o.632
p<0.02
ACGT
M
O
r r.-O.358
p N.S.
% CGT
K
O
r =-0.682
P<0.01
A (CGT/PBG)
|
o
r*-0.594
p< 0.02
CGT/PBG
•o
>.
X
x
o
r *0.875
p<0.01
PBG - A CGT
x
o
r =0 696
p<0.02
U PBG
x
o
IS*
r — 0.530
P<005
PBG « L CGT
-------
TABLE 25. KFO COJJCUrrRATlONS AliD PEAK LOCATIONS FRCM
cx/:JTftOL AND TCB TREATED MONKEYS
Treatment
Eaulphor
Vehicle
90 tag/kg
TCB
125 (ogAq
TCB
174 BKj/Xg
TCB
Monkey
No.
573
483
366
495
634
505
110
500
CIS
C14
C12
253*
287*
39*
343*
276
r.M yTO
g liver
25.5
18.0
29.9
24.2
56.3
60.0
61.0
40.2
54.9
48.8
48.8
-
-
24.2
13.8
34.7
nM MFC
rn
451.0 -
451.5 -
451.0 -
451.0 -
451.0 -
451.0 -
451.0 -
451.0 -
451.0 -
451.0 -
451.0 -
-
_
451.0 -
451.0 -
451.5 -
-------
indicated "f" ar.d "j * values wh.i.::: w*r« calculated us 11.1.3 st.Aii.iari pie-*dur«t»,
it is ir.triguiny to reccgr.ize that t'.ese ccrre. itic:-.* are ail ir. "he dir«?c-
ticn we wculd h*ve ar.tici; atei bas.«ri on tr.e hy%<. thesis t.-. j.-. increase ir.
r.epatic I'J-'C activity ct t.';e P--4t>0 *_•/:« s:,r-uld ir. crease Ptv_, «'xcr«-* : or. <«r.-J
reduce CCT excretirr. ar.ci possibly C "."- as well.
These data have also beer <»v .tiuited rcr-.p-araaaetrieally using the « i 1 c
Signea RarJc. and the Wilcoxo.--. KirJc Sur: Tests ( IS!. Mthcuqh th« charts
sho*.ir;".e treatment wit^i TC'S
and chin^es ir. the -rir.ary excretlcr. ;. jtterr.s of C-1 netabolxtcs. "r.ese
ar.alysas car. be kjicie ivjiiaiile if desired. Tr.es* results in conjunction vit
the ANCVA analyses sh-cvr. creviccaly i .:rr--ort the ccnclusion that daily sdaur,-
istraticn of 7CT to norJceys in doses cf 90 to 174 nq/k.-T for 2 or 3 r\cr.ths
causes a definite iruiucticri of hepatic MrO activity and this ir.creased a-ti-
vity can t« rather accurately predicted usir.g ti;e new in vivo OS excretion
procedure descried in this j
II. EXTRACTION OK TCB KETABC'LJTES FFC*4 WIT URINE
Prellair.ary Sata
A pooled ;4-h<^'jr treatment urir.* sAi^plc ccntaining 1 N HC1 was refluxed
tor one hour. Thi.. hydrolyred urine p-ool was then buffered to pH 2 with
0.4 M phosphate And 1 tsl aliquot* w«>rc extracted with 10 r,l of various or-
qAnic solvents* listed in Table ."*7. A duplicate srt of aliquot* was ru.'i which.
in adj'tion rontaii»ed 1 «<"*» o' (KH.i^SO .
i . 4
As shown in l.thle 21 a.'.vlition cf » »' rtliyl h*xar.ol > ETXT » Kt)!»«x >
r.CC • ethyl nthur * CKCl j > L«r,ror,« • r?lo»T.c > CC14 > s>«troleur» «thcr >
cyclch«x.tne. At ;.-r«scnt th« hast *oi*.vr.^s ,d Tea r«-r ju.>ol» tcs R'jch as I'-^nol* and dlhy-iro-
diols And c.tt«chol8. Th«»c wore prr-to£ly present in th« original urir*1
Darples a* conjugates.
W» also carried out c-hror-sT.o<;r«r.v.ic studio* cf hydrolyrrd urine *"\ !
urine extracts u:»lr.j both TLC .ir.d pA}«»t chior.atcir.iphy. Th* ar.tir ij.---»te J TCB
r*. t ,»t--ol i teis wt?rc : ^ , j , S-t 1 1 r;ii-7vri, ^ , 4 . S- tr i chloroph*nol
(.' ,i,S-T'.-p) , J, 3.«.-trlchlr fophenol i . . ' .1--T-,"! ! . ,v -J i .4 ,'.-»,r IchJoiothtcph.onoi
{* ,4,i-TCT«') . ';h*v could tx? v. -; ;al : ,-n-i u-iir.} d: arot iicul p-ni t roar.i 1 Ire
di.izotii<"d sulfanllic acid .v.rl »vo ' .' c •-••- ".'*>> . -vn in T.xtle *8.
-------
TABLE 27. LXTKACTION CF C-1ABELTD METJBCLITES OF
7?:CHLCF.CbEN;:i:.VE IN HYDfCLYCEO RAT VRISE
BY VA.'.IO','S CJCANIC SCLVTNTS
EFFECT CF AICED [hH,),SO,
t . 4
Solvent Vooled
(10 tl) Urir.e
n-assyl alcohol 1 rsl
2-ethylhexanol
i'DC * 2 etr.ylhexar.ol (8:2)
ethylen* dich.ond* (EtXT)
ethyl ether
OfCl 3
^r,r.r,*
to lucre
CCl .
«»
^t.ol..^ .0,-r
cy c lo^vc xAf(<4 "*
% 14c
No Salt
67.7
77.8
64.5
W.7
45.'.
js.:
26.5
23.1
1 S . t>
«. , H
9. J
Extracted
INH4),S04
90.9
62.6
73.8
S3. 9
49.9
3>.<;
2 3 . i<
27. a
19.5
10.;
10.0
-------
TABLE 26. CHRCMOCENIC Ki'ACi'
IL . . .1
Star.
2,3,
2,3,
2,3.
2,4,
iard
5-TCP
5-TC°
t-rce
5-TCTF
: - r. 1 1 r car. 1 1 ir.e
Crar.-^e
Violet
Magenta
Yellow
* vl*dl"1*^c AC * d
Cran«je
Violet
Yellow
Yellow
The solvent systems employed were (A) benzene - acetic acid - water (1:1:2;
v/v/v) Ar.a IB) N-hutanol - ethar.ol - 1.5 S dzsaonium acetate (40:11:19;
v/v/v). Ttve Hf values for the postulated metabolites in the two solvant sye-
using either cellulose or silica 0.69
,4.5-TCTf O.'l
Silic*
Ce 1
0.2')
0.29
0.23
3 , t' f>
i'olvpr.t Systc» H
Silica
C>Hul<.»« '.>!
0.,* 0.6)
0.71 O.C6
O.BQ O.C1
O.HJ 0.70
An interfering red t.tck'irouf.d in &olver.t -y-tfa B cot>)J r>« r«?r>over«yin<) with 1 N !!,:.O »«»\l>or,»l r-ir,ut«8 a£t«t '-'^ ^ spraying.
fr>-)«r. N>th the TIX .tnJ j .t(x>r chr< pwit ot« not
sr. H<. ^"^d the f'rp&«fr.c* of .tt Ic.iKt two r»»t Jl^Jlilc* {ur ciasfs tj
tnet*i>o!ii«e j wfucrt chroPJito'jr.iij'h it»f>rav h«d on cclluloa* f-latc* in nolvor'.t
&>'ftt0tr. A (r.or.j'oiar ) »f>r,w«d con»i Jor juLIc r»'Uo*cl ivit y at tf,« uil-un, 7T.i*
•t"tt«'Jty co-^id r.ot l/« '^iucutoftlde or nuJfat* con }u )
-------
had been acid hydrolyz«-J 1 ?r or.e ho>_:r. ""h^reicre . it sj-j:0.
TABU: 30. GC FXTEJ.Ti.ON TIKES
corresponding
Standard K~ (rin.) fron ether extract
2.3.5-TCP 4.09 y*>«
2.4.5-TCP 4.S8 yes
2,3,6-TCP 5.20 no
2.4.5-TCTP H.Oi yes
tcd ether extract* (!£>-«) of ur.hydiolyzed ui ir.« i'r-«Kl
to 2.J.S- ,u-,d 2.4.5-1XT. No f« j « iiK %.ith an identic*! r*t«ntlor> tirse .!• th« 2,4.5-
TCTr w«* alsio ol--»*rvw.1 j f-,c»™?vtir. thi* ««• ur.«kfi«cte'i from ur.hydroi yted urir.e,
bittc* thlojh«riOl ic net Afc«l it«>» ar« usually ccfl»id*r»d tn t*» 'if'jra i.«* ion pro-
ducts frop» r«rc.icturat»« after (.y.irolysi* und«r »cror.<;ly aclilic or La*ic
rfflux conditions. :t was surm6»d th.»t hi'jh tnl*t jx^rt t«»rs}-*r»tur« tculd
bo dcqradini r«rcaptur*te which had t*<»n «xtrat:t«d into wth«r (Th«»« ccnlu-
arc slowly extractabl* ir.to ether). When the inlet j-ort tprj;-et aturc
^vi^d frcn 300°C to 13J°C, th* rrourtpttv* thiov^nol i-«>*k dinao^arctl
t »uc(-h«r.ol j*** K*» still visible at the a.\ra* hr. 7h« lr-i«t
r» <'f 1 3./-T stsll left the two f«4k' ccrr««;>rT.,Jir.<7 to tr«*
r.ed ih^tioln ir.t.v.-t *r.d at th^ir rp«.}-«Jct iv* K»'a, Jt i«. ^.V.«r»*
Joto, tontativw'.y tv r.clu i«-»l ly rt«..' KA
<*>e ml *ach of 0,1,2. ard 4 hour ;>1»BS&A «a)K|>i«» fro» * i*':«r.k*y r«"C*>ivirn
-------
I:- »xj/*3 roi was added sic-wly t_ 4 r.i cf.ir-% ptrc.il one- acid ifC-ft) and
~_ixe,i rapidly cr: a vortex to .-.-.sure i.rfe .- ;;. iratior. cf all prcteins. 7 he
j- CA-precipxtatei plasra wia extracted vitr. i ?-.! hexar.e 3 tiroes. I'ach hex-
i:.* extract was analyzed ;'c-r total riiioact ivity ej-;d t-'^e percent *"*C ex-
tracttd was calculated. iollcwir.g t:.e third r.exar.e extract, the plasri4/*CA
residue was extracted 3 tir*s with S ni ^-orticr.s ot" ethyl ether.
After .drying ov«r A.'.r.vircjs Ka^Sc^, the hexar.e extracts were an*lyr«d
by .'C-£C J!.'3.,--i*tector> cr. a fc' K 2 •m, i.d. coli?sn tacked with 1CO-120
nesh Verjport>> coated wit". 3% SE-31; carrier cas , !;> at 30 el/∈ colunn
terrier ature lOC'-'C; iroection port, 3JO"C; detector over, 2'>00C; sensitivity
2 x iJ~*J ar^js/ev.
P^sul.ts
Recovery fc^sed on ra-Hojctivity analysis of the hexar.* and ether ex-
tracts is shown in 7ai>le 31.
TASIX 31. fiCC-.TfV (»• KAriCACTIVITY KJOM
v .-r.w.y riAsxA is IEBCEXT
7
ine
'; hr
1
i
4
; fXAT.e
Sclui>le
52.5
41.',
n.*.
j - - i n im
i t;-.er 'lotal
Jciufcl* Exrra.-ted
46 100. S
V3.S M>.0
-Kr- »» of th* fir&t h*x-*r,« nxtractai revealed only cr.e [^?Ak r.ot present
in 5 arallel extracts of j'rrtr*atp*nt (•l.is-rj. '.'.'u» i-e»k h»i1 th* s,\rw ret^n-
tiv«i ti»o «* i ,»',4-tt ichlcrv b«-nrcn« (TCBl . Attprj'.s to rd*r't»hl» rwteridi t^y GLC «rt?re not «ucc«»sfui .tnd further research
in ".his area was no? carried out.
tv. INHIBIT; --N w;r> VvTr.NTixTioN cr nu; I-'J-TAT-^TOXICITY cr I.J.A
TT.e f ."-I loving study V3=> un.Jert .tk. er, to cordate t r
-------
(ir. corn oil) .
TASLE 22. Tr^AT^-TNT PROTOCOL
i retreat-ner.t
Gioui Pretre.itrier.t 2fj'sc per day
1 H,0 2 r^lAg
2 PB in H.,0 75 »gAg
3 Corn Oil 2 rJ.Ag
4 PCS in Corn Cil 50 sngAg
5 3-MC in Corn Cil 20 mqA?
So. Days
Pretreataent
4
4
5
5
2
TCB was administered 24 hours after th« last pretreataent and the rat*
vere sacrificed six days liter for histoloqlcal examination. Aniaals were
v«ighed daily throughout the entire *xp«riner.t . The two hiqh dose (1000
r*j/Kg) I'CS-pretreated r.its c!ieJ withxn ;-4 hours after TCB aduinistration .
^'ne low cose TCB rat ir.d cr.« hi;jh dose 3-HC- induced rat died four ar.d three
days after TCB adwir.ist-Jtion . respectively. All other r«ts survsvert t;.«
i«riod of observation. i'C.M uutl 5-MC pretreated rats lost wore weiqht than
either li^O or corr. oil f retreated cciitrol r^is. i her.obariutAl («'l5>«ctlve controls.
All rat* vhith «urviv«.1 h,i,J -jxir.il vxsi-jht loss at days three or four ar.cl
then lt>'Mn to .ination indicated that the post »<*vere
liver cKjraae occurred in '.-.cse ijioups which lost t'iia no«t weight. IB-troated
rats chov«,i 1«8» liver d.w^n;* than th* H,C~t»«ated controls.
th-»t JB pret »o<«tn*nt protected aqatn«t TCB
city Is txir.tr ary to th« fir.'S>nqs cf F0 ; .1 «rt a 1 . ( It ) vho found that PB-
iriJuction rotentl atpcl the •oxit-jt.y of low«-r halmx-nated conxjurds fluoro-,
c-yiloro-. brcno-, sr.-ia-, i . ." . ii chl. c- - . and 1 , 3-d ichloroberscrse . Totentlation
of 7CB tsjxlftty t;y 5-VC j-t ct r»>.»rnent in pyr »?«.<>• i« al«o fJusctly c}-;-osits
to the c bscrvat lc*-, s cf ; r.r.ey , .Miil«>r ,->.d Killer ( 17) or. f hlorpt*n«cne And
,:ollo»t, r.as^-a^l ior.o AJU! -.,1 1 lrtt»- It-) for brcrolwn:«>n« . These lr.v*»t i<)«-
tora found that J-KC lr:iu-.;t:c«i t-.^.; « protective effect on the hepatot.oxicity
(•f chloto- or btonot>«jriz«tn«.
ihenob.arbit.il
stntle ot.ll .Jos«» of TtB.
J-es^thylchoJ *nthr*n« iricr«4««J th« toitlctty
inn» on tho histolrwjy Of lh« livorn of
-------
the vari'-us groups suggested that the tasis lor the inhibitor a (FB) or
pctentiat i-r. (3-MC) «as pro&auiy relat^j to the changes in hepatctoxicity.
~jr;ccxiciTY AND KUTAGKNICITY CF res KLTABCLITES
Xith the assistir.ee, of Kr. Steve Glasser ir. Dr, John C. Leper's labor-
atory w« have exar.ir.ed the cytetoxieity And tutagenicity of son* of the knovn
or jxjtential nfetabolites of 1,2 .^-tri^i-.lorobenrer.e (7CB) using two of the
sensitive Aries Salnor.eIIa t_yj:hiaur 1 ua tester strains TA98 and TA100 ( 20 ) .
A dose-respor.se cvvrve %as camec out for each ccrpound both with and without
idded liver Bicrosor«s (S9) derived frca rats treated optiBilly with FCB's.
The data obtained fro» delicate plates are summarized ir; Table 33. It
is obvious that although the conpouiids are generally toxic in the highest
concentrations used no increase in nutation rate was obtained with non-toxic
concentrations. Also. TCE, the parent compound, had no outager.ic activity
with or without added liver nicrosoaes based on data obtained previously in
2r. Leper's laboratory.
The data in Table 34 are presented only to show that the tester strains
are sensitive to the three known irjta jers, Mthylnethar.a sulfor.ate, nitro-
quir.olir.e N-oxide and 2-aBunoai-.thracer.e.
We aust conclude that TCB cr its kncwr-. or jxjter.tial phenolic mtatolltes
tre lractiv« as rutageni;. t'urthenaor*. ccrj:ir.dt ion of TCB ,».rici active liver
"icios;or*» bailed to f rex!we
-------
7ABLE 33. TCXICITY {T) Ck ACTIVITY CF TCB KE7ABGU7ES
in CKSO
:o i .01
733 T1CO 798 TlOO 793 710?
2.3,4 Tnchlorophenol 7T __ __ __
2.3,5 Trichlorcphenol 7T -- -- - -
2,3,6 TrichlorcphenoX 7T 7- -- --
2,4,5 7richlorophenol 7T -- -- _.
2.4.C Trichlorophenol 7T -- -- - -
3,4,5 7richlorophervol 7T -- - - - -
2,4,5 7richlorothiopher.ol T- -- -- --
2.4 Dichlorcphenol «_T - *7 - «_7 -
2,5 Dichlorc5>h«nol. 7T -- -- --
2.S rs.chlorchydrtx:aincr.e 77 77 77 -7
7 • toxic
- «» riO ir.cro«s« in rutatxon iat« over th« Bpont*n*ou» control i*t«. Pr«»«nc«
or Atn«r.?« of s / fraction h.»di r;o sitrru i itar.t effect en the rcnuita «nd
therefore le not ir,.Ucate
-------
TABLX 34. CCNTKCL MVrAGEMCITY TKST ~ATA I'SING
AMIS TESTER STRAINS TA98 AKD TAIOO
Cheaical .Vddej -S9* *S9 -59 *S'J
Spontaneous nutrition rat« 23 32 117 115
Methyl nethan* sulfor.ate 1644
Nitroquir.olin* N-oxide 156
2-iair.oanthrac«n« 3079 2114
•Liver aicrosoraes (S-9) froa rat liver induced with fCB.
-------
5LC7ION 6
XTTABCLISK CF BROMOOiaiICf£.«ETHAME IN KATS AND RHESfS f.Ct.'
I. ISTKCDCCTICK
isronodichloreaethane (BDC) is one cf the halo-crgamcs found cortnanly
in potAble water following cr.ionnation < 19). It is an analogue cf
chlorcfcra and very i-\t-.le data are available cor.cerr.ir.g its netabolic fate
in laboratory aninals. This report contains data or. trie pharaacckir.etics
tissue distribution of BDC in rats *r.d rhesus aorJieys.
II. PRELIMINARY FHAMUICCK1KETIC STUDIES ON BRCMODICHLORCHETHANE (BDC) IN
f0 rsj of a:c ar.d i.O ^Ci of labeled cos^xjund t*r r«i.
Tt:9 ir.t ravenous doso was fjiven vi* th« forwrai vein «x;.ose«J un !er light
w«r«
To treat rats orally, a 1:5 dilution of the treatment solution described
aix;v« WAS prepared. The solution cortatr.ed ^.0 B»7/r.l &r,d 0.4 uCi/sl of
tro»odichloro«otn*r.e . The oral done was .'0
ouvod irs stainl*»* «t**l cwt alioli&n ra^jes. Crincs
collected in iced containers until tr.o AriicvAli* w«te killri) no foc«»
excreted.
fd* not«« froo t.h« yB, and iusvjs. Tf.«re
»t" srwill i.ui irjK.rtAnt «iv>ur>t.« in t>v« til t».i.- t ,. »u'M«*t tn
-------
cr.e observes that the peak rcr.cer.tratio.-.s usually occur at three hours but
in biocxl ar.d plasr-a, liver, Xii-'.cy, muscle, oj-.^ Gl tract the opposite hold*
true. Jr.- terns of percent Jcse, t.-.e tat at: counts for rore than 5C» of the
dose recovered in the tissues. Very little -"V is excreted in the urir.e or
ir. t' » i'eces. even if or.e irclu.i«js the cor.te.it5 of the GI tract.
K.-.en BDC was administered to rats orally the data in Table 3€ indicate
at quite different distribution pattern. the cocfjxjur.d was slow to leave the
stcs&ach ar.d concentrations ir. fat were hiqh but lower than whe.i the compound
was administered intravenously. The ^"*C ccr.ter.t of the liver WAS high (pro-
bably because of first pass effects) but wr.en one cos--pares oral with ir.tra-
%-enous administration one sees that the tissues accounted for isuch les*
of tij« close when the coKpour.d is «jiven orally.
III. PHAJy-ACCKISETICS OF &VC IH EKESUS MOKKTYS
Two feiaale rhesus loonXeys were used in a "crcss-ov*r" study of blood
levels, excretion, and tissu* distribution of BDC. Each aorJcey received
sir.qle oral and intravenous dcses of 10 r*?/)cy. !Jo. 3 received the intra-
venous dose initially and an oral dos* 3 days later. Finally after 82 day«
another oral dcse was ac.'r_inistered and 24 hours later the ncnxey was nec-
rcfsied for tissue distribution of BT/C- derived *"*C. Jio. 13 received the
oral dote initially; single intravenous cksses were injected 9 arid 62 day»
later. After 24 hours the tsor.key was r.e crops led *nd tissue levels of **€
\««r« det« mined.
alt ar.d Methods
TT;« intr.tvcr.ous trcatrwnt ftolutlon vas prepared by adclvnij iOO u 1 of
l.ulphor*-et hoir.oi (lil.
v/v) or.d dilutinfj to ^0 ni v»th diet i lie. ,J w.»l«r. Th« resultir.-? solution
contained <>.•> r-j/r,l ar.d 1.34 i-Ci/ril of HDC a« d«t«r»tr.«<1 by replicate
*"N
Thii solution waa diluted !:•> with Inuli hor^^thanol :wator (1:1, -
v/v/v) to prepare the oral tr«»JtPX>nt *o)uti,^n «*hich contained 1.98
and 0.147 yCi/»l of UIXT .is doternnocl >.y replicate
for th« tw> initial treatmer.t* both ncr.ke-ya v<«re tranquil it«»d with
KetAKine liCi (S ir*iA-.>l . Tor the terr.lr.jil ttcatrxjnt* both r»?nkey» were
sir-ply restrained by the anlcal t«chr.ici-an curinq th« tr»at *x?nt proc«1ur«».
r«o. J w«>j'jhinc) 4,'t'i k'j received ^ rl of th« ir,t ra-."«r,o«» tr<*at-
nolution and r«n«ikcy >K>. 13 v*l'.jhin'} 4.b K-j r^ccivfld 4.6 rl of the
intravenous tre.itrwnt R• ri. for t!^e f»n«l intravwnoua tr*»t,p««nt tto. 1J
K wi>)h«in. Thu
fir.*l ir,tt4V*i-,ou» i of tfn» crAl tr«»tncr,t, solu-
tion .1 t~iiril sT.crti-". ir.tr »'}••» «*• r leal ly us»ircj « tut>*r c«th«t«r. Th«> C4ih«ter
f 3
-------
TABLX 35. TlSifi: LISTKIELTION OF 6FCMCCIOilXPC-METHANE
(AS 14C) IN MALE FATS
SINGLE IN7RAVESOCS DOSE 10 MG/KC
Tissue
Blotxi
Masr-a
Liver
Spleen
Kidney
Lur.
H5 " No
4")%
-------
TABLE 36. TISSLT CICTJUBLTION CF
(AS 14C) IK MALE FATS
CRAL DCSE 20 HG/KG
Ferc^r.t. Dose -9/i/
Tissua
blood
Flasiu
Liver
Spleen
Kidney
Lur.qs
Heart
testes
Brain
Fat"
Muscle ••
Skin*
Total Ti*cu*
r-to*^ach
Snail Irtestin*
Otcua
Lar<;« Int«-stir,«
CI Tr»ct I Cejit«r.t»
frln*
3 hours
1.16
-
1.8
O.C1
0.15
0.03
0.02
J.03
0.02
2.e
I. it
0.75
8.6
21.5
1.45
0.31
0 . 14
" J'4
0.06
NS
6 hours
o.sa
-
1.4
0.01
o.ia
0.02
0.01
0.02
0.01
0.71
0.62
0. J6
3.9
35.9
0.67
0.37
0.11
J7.1
KS
3 hours
2.6
2.1
9.0
2.1
4.2
1.5
l.fcO
0.82
0.89
6.9
0.91
0.88
2R4
9.5
3.2
1.9
10.fi
KS
6 hours
1.3
1.9
7.3
1.1
4.7
l.CO
0.97
0.4fc
O.S4
1.8
0.31
C.42
202
t.i
5.0
* • *
11.1
KS
SS « Ho «*rvl«.
T«t * <|u«intii.at*!y wt.
;>kin -
a* 17% bo-iy wt.
-------
w,»s flubl-.ed with 20 r-1 cf H.,0. T*je rvor.Xeys were Reused ir. netabolisa cages
r*iir.tdir;fc j WJL-.^XI; exj-,r the f'.rst a hours. tT.cn tho tloocS level of •'••C falls stuch more
slowly ior the next 40 hours drc^pincj finaiiy to about 25 u7/nl> The values
for ».>. J cr, H-I>-?O seea atr.uirr^lly Kw su«
j >»J'j«»t cf t. :n> *JC iro« »>tr<; t--j;,t be «rx)i*l«'d t-Y the lur.g . The ov«>r-
all siailarity of The bloo<5 2*»vel cur\-os .ir..i t;rir,Aty Ar.d fecal excretion
curve* »utraHon j'layn little role in thin
loss, via the Ju,v;.
T|KR,;P ctistr il/ut ion data in T-Me 40 j'slnt u;> fh» Bmall aftnunta of
*d»ini»t«np?5 ftA; that apt^ar to r.«» n^'fynBtonid in .^ny ttusue. iTi a wj/'
kaeie on« r.ot«» that tho highent concefttrationa after ar, cral do?se w*>re
-------
fc.ur.ci in the bile, recur, and liver su-jge-t ir.-j t.'iat sone ef t:.o " C is
£->.-rettJ via the tile- ^r._i r v/ not i-« entire!'. re,iL^ort>ei. Bile, liver, an.J
teci^s were .'ii-j.'iest iltc Alter intravenous a.-s-.ir.istraticr. ~ut vere r.ct as
.-—gh as t;.e oral v>.^es in these two rscr.f-.eys. . t seezis siie to rcr.rluJe
that less tr.An S% ct -.:.e adr-.ii;: stere- *"*C : rcr i-TC is ;.rc=tnt tr: t.'.e tc-iy
--1 hours alter a si'.-j*e oral or ij-.tr4ver.cas iose c{ 10 r>^ "*•••.
We c^n conclude that although ccnkeys apc^ear to r*tiT'Oliz. E^" slightly
r<,re extensively than !-U=!AT.S eietatoliii chlcro::>rrs t.ne ccspo-jr^ » chlcro-
fcrc is excreted texhaiej) prirani. via v.'^ lur.q. The jreser.c . ca ;.S%
of tr.e ^""C in the uri.-.e su99ests >:iat scsae aet&tolise .1 =IC o_. jrb but
prctaJDiy account? irr r.o rcre th-i-i atout 5% of tr^e c!^*«. :*: dita ate avail-
able to ir.dieate whether tr.e ***C is exhaled as j-arcr.t cowf-oi^n-i or as CO-..
Ir. the case of hurvar.s, About 50% of the expired A JC froa stafcle-Iaiieled
chicrorcra aj^earsd as cr.icrotora and t>0% as •'-'COj.
iv. MUTACExicinr TESI-S
Urir.e SAisf>les frca the 12-24 hr. period foi' both ncrJceys 3 and 13 werr.
filtered as«ptic<«lly ar.d us*»fi for rutagenesis assays u»i-cj the 5alr-phiruri'jryaaaBuallar. ricrcsoaae bacterial assay !stra -.s TAv8 ana 7A1UO)
(-3) ir. t.-.e laboratory of 5r. John Loj^er, Professor of Kicrotiolcqy, t'niv^r-
sity of Cir.ciRT.Ati, College of .Kedicir.e. The f;ltraticr. through Millifcre*'
filters reaoved no radioactivity. The analyses iridicated that theiM urine
SAPi{>les exr.itited no cutas'er.ic activity toward «nh*r ba--t«rial strain.
-------
14
tAsii 3". BLOCO coNCTJrrf'.ATiL'Ki, ./ BDC ;AS c> :r; XC-NXEYS
SIN^'-E ::.7KAVL?«CeS >" 5E CF 1 j WJ/iCG
TIE* lest
Treatiaent.
(.--.ours)
i
*
4
6
a
12
24
4H
3
:-rt -.g/'j
1" 1.8
47 1.6
45 1.1
45 0.7
45 0.9
45 0.5
42 D.4
13
—
42 2.1
41 1.7
42 1.2
41 0.9
41 D.c
42 0.4
41 0.2
1 3R
H.ct . 9
4€ 2
46 1
45 1
4J.5 1
4? 1
44 0
4-
-.
/(i
.1
.6
.5
.1
^ Q
•J
.4
-
>*crif J c*(J at .'4
CF 10
1J
4 4> 2.2 *0 0.4 4«
1, 42 1.? -- C.<, 4fc
H V? O. 'i V» 0. ,t 4<-
32 41 O.'i rt 0.2 4^
;4 V.» 3.', 40 O.I 44*
4« <
>••* t If if Oil At. .4 !>•--!<• 9.
-------
BLOOD CONCENTRATIONS CF SIX (AS *;)C) IN FU1ALE RHESUS f»IYS
SINGLE INTRAVENOUS DOSE 10 MG/KG
30
2.5
2O
C
O
O
IS
to
D 3 8-10-70
O 13 8-19-73
13 11*9-78
12 16 20 24 28 32
Time in Hour*
36
-44
-------
FIGURE 9
£lu:3 CCXENlRATICJiS Of BDC (AS lHC) !>•» i
SINGLE ORAL DOSE 10 K-.
c
30
2.6
20
o
5 V5
• 10
13 8-10-76
3 8-19-76
3 11-9-76
12
16 20 24 28
Tim« in Hour*
-------
TABLE 33. CUMULATIVE EXCFETICH OF BZC CAS '"o IS M'.^JKEYS IN * DOSE
SINGU: i!*T RAVI sees DOSE 10 MG/KG
- _ -_ __
Hours L'rir.e races I" i F Urine i'ec-»s '„' fc F
1 — -- — — — — <0.01 — <0.01
2 -- — -- — — — 0.14 0.02 0.16
4 0.29 — 0.29 0.51 O.C4 0.55 0.29 0.06 0.35
6 -- -- — -- — — C.38 — 0.44
8 -- -- -- -- -- — O.S2 -- 0.5'j
12 C.fc4 -- -2.C4 " -- — 0.67 0.0* 0.9«
24 1.54 0..'0 1.74 2.11 0.15 2.26 •!.?;, 0.48 2.2fi
4a 2.10 -- +.1'i S.j'l O..'l 6.20
•
f-acrific»J *t 24 hour*.
-------
TA3LE 39. CX'yjLATlVE EXCRETION CF BDC {AS C) IK KCSXEYS IN % TCSE
Sir.'GU: ORAL tCSE 10 MG/KG
_ - _.
lTir.« Feces 'J t F Vrir.c Feces U t F frir.e Ffces U t F
1 «- - *'jW*'^*—.*•('"'.*^1
2 -- — — — -- -- 0.04 — 0.04
4 0.10 — 0.10 0.10 0.03 0.13 0.23 — 0.23
6 — -- — 0.16 0.05 0.21 0.72 — 0.72
tj — -- -- — — — 0.94 — 0.94
L2 — — — 0.20 -- 0.25 l.Ce -- 1.08.
:4 l.l-.» 0.21 1.40 0.75 0.24 O.V> »2.ti 0.0* 2.67
;rt 1.0y I.i2 3.40 i.4;> 0.4-j i.'>7
i.CS 'S.SJ
sil at «4 hour*.
-------
u
FIGURE 10
OHJLATIVE URINE .EXCRETION OF BDC IN MONKEYS
SINGLE INTRAVENOUS DOSE 10 KG/KG
o
o
«• 2
8 12 16
20 24 28 32 36 40 44
Timt in Hours
48
-------
FIGURE 11
CUMULATIVE URINE EXCRETION OF BDC III MONKEYS
SINGLE ORAL DOSE OF 10 KG/KG
u
»
••
5
o
o
O 13 8-10-76
Q 3 8-19-78
B 3 11-9-76
32 36 40 44 48
Time in Hour*
-------
TA8I.S 40.
:i£SV£ DISTRIBUTION OF EDC (A3 * C) I'.
Sl!i.Z,E DOSE OF 10 H3/K.G
Tissue.
Blood
Plasoa
Liver
Gall Bladder
Bile
Pancreas
Spl*«n
JO.dr.ey
Adrenals
Lungs
Heart
Skeletal Muscle t
Abdcear,*! Muscle tt
Sinn tl*
Fat *r**
StOBACh*
Saall Xr.testir.**
CCCVSB*
L-ir-.-jo Ir.testifM*
Ovar t*»
Sex Tract"
t'rindry blaci^er
I'rai;.
L'y««
Lorv* Harrow
Jr. Crai
%
0.66
—
2.38
0.01
0.'06
0.0*
0.01
0.05
<0.01
O.C4
O.C2
0.97
0.53
<0.01
0.48
o.os
0.17
Ci.^J
1. J>
,). 01
0.07
o.ci
O.Oi
13R i.V.
cf Icse
0.41
i.e*
4
1.7
15.4
*."
1.1
0. V
o.t.
0.5
'v « Mi
J.u
13K I.V.
0.43
O.C3
9.0
2.3
1C. 2
0.5
0.6
0.5
1.0
C.5
0.4
0.2
0.2
0.3
1. J
0.7
1.1
4.t,
•> •>
* » *
o.<>
0.4
0.5
0.3
0.2
1.7
Skeletal nuscl* calculated as ?C% of body
Al>it*tir.ai H"jacl« calculated »» 1~\ to^iy «ci
-------
SIX? ION 7
S7VDIES CS bli 12-CHIX.F.CISCPFCFYL LTHEB)
jhA>;vAcoxiKrrics cr sis(2-CK:x>5i'isc?ROpyi.) ETKTR-'C IN f.Hrsus MONKEYS
FC H>C%iI!« SI!iGU. I!fTRA\TNOCS OR ORAL ICSES
Introduction
Bis{2-chloroiscpropyl) ether (BCIE) is one of several halogenated or-
ganic coepour.ds which have been detected in potible water supplies. Our
early preltainaxy studies in rats and aonkeys were published in 1977(21).More
recently we have extended these studies to include additional observations
in enorJteys giver, both single and multiple doses.
SINGLE DCSE S7X.T3IES OT BCIE IN HOTKEYS
A total of seven fertile rhesus ror.keys have been used in defining the
single dose toxicity of BCIE Three v*>re dosed intravenously and four
orally &r.d sacrificed as follows:
Intravenous dose-after 4. 1C8, and 169 hours
Oral dose-after 4. 6. 16#, dr.d 1*>« hours
KAt*rl«l* Ar.d Methods
h* tr«at»x«nt solution for intra\* «isiniatr*t '<.>n was ; rcvjtec! by
1.3 »l of fci» (i-chlos-CKli I sorrow 1 ) ctv.«r and ;X >l of t-is (i-ctiloro-
rcpyl) «Ux.r-i4C (0.5 i»Ci/sil) to IS wl of r»ulv-^r!N- (TL-e 20} iwthar.ol
(ICC*) (1:1. v/v) «.nd diluted with deic^m^d dintilied water to SO »l. TT^
resulting »olution contained JJ.l s»--j/s>i and 1 . )J uCi/«l with a »j««cific
activity of Ht«.t8 di^ss/uq, «8t*iilish«d k.-y tvplicate assays of the treatment
solution.
The oral trcatrwnt solution wa» pt*p«red l;y addlj-.q 20 r.1 of th<» intr.*-
ver.o«» «r)lutio« described above to ^0 nl of IztuU-hor^-ethanol (1:1, v/v) q.».
to 100 el with n?O. TTi* r#»-jlt ir.fj solution c«>'-it«in«?y rr>plicat« a**«y» of th* oral trpat**nt solution.
Three fe»j>l« rJ.«eu *Jr,-jl* ir.tra-
v«r.c-u» do«.«« of )0 rvj/k I. < 'tie r«.r.**y, .'•/?• f «.f< k-j!, WA» ir. *»ct»J via t.'.o
(.wjhAlic •-••in o,f the loft f':-rp*rf!i| t *«> m-r.k*y« J >« (S.t k'jj »hd 10:i (^.7 kq>,
w«r«» if, ;«»ct*'S vi* h«r.ou* vein c.>f the IpJt !oj. f .** of ?fi* ri-wf-.tM ty
c«thet«?r. l.f»cr the t*'>«« w4» 'jivwn the c
(Jr. cc-1 iy j>>J*iiil«it»rin«| ."J »i of
-------
T*.e ucrJceys were r.ousea in r^ti&oiitiL ca?«s maintained within exposure
char&ers wr.ich exhausted all the ex.1-. alecs ccrj-.cur.j. T.-.e norjtcys vore cellars
with chains that hooked to the cd'-je CO.TS.. This facilitated obtaining blood
sarnies. They received water aa lj.i._it-_3 AT. 2 w«re fed 1J-15 ;«llets of
rx hr.
Urine sarpies when availajle were collected at 4, 6, 3, 12, 24, -.3, 72, 96,
125, 144, and IbB ho-jr» for the seven day studies. In addition, ar.y urine
lost during bleecur.g was caught en a tray over which the rjo.-Jiey was held.
Recovery of •t'*C in the tray wash was tleterruned and added to the urine
figures. Feces w«re collecte-d at 24 boar ir.t :rvals in the seven day studies,
and, whenever available in th« short- tern studies.
HeKatocrits were deter raided on each blcxxl sar-ple and whole blood, urine,
feces, bile and tiss'jes were analyzed for total ''•C content ersployir.q the
standard procedures u&ed in this laboratory. Ir.terr.il standardization was
used to correct lor quenching.
Results
When this cor-pound was a-lrunistered intravenously , there was a rapid
drop frc«E an estiPiated rero l«rvel of about J30 'i-9/q to i2.7 -*g/ hour* ar.d about 2.5 t9/-; (as **C!
at lt,H hours. Vljs.*ji )rv*la of RCIK ax ,5 r«t*lolif«> wwr« s3j<,'M}y ht'7h*r
than valu** for whole tl.l vip to 120 J^ur* but «ft«r that ties*, th* v«lu«»
for whola blooJ ar-1 {>la»iw w*rc about ^.jual {Table 42).
T>i« orally treated noiJ^eys studied for »«v«n days showed j>c.»k Moc*.i
l«v*l« at 2 hr aftor tieatwr.t , th« B.v,ort-t«rei t.*.:r>cy» »t. 2 hr ar,d a?. 4-6 hr
(7aM» 43 and f"i pir* 13). 'rr* 2 hr t Itod levels in the 4 rtsn**Y« rarqc!
frc«s 20.1 to *<>.! i.'j/vj i.-.d th« 4 hr ltv«ls rT.«i*di trots lfc.8 - 24. 5 u-.j/q. A*
was ctservvd AtxiN** In th» I.V. nenk«ys *h« blcod l«v«la (ironed sloviy to an
,»vt> ra ;* valu« of 2.4 t,':.-'<) at «4 hour* ar.d to about 1.5 i-f;/ol« lj;c«c-'i nr.4 v~l*s»« i?.-!ic«r.ir»^
that HCir. 4r.-5/or tt» rwr .vl/el its* *r* »,c-t st»or>';ly •*rl-^l«d ft am or n
cd in ir,« re-S cell (T»fcle 44».
Th^» two ««•.-«« itay r» nkcy« or. lrtr»v»r.r»y» tr*«t*Hrnt »xcrft«tJ IS *r.<1
of ihtir .;-JM» ir. *h« urin« ("Tati* 41> ar 1 J'l'j-.i* 14). fr.-ur tx • ur cu»«Ml at t
urinary «*«<,-r»it.»<«i v«rto4 tt<» !).•> to i )* rr-ptwB^niir.'i »l^-«j? :,n% cf » «•>•
lr-*'; w*a ra;at*i 1.1 in*ry w»'.:r««t ion * ftiit>te--l for only « o*w»}l j'4it of «h» t.-,t*J .io««
-------
• ecal excretion over the s*v«rn day terioj aaour.ted. to or.Jy 1.2 tc l.E€%
of the .iose (Table 45). Iron this cr.e cin .-on elude that excretion In urir.e
arid feces accounted for only arout cr.e-t_r;ird of the atisu-r.istered dose when
the confound was ir. ^ected ir.tr aver.c us iy .
x*-*n the corfour.d was a. in postered orally total urinary excretion after
163 hours accsur.ted fcr 24.1 and ?:. i% cf the ,5ose {Tihle 46 AT.d figure 15).
A&out 53% of the total urinary excretion occurred by 6 hours. Total fecal
excretion ajw.tr.ted to l.bv to 1.91% of the dose (Table 46). values very
sir.ilar to those observed when bCJE was injecttd intravenously. Thus we can
conclude that following either oral or intravenous adrur.istration cf labeled
BCIE only a third or less is recovered in the urine ar.d feces. Very little
additional radioactivity was recovered in the tissues as notei below.
Cat* en tissue distribution of BCIE ar-d netabolites in 1 »er.k«y wa«
included in the Butr-uscrtpt entitled *Cotparacive Metabolis* of Haloethers",
a copy of which is attached. Cur additional data on tissue distribution in
this report (Tables 47/>48. 49, 50) suggest that the only organ* showing
significant upt'axe of A"C free BCIE are the liver arid kidneys. All the
other tissues showed lower concentrations of *"*C which were about equal to
the levels in blood. Fat has slight sequestering activity but always less
than liver. Theie appears to be scne biliary excretion of BC!E and/or its
saetacolites but at 7 days the concentration or total aoour.t of ^C in bile
is r.tjt renarfcable. Although no data for 24 hours are available it can b»
deduced that there is very little sequestration of the coppour.i or lt» raet-
abolites in ar.y ti**ue except *:>.Jt observed in liver, fat and sX*iet«l
The tutal in the»e thr«« craino, at tf^ er.d of 7 days, is or.ly Aiout 4% of
the aiRinl»t«red do*e (of ^""CJ .
*r~i.t*vLr. usr sfvtr ci- Eisi^-CHUKTiSfji'^ci'YDrTiirK IN
The f -.jri'-osc cf this «xT*rie^nt was to deternin* th«> wffpct of r-ultipl
cr*l dally 'l ) ether (BCIt) In ferjil* rhesus
nw.ii«»y«. Ir. particular , we wi»,*:»d to «tKAi»ir.« th* phjinR-jcr.Jrinot ic« of K'J
follwwtnc| multiple oral
The bCIS" trp»ta«»r.t solution w»» prrpjircl f rcn» C labeled ftCIC *r.tl
stable IK"*!; «• f->llov«: to 4^ Pi of »sul[. hoi*^ and 40 wl of absolute ethanol
was a-J.i«d ^.-W r-1 or ):>J r»i of cold BCir *r.d enou-jh i4C l«li*J»
-------
TABU: 4i. BLOCD cc:iCE!/>ATict;s or tciE i^s c) n; HC:;rrys
si:.ci£ i:,"r?Avr:iCcs DOSE 30 HG/KG
Tiss*
tost
Treatment
Hours
.1
2
4
6
S
12
24
48
72
-x.
120
U4
US
136^ 295
4 Hr 168
% Hct yg/g % frict
39.3 30. fc
36.8 2S.9 41
35.5 19.0 42
42
43
35
40
39
J7
33
j3
36
3'.
1$
Hr
'-9/9
32.7
24.5
20. 7
15.2
8.4
4.4
4.2
2.7
2.9
2.7
2.9
2.4
100
lea
% Hct
33. S
40.0
38.0
39.0
33.0
38.0
37.0
><>.o
37.5
38.0
37.0
37. S
J7.S
9
Hr
y
-------
te
o
FIGURE 12
BLOOD LEVELS OF BCIE IN MONKEYS
SINGLE INTRAVENOUS DOSE 30 MG/KC
O
138-4 Hour*
100-168 Hour*
2051-168 Hours
-------
42. PIAS.«A roMcrwrwiTrciis cr ^CIT. IA.S "4c> IN M
'S :XS£ C? 30 MC/K3
Tin*
rost
Hours
1
2
4
6
8
12
24
48
72
<*
120
144
I6a
138? 295 j? :;c?
4 lir 163 Hr Ita Hi
ug/9 ^9/9 '^9^9
33.2 32. S
27.3 35.0 2d.l
19.4 27.2 19.8
21.6 17.7
1S.O ?4.4
10,1 11.7
6.2 6.3
S.O 4.7
3.3 3.9
3.0 3.5
i . 6 J . S
2.7 2.4
2.4 2.1
til
-------
TABLE 43. BLOCD CONCENTRATIONS OF ECIE (AS 14C) III MONKEYS
SINGLE ORAL DOSE 30 MG/KG
Time
Post
Treatment
Hours
1
2
4
6
6
12
24
48
72
96
120
144
168
111? 116? 290 F?
4 Hr 6 Hr 168 Hr
% % %
Hct wg/g Hct vg/g Hct
37.8 18.9 28 12.3
37.5 26.1 29 20.1 46
36.0 23.4 28 24.5 44
28.3 24.4 42
40
40
39
40
39
41
40.5
40
39
ug/g
-
21.3
16.8
18.4
13.1
6.9
3.5
3.1
2.3
2.2
2.3
2.2
1.9
75-6?
168 Hr
%
Hct
-
45
-
45
44
43
45
42
42
42
43.5
43
43
ng/g
-
25.1
17.6
13.1
6.7
2.5
1.3
1.6
1.5
1.3
1.3
1.3
1.2
82
-------
25
20
15
a>
-?
en
U
111
U
CO
10
. FIGURE 13
BLOOD LEVELS OF BCIE IN MONKEYS
SINGLE ORAL DOSE 30 MG/KG
£ 111 • 4 Hours
Q 116 • 6 Hours
290F-168 Hours
75-6-168 Hours
•-••o
.....^.-•Q. -v Q.....
~~O
6 12 16 20 24 28
Time in Hours
48
72
96
-------
TABLE 44. PLASMA CONCENTRATIONS OF BCIE (AS 4C)
IN MDNKEYS
SINGLE ORAL DOSE OF 30 MG/KG
Tiine
Post 111? 116?
Treatment 4 Hr 6 Hr
Hours Pg/g Vg/g
1 20.3 12.4
2 27.0 21.7
4 23.1 23.0
6
8
12
24
48
72
96
120
144
168
290F?
168 Hr
Pg/g
23.8
17.2
13.7
13.0
9.2
4.7
3.4
2.6
1.9
2.2
1.9
1.8
75-6?
168 Hr
vg/g
27.9
-
12.6
6.9
-
1.9
1.5
1.3
1.3
1.0
0.9
0.7
84
-------
14
TABLE 45. CUMULATIVE EXCRETION OF BCIE (AS C) IN MONKEYS
SINGLE INTRAVENOUS DOSE OF 30 MG/KG
Time Post
Treatment
(Hours)
1
2
4
6
8
12
24
48
72
96
120
144
168
Percent
138 100
4 hr 168 hr
Urine Feces U & F Urine Feccs
<".22 ~ 6.22 4
13.21 — 13.21 9
23.01 0.01 23.02* 16
20
21
23
29
32
33
34
34
34
34
.68
.61
.85
.02
.48
.51
.81
.49
.53
.03
.37
.59
.76
—
—
<0.
<0.
—
~
0.
0.
1.
1.
1.
1.
1.
01
01
44
97
27
36
45
52
56
Dose
U ,% F
4
9
16
20
21
23
30
33
34
35
35
36
36
.68
.61
.85
.02
.48
.51
.25
.46
.80
.39
.82
.11
.32*
Urine
-.
-
13
17
18
21
25
27
_
-
.88
.84
.71
.40
.49
.33
27.98
28
28
28
28
.24
.40
.52
.62
295 I
168 hr
Fec'is U & F
0
0
0
0
0
1
1
1
1
—
—
.01
.01
—
~
.02
.08
.88
.05
.13
.18
.20
--
—
13.89
17.85
18.72
21.41
25.51
27.41
28.86
29.29
29.53
29.70
29.82*
Sacrificed
-------
co
LU
to
o
35 r-
30 -
25 -
20
15
10
5
FIGURE Hi
CUMULATIVE URINARY EXCRETION OF BCIE IN MONKEYS
SINGLE INTRAVENOUS DOSE 30 MG/KG
D 133 FEMALE - [\ HOURS
• 100 FEMALE - 163 HOURS
O 295 I FEMALE - 163 HOURS
0
12 16 20
23
72
96
1 ..// 1
120 163
-------
CD
TABLE 46. CUMULATIVE EXCRETION OF BCIE (AS 14C) IN MONKEYS
SINGLE ORAL DOSE OF 30 MG/KG
Time Post
Treatment
(Hours)
1
2
4
6
8
12
24
48
72
96
120
144
368
Percent Dose
111 116
4 hr 6 hr
Urine Feces U & F Urine Fcces TJ & F Urine
0.96 -- 0.96 0.46 — 0.46
2.91 — 2.91 1.21 ~ 1.21
14.20 1.55 15.75* 7.69 — 7.69 7.
13.69 1.42 15.11* 9,
12.
16.
19.
22.
23.
23.
23.
23.
24.
91
22
22
49
43
95
35
64
85
95
05
290 F
168 hr
Foces U &
0
0
0
__
—
—
.01
—
—
.30
.39
NS
0.46
1
1
1
.46
.66
.91
~_
—
7.
9.
12.
16.
19.
23.
23.
24.
25.
25.
25.
F
91
23
23
50
73
34
74
10
31
61
96*
Urine
_
-
13
13
14
26
28
29
29
29
29
30
30
_
-
.19
.72
.10
.22
.56
.42
.70
.88
.97
.03
.09
75-6
168 hr
Foces US F
_
-
0.
-
0.
-
1.
1.
1.
1.
1.
1.
1.
_
-
01
-
08
-
08
21
39
49
52
55
59
_-
—
13.20
13.73
14. IB
26 . 30
29.64
30.63
31.09
31.37
31.49
31.58
31.68*
Sacrificed
-------
35 r
30 -
25 -
< 20
15
ai
in
o
10 -
5 -
FIGURE 15
CUMULATIVE URINARY EXCRETION OF BCIE III MONKEYS
SINGLE ORAL DOSE 30 MG/KG
• 111 FEMALE - 4 HOURS
O 116 FEMALE - 6 HOURS
A 290 F FEMALE - 168 HOURS
D 75-6 FEMALE - 163 HOURS
0
12 16 20
00
Zo
72
96
120
163
TIME IN HOURS
-------
TABLE 47. TISSUE DISTRIBUTION OF BCIE (AS
IN MONKEYS
SINGLE INTRAVENOUS DOSE OF 30 KG/KG
14
C)
Tissue
Blood
Plasma
Liver
Gall Bladder
Bile
Pancreas
Spleen
Kidneys
Adrenals
Lungs
Heart
Skel M.
Abdom M.
Skin
Fat
Stomach*
Small Int.*
Cecum*
Lg. Int.*
Ovaries
Sex T.**
U. Bladder
U. Bl. Urine
Brain
Eyes
Bone Marrow
Aorta
Orbital Fat
Cardiac Fat
Mesenteric Fat
138?
4 Hr
19.0
19.4
73.8
64.2
98.9
26.7
23.3
48.6
24.9
18.2
20.0
12.5
10.0
17.1
26.4
17.9
38.6
73.1
16.7
15.6
17.7
***
-
20.1
13.0
24.6
11.8
-
-
26.2
yg/g
2951?
168 Hr
2,4
2.4
28.8
5.0
6.0
3.8
4.5
5.0
6.6
3.0
3.0
3.3
2.4
3.0
3.2
2.6
3.5
1.8
2.1
2.3
1.95
1.8
0.5
3.3
0.3
3.3
1,8
-
3.0
—
100?
168 Hr
2.5
21
17.2
1.8
9.0
3.1
10.5
5.2
8.9
3.1
2.6
2.0
3.3
3.8
4.5
1.5
4.1
2.6
2.6
3.6
3.7
1.5
2.5
2.4
0.5
3.7
-
_
3.4
—
* Tissue plus contents.
** Uterus, vagina and fallopian tubes.
*** I5o sample.
-------
TABLE 48. TISSUE DISTRIBUTION OF BCIE (AS 14C)
n MG!!KEYS
SINGLE lOTPAVEtiOUS DOSE OF 30 KG/KG
Tissue
Blood
Liver
Gall Bladder
Bile
Pancreas
Spleen
Kidneys
Adrenals
Lungs
Heart
Skel M.t
Abdom M.tt
Skin ttt
Fat tttt
Stomach*
Small Int.*
Cecum*
Large Int.*
Ovaries
Sex T.**
U. Bladder
U. Bl. Urine
Brain
Eyes
Bone M.
Aorta
Eye Fat
Heart Fat
Mesenteric Fat*
138?
4 Hr
5.76
4.83
0.04
0.05
0.10
0.09
0.61
0.02
0.42
0.24
7.58
3.04
0.01
6.40
0.37
1.46
1.27
0.49
0.01
0.31
N.S.
-
1.00
0.06
0.01
0.01
-
-
0.01
% Dose Administered
2951?
168 Hr
0.74
1.34
<0.01
0.01
0.02
0.01
0.05
<0.01
0.05
0.03
2.01
0.75
<0.01
0.78
0.05
0.11
0.03
0.09
<0.01
0.01
0.01
<0.01
0.18
<0.01
<0.01
<0.01
-
<0.01
~
100?
168 Hr
0.75
1.0
<0.01
0.02
0.01
0.02
0.04
<0.01
0.04
0.02
1.17
0.96
<0.01
1.05
0.14
0.14
0.05
0.10
<0.01
0.02
<0.01
<0.01
0.11
<0.01
<0.01
-
-
<0.01
t Skeletal muscle, calculated as 20% body weight.
tt Abdominal muscle, calculated as 10% body weight,
ttt %/g or ug/g.
tttt Fat calculated as 8% body weight.
* Tissue plus contents.
** Uterus, vagina and fallopian tubes,
90
-------
TABLE 49. TISSUE DISTRIBUTION OF BCIE {AS
IN MONKEYS
SINGir. ORAL DOSE OF 30 MG/KG
14
C)
Tissue
Blood
Plasma
Liver
Gall Bladder
Bile
Pancreas
Spleen
Kidneys
Adrenals
Lungs
Heart
Skel. M.
Abdom. M.
Skin
Fat
Stomach*
Small Int.*
Cecum*
Large Int.*
Ovaries
Sex T. **
U. Bladder
U. Bl. Urine
Brain
Eyes
Bone M.
Aorta
Orbital Fat
Cardiac Fat
111?
4 Hr
23.4
23.1
82.0
71.7
293
11.2
17.0
48.2
18.7
17.6
19.8
16.6
10.4
14.3
-
35.4
59.2
76.7
16.0
17.1
23.2
55.1
-
18.3
16.5
20.1
-
-
—
ug/g
116?
6 Hr
24.4
25.4
74.5
22.4
50.6
35.1
1.89
59.1
22.1
21.1
16.5
11.6
12.3
13.3
-
180
45.6
55.1
16.9
-
26.9
34.1
-
26.2
13.6
24.2
-
-
19.3***
290F ?
168 Hr
1.9
1.8
8.6
2.9
7.2
2.8
3.0
3.5
8.0
2.0
2.2
1.8
1.2
2.5
5.7
2.2
2.6
2.9
3.9
1.9
1.8
1.2
0.1
0.7
0.0
2.3
1.5
4.1
3.1
75-6?
168 Hr
1.21
0.7
15.6
3.8
4.4
1.3
1.3
1.9
3.2
1.2
1.2
1.8
1.5
1.3
5.4
0.6
0.9
0.6
0.9
1.3
0.9
0.4
0.1
0.7
0.0
1.6
0.7
-
2.6
* Tissue plus contents.
** Uterus, vagina and fallopian tubes.
*** Brown fat.
93
-------
TABLE 50. TISSUE DISTRIBUTION OF BCIE (AS 14C)
IN MONKEYS
SINGLE ORAL DOSE OF 30 MG/KG
Tissue
Blood
Liver
Gall Bladder
Bile
Pancreas
Spleen
Kidneys
Adrenals
Lungs
Heart
Skel. M.t
Abdom M.tt
Skinttt
Fattttf
Stomach*
Small Int.*
Cecum*
Large Int.*
Ovaries
Sex T.**
U. Bladder
Brain
Eyes
Bone M.
Aorta
Orbital Fat
Cardiac Fat
111?
4 Hr
7.13
6.44
0.12
1.35
0.09
0.04
0.63
0.02
0.41
0.20
10.16
3.18
0.01
-
O.b8
3.43
2.81
1.95
0.01
0.27
0.13
1.14
0.13
0.02
-
-
—
% Dose Administered
116? 290F ?
6 Hr 168 Hr
7.44
6.^9
0.02
0.30
0.1^
0.08
0.83
O.C2
0.41
0.20
7.09
3.75
0.01
-
5.02
1.87
3.93
4.21
-
0.63
0.18
1.23
0.08
0.02
-
-
0.01***
0.57
0.48
<0.01
0.01
0.01
O.C1
0.03
<0.01
0.03
0.02
1.07
0.35
<0.01
1.4
0.06
0.13
0.07
0.32
<0.01
0.03
0.01
0.04
0.00
<0.01
<0.01
<0.01
<0.01
75-6?
168 Hr
0.34
1.12
<0.01
0.01
0.01
0.01
0.02
<0.01
0.01
0.01
1.10
0.44
<0.01
0.96
0.04
0.04
0.02
0.06
<0.0t
<0.01
<0.01
0.06
0.0
<0.01
<0.01
_
<0.01
t Skeletal tnuscl", calculated as 20% body weight.
ft JUbdominal muscle, calculated as 10% body weight.
ttt %/g or ug/g.
tttt Fat calculated as 8% body weight.
* Tissue plus contents.
** Uterv;*, vagina and fallopian tubes.
*** Brown fat.
92
-------
Blood samples were taken before treatment (0 blood sampler) and at 1, 2,
4, 8 and 24 hours after treatrvent on days 1, 2 and 3. One aniral (No. 9f)
received only 2 doses and this is indicated on the tables and oraphs. ON
days 4 to 10 blood samples were collected at the same time on each day
equivalent to the 0 sample. Hematocrits were obtained on all blood salaries,
and after two aliquots were prepared for LSC the remainina blood was centri-
fuged and one or more plasma aliquots were counted (one monkey, No. 1, was
also bled on days 13, 15 and 17).
Urine samples were collected whenever the monkeys were bled if a sample
was available. On day 1 urine was collected at 12 and 24 hours and there-
after at 24 hour intervals.
Feces were collected whenever available during the first 3 days, at 12
and 24 hours on day 4 and 24 hour intervals for the duration of the experi-
ment.
14
Whole blood, plasma, urine and feces were analyzed for total C con'^ent
employing standard procedures used in this laboratory. All samples were
corrected for quenching using internal standardization.
Results - Physiological Effects
The oral treatments with BCIE i reduced different effects in the three
monkeys. One monkey, N'o. 96, exhibited no reaction while the other two
animals both developed evidence of local or systemic effects in the tissues
around the eyes (Table 51). These ocular effects appeared to be roughly
dose-related in the sensitive nonkeys (1 and 98). The ocular symptoms were
most prominent in Monkey 98 and, in fact, this animal was sufficiently toxic
to make blood sampling a problem, and therefore BCIE was discontinued on
this monkey after the second dose. After the first dose had been cLdminister-
ed glucoauria had been verified in her 0 hr urine by Bililabstix* tests.
This glucosuria persisted in all subsequent urine samples. Hyperglycerua
was then verified by SMA 12/60 assay. Her general weakness, ocular changes,
poor nutriture and urine flow rdlitated against further dosing. It w
-------
with 75 ml of h;>0 on days 2, 3 and 15, no improvement in urine flow was
observed except on day 16, when 118 ml of urine was excreted. Monkey S3
also showed reduction in urine flow which, however, was corrected as soon
as dosing was discontinued. Details of the urine excretion of BCIE will tx»
considered later.
Blood levels of BCIE in the 3 monkeys are detailed in Figure 17. Peak
concentrations ranging from 17 to 34 ug/ml occurred 2 hours after treatment,
after which the levels dropped rapidly for the next 6 hours and more slowly
between the 8 and 24 hour period. One observes increasing residual radio-
activity in the blood with repeated dosos (Table 52 , Figure 17). If all
three monkeys had received 3 consecutive doses, one could conclude that the
average trough level at 24 hours after the third dose would have appreciat-
ed from 50 to 100%.
On the other hand, there is no clear cut indication of rising peak
concentrations with repeated doses, indicating a reasonable maintenance of
the early "a" phase of the dieaway curve; however, even the limited data
obtained in this study suggests that the slope of the dieaway curves is
flatter on day 3 than previously.
Plasma levels of BCIE were regularly higher and follow essentially the
same patterns observed with whole blood (Table 53 and Figure 18). The evi-
dence of toxicity in Monkey 98 on the morning of day 3 correlates with her
high residual '^C activity from the previous dose as well as her high peak
reading (41 ug/ml) observed after administration of the second dose. After
dosing was discontinued the levels in plasma fell, but even at 7 days all
levels were still above 5 pg/ml (as ^C) .
14
The data on urinary excretion of BCIE expressed as C are presented
in two ways. In Table 54 and Figure 19 the c'ata on urinary radioactivity
in "Kg of BCIE equivalents indicate the variability in response observed.
The low recovery in Monkey 1 on day 2 is, of course, due to the animal's
having vomited an unknown, (ca 50%?) but obviously significant, portion of
its second dose of BCIE. The omission of the third dose for Monkey 98 pre-
vents comparison with the "3 dose" monkeys. As discussed previously, this
monkey had poor urine flow. When dosing ceased, urine flow improved
promptly but only small additional amounts of C were recovered in the
urine.
If we consider the excretion of drug in the urine in % dose one can
generate the upper and lower limit values (single dose vs. additive dose
calculations. Table 55) but the absolute values for % dose would depend on
one's knowing how much residual BCIE remained in the monkey at the end of
the first day, the second day, etc. Since much of the BCIE is lost by
exhalation we cannot establish this residual from data on urine and feces
recovery. Nevertheless, the data in Figures 20, 21 and 22 support the
conclusion that although only 30-45% of the dose is accounted for in the
urine (and feces) the rest of the radioactivity must have been lost by
another route, exhalation.
94
-------
In the case of Monkey 1, (Figure 20), the data are rather vague (be-
cause of the vomiting of the second dose). Nevertheless, one is inpressed
by the small amount of * C recovered during the 3rd day, less than 15%,
based on single dose calculations. Thus, this may indicate that excretion
of BCIE in the urine is dependent on urine flow. The data on Monkey 1 whi^.
had very poor urine flow on days 1, 2 and 3 and thereafter suggests that
urine flow is critical.
In the case of Monkey 96 (Figure 21) one observes a fairly consistent
urinary excretion pattern with somewhat lower recovery figures on the secon
day, possibly related to the relatively lower figures on this day for BCIK
in plasma (Table 53, Figure 18).
Monkey 98 whose data are shown in Figure 22 has very consistent values
for urinary excretion of ^4C, roughly 26% of dose based on single dose cal-
culation; however, consideration of the plasma concentration data in Tai>l«
53 and Figure 18 indicates that this monkey had relatively high residual
24 hours after the first dose of BCIE and the highest levels of C of any
monkey after the second dose. Concomitantly, a great reduction in urine
flow was observed. These findings probably account for the high degree of
ocular changes observed and the overall toxicity observed which necessitated
discontinuation of dosing.
14
Turning to the figures for fecal excretion of C from BCIE (Table 56)
one notes that excretion via this route never accounts for more than 4* of
the dose (based on single dose of 30 mg/kg)• It is obvious that at least
a small fraction of the dose in every case is still present as residual ^*C
24 hours after the BCIE was administered. Therefore, one can be certain
that no more than about 3% of the ^C from BCIE is excreted in the feces
even under the extrene conditions of low urine flow observed in Monkeys 1
and 98. One can conclude, in fact, that fecal excretion is a very winor
pathway with BCIE and that whatever ^C is excreted in the bile is either
relatively little or is well reabsorbed via the enterohepatic circulation,
Discussion
This experiment points out some very interesting findings. One, that
some monkeys are highly susceptible to the toxic side effects of BCIE on
the ocular and renal systems and possibly on other organ systems not stu-!i«'J.
Some monkeys (like q6) , however, exhibit neither the ocular nor the renal
effects, and show little effect on appetite or weight. Whether or not thom
is any direct connection between these toxic manifestations is not known.
14
It is obvious that only a small fraction of the C from BCIE is bour,60* of the dor-;) must be exhaled. However, it is difficult to
understand how the pulmonary clearance of the compound can account for the
eye irritation, since in the previous animals which received 30 mg/kg and
in 96 in this series the same proportion of the dose was apparently exhalod,
yet only 1 and 98 exhibited both the severe eye toxicity and effects on
urine flow. We maintain careful records on menstrual cycles of our rhesu«
-------
stock. The menstrual cycle of Rhesus 1 may have been affected by BCIE.
Her 5-day cycle ended 6 days before dosing was instituted and menses recurred
on the fifth day of this experiment - some 14 days before it. was expected.
No other menstrual deviations were observed.
No explanation is available from these studies to account fou the large
differences in the way different monkeys respond to tnis compound, although
ona monkey exhibited hyperglyceriia and glucosuria before and after the ad-
ministration of BCIE. How this affected the response to BCIE is not known
at this time.
Conclusions
1. A single oral dose of BCIE (30 mg/kg) produced marked toxic effects
on the eyes of 2 of 3 monkeys studied and these changes were intensified
with repeated doses.
2. The same monkeys exhibiting ocular toxicity also showed very great
reductions in urine flow. These were readily reversed in one monkey follow-
ing cessation of treatment but persisted in a second for 12 days.
3. A third monkey exhibited no apparent side effects from 3 oral 30
mg/kg doses of BCIE, but residual blood and plasma levels of ^4C rose with
each dose, suggesting the possibility of subacute toxic effects if the
dosing had been continued ao planned.
14
4. As noted in previous studies, very little C from BCIE is excreted
in the feces indicating rapid and complete absorption of oral doses and
apparent facile absorption of any *4C excreted via the bile.
5. One monkey may have had an early menstrual cycle as a result of
BCIE.
-------
TABLE 51. OCULAR EFFECTS OF BCIE
Time Post
Treatment
Day Hr
0
1 4
12
2.4
2 4
12
24
3
4
5
6
7
8
9
10
11
12
1 ? 96 ? 98 ?
000
++ 0 ++
+ 0 +
± ° ±
+•++ 0 ++++
++ 0 +++
++ 0 +++
+++ 0 +++ (no treat-
ment)
+++ 0 -M-
-H- 0 ++
+ 0 ++
jf 0 +
_+ (0.4 kg wt 0 (0.1 kg wt + (0.4 kg wt
loss) loss) loss)
+ 0 +
00 +
00 +
00 +
i Minimal changes.
0 No visible effects.
jf Regressing symptoms.
+ Edema, discoloration.
•M- Prominent edema, si. hemorrhage (?)
•M-+ Marked swelling, edema, prominent subdural hemorrhage (?)
++++ Very intense effects, eyes essentially swollen shut.
97
-------
CD
FIGURE 16
URINE VOLUMES IN RHESUS MONKEYS BEFORE AND AFTER
3 ORAL DOSES OF BCIE 30 MG/KG
400 —
O 1?
A9G J
D 93 J (ONLY 2 DOSES)
75 ML HoO BY GAVAGE
TIME 111 DAYS
-------
TABLE 52. BLOOD CONCENTRATIONS OF BCIE (AS 14C) IN MONKEYS
THREE CONSECUTIVE DAILY ORAL DOSES OF 30 MG/KG
Time Post
Treatment
Day Hr
1
2
3
4
5
6
7
8
9
10
13
15
17
0
1
2
4
8
24
1
2
4
8
24
1
2
4
8
24
1
% Hct
48.5
48.5
48
46
50.5
46
46.5
41
44
42.5
tO
40
39
38
39
36.5
35.5
38.0
38.5
38
43
39
44
48
43.5
-
? 96
•jg/g % Hct
0
16
17
17
17
8
16
13
12
10
7
30
31
27
23
14
11
10
8
8
8
7
7
5
5
4
.5
.2
.1
.9
.5
.7
.2
.4
.8
.7
.7
.6
.2
.9
.6
.6
.8
.9
.0
.3
.0
.7
.5
.9
46
46
44
46
45
44
43
43
-------
30 -
z:
o
20
o
o
CO
-------
14
TABLE 53. PLASMA CONCENTRATIONS OF BCIE (AS C)
IN MONKEYS
THREE CONSECUTIVE DAILY ORAL DOSES OF 30 KG/KG
Day
1
2
3
4
5
6
7
8
9
10
13
15
17
Tine Post
Treatment
Hr
0
1
2
4
8
24
1
2
4
8
24
1
2
4
8
24
1?
yg/g
0
19.1
19.0
15.7
20.3
13.2
24.1
19.3
18.1
35.7
10.6
37.7
37.7
31.4
28.5
20.0
14.5
12.0
9.5
7.5
7.1
6.4
5.9
4.5
3.8
3.3
96 ?
yg/g
0
25.5
32.0
21.1
18.2
6.6
28.2
26.5
21.9
14.7
10.4
36.1
37.5
24.9
19.6
13.4
10.5
9.8
8.4
7.3
6.4
6.0
5.4
98?*
vg/g
0
28.4
32.1
21.9
16.5
12.4
31.2
40.7
32.0
-
16.0
_
-
-
-
10.2
8.8
10.1
7.1
6.0
5.1
4.8
4.8
* 98 ? received only 2 doses.
101
-------
FIGURE 1C
CONCENTRATION OF BCIE (AS %) IN PLASMA IN RHESUS MONKEYS
3 ORAL DOSES OF 30 MG/KG
30
o
A 96 J
D 98 J (OILY 2 DOSES)
O
20
co
CQ
10 -
0
TV'C \Y>
1 i. •£. i i y
-------
TABLE 54. RECOVERY OF ECIE IN MONKEY URINE
30 MG/KG ONCE DAILY FOR 3 DAY'S
Day
1
2
3
4
5
6
7
8
9
10
Time Post
Treatment
Hr
12
24
12
24
12
24
12
24
24
24
24
24
24
24
-1?
59.69
5.19
12.00
2.45
20.02
5.78
2.65
2.23
1.84
1.10
0.87
0.60
0.44
0.?9
14
Total mg as c
96 ?
82.00
16.49
49.04
19.85
66.26
8.58
NS
3.49
1.28
0.84
0.54
0.48
0.35
0.32
98?*
45.56
4.35
42.12
4.71
1.72
1.23
0.39
0.38
0,57
0.65
0.37
0.30
0.27
0.21
* 98 ? received only 2 doses.
10 3
-------
SO -
FIGURE 19
RECOVERY OF URINARY BCIE (AS l^C) IN MG IN RHESUS MONKEYS
A 3 DAILY ORAL DOSES OF 50 MG/KG
60 r-
o
*>
20 »—
A
\
0
01?
A 96 J
a 93 J (ONLY 2 DOSES)
* VOMITED (1 J)
U DAYS
-------
14
TABLE 55. CUMULATIVE RECOVERY OF BCIE (AS C) IN MONKKY URINE BASED OH
SINGLE DOSE (30 MG/KG) OR ADDITIVE DOSE (30, 60, 90 MG/KG)
3 CONSECUTIVE ORAL DAILY DOSES OF 30 MG/KG
Time Post
Treatment
Day Hr
1
2
3
4
5
6
7
8
9
10
0
1
2
4
8
12
24
1
2
4
8
12
24
1
2
4
8
12
24
12
24
24
24
24
24
24
24
]
Single
Dose
0
NS
6.86
10.96
22.19
28.70
31.19
NS
NC
4.93
5.09
5.76
6.93
NS
NS
3.73
9.63
NS
12.41
13.69
14.75
15.63
16.16
16.59
16.89
17.10
17.25
Additive
Dose
0
NS
6.86
10.96
22.19
28.70
31.19
NS
NS
2.46
2.55
2.89
3.48
NS
NS
1.25
3.22
NS
4.15
4.57
4.92
5.22
5.40
5.54
5.64
5.71
5.76
% Dose
96 ?
Single Additive
Dose Dose
0
4.02
10.85
26.28
36.39
36.58
43.93
NS
0.12
12.15
12.38
21.90
30.75
4.20
12.38
23.08
29.35
29.44
33.13
_
34.68
35.26
35.63
35.87
36.08
36.23
36.37
0
4.02
10.85
26.28
36.39
36.58
43.93
NS
0.06
6.07
6.18
10.94
15.37
1.40
4.17
7.74
9.83
9 86
11.09
_
11.61
11. GO
11.92
12.00
12.07
12.12
12.17
98 ?*
Single Additive
Dose Dose
0
2.10
8. VI
12.37
20.17
23.72
25.98
1.57
5.81
10.57
20.75
21.93
24.38
_
-
-
-
25.27
25.90
26.11
26.31
26.60
26.93
27.12
27.27
27.41
27.52
0
2.10
8.71
12.37
20.17
23.72
25.98
O.79
2.91
5.29
10.38
10.97
12.20
_
-
-
-
12.64
12.97
13.07
13.17
13.31
13.47
13.57
13.64
13.71
13.76
98 received only two doses.
10S
-------
60
FIGURE 20
CUMULATIVE URINARY EXCRETION OF BCIE (AS WC) IN 1 J
3 ORAL DOSES OF 30 MG/KG
CO
B
o
o\
• BASED ON SINGLE DOSE
o BASED ON ADDITIVE DOSE
+ VOMITED
30
c/o
CQ
15
0
L- /
i> i i
, , I //
T> t'r
I..L
-------
UJ
t_>
•=r
oo
UJ
CQ
30 -
FIGURE 21
CUnULATIVE URINARY EXCRETION OF BCIE- (AS 14C) IK 9Gj
3 ORAL DOSES OF 30 MG/KG
A-
A
-/
1
, t
A BASF* ON SINGLE DOSE
A BASED ON ADDITIVE DOSE
•/ ^ _.-.-.
x* /*
W .--"^ A.. .^ A— -^-— A
LnA/ /f
rr ^
/* A' ^
1 i i 1 A' i i ! i i r^ i i * i f 1 ^f 1 ' 1 '
Y
1 2 3 4 5 G 7
Till K; BAYS
-------
o
00
FIGURE 22
CUMULATIVE URINARY EXCRETION OF BCIE (AS !/lC) IN 939
2 ORAL DOSES OF 30 MG/KG
60
UJ
CO
CD
I—I
£ 30
*—*
LU
00
15
• BASED ON SINGLE DOSE
a BASED ON ADDITIVE DOSE
-B
I
i
L
-D-
•n- —
•D-
, L J I . I . .
0
2 3
TIME IN DAYS
-------
TABLE 56. CUMULATIVE RECOVERY OF BCIE (AS C) IN MONKEY
FECES BASED ON SINGLE DOSE (30 MG/KG) OR ADDI-
TIVE DOSE (30, 60, SO MG/KG)
3 CONSECUTIVE ORAL DAILY DOSES OF 30 MG/KG
Time Post
Treatment
Day
1
2
3
4
5
Hr
12
24
12
24
12
24
12
24
1
Single
Dose
0.45
0.08
0.17
0.77
1.80
2.13
2.51
% Dose
? 96?
Additive
Dose
0.45
0.04
0.09
0.26
0.60
0.71
0.84
Single
Dose
0.90
1.25
0.92
2.56
0.83
1.16
2.52
2.81
Additive
Dose
0.90
1.25
0.46
1.28
0.28
0.39
0.84
0.94
98
Single
Dose
0.66
0.71
2.31
3.32
3.41
3.65
3.71
?*
Additive
Dose
0.86
0.36
1.16
1.66
1.70
1.82
1.05
• 98 ° received only 2 doses.
109
-------
SECT!' i
PHARMACOKINETIC STUDY OF BIS (2-CKLCI-.. ./THYL)ETHER IN TWO RHESUS MONKEYS
Introduction
Bis (2-chloroethyl) ether (BCEE) is one of a group of haloetliers which
are widespread environmental contaminants and have been detected in drinking
water. BCEE is used as a dewaxing agent, solvent, cleaning agent and pene-
trant (EPA Criterion Document, July 9, 1979). Lingg e_t aK (1970) reported
observations on the metabolic fate of BCEE in rats. Smith and Lingg suiwvar-
ized the current information of metabolism of B-haloethers in rats and mon-
keys at a recent symposium.(22). BCEE has been studied pharmacokinetically
in two rhesus monkeys and these data comprise this report.
Materials and Methods
The treatment solution for oral administration,was prepared as follows:
4.0 ml of ethanol was mixed with 4.3 ml of Emulphor^'. To this was added
0.39 g of cold BCEE, slightly more than 0.02 ml of 14C-BCEE (0.56 uCi/ml),
and 31.7 g of deionized distilled water. Liquid scintillation assay of thin
solution established the specific activity to be 0.49 uCi/ml (1,090,600 dpm)
and 106.27 dpm/\ig of BCEE.
The two female rhesus monkeys weighed 8.9 and 9.75 kg, respectively.
The monkeys were dosed by oral gavage with a sinal<- 10 mg/Xg dose in the
A.M. Each was housed in a metabolism cage maintained within an exposure
chamber which exhausted all the exhaled compound. Each monkey wore a collar
with a chain that hooked to the cage door. The monkeys were fed 10-15
pellets of Purina chow per day and provided water ad_ libitum. Urine sample*
were collected (if available) at 6, 12, 24, 36, 48, 72 and 96 hours after
treatment. Collection flasks were iced over the entire collection time.
Feces samples were collected at 24, 48, 72 and 96 hours.
Both urine and leces samples were processed by standard procedures of
this laboratory for liquid scintillation counting. In addition, all urines
were tested with Bililabstix at the time of measuring samples and taking
aliquots for LSC counting.
Results
Data on cumulative excretion of bis(2-chloroethyl)ether in urine and
feces of the two monkeys is presented in Table 57. The 6-hour urine sample
on Rhesus 1 contained 17% of the dose. At 12 hours this monkey had excreted
ca 30% of the dose in urine and at 24 hours this increased to 43%. It is
interesting that Khesus 96 also excreted 43% of the dose in urine by the
time lapse of 24 hours, but this monkey voided only 26.5 ml of urine beforo
the 24 hour collection time (see urine volumes for both monkeys listed in
Table 58. By 72 hours we recovered 53% of the dose in urine for Rhesus 1
and 63% of the dose in urine for Rhesus 96. '.Ve did not collect 96-hour
110
-------
urine on Rhesus 1 since she was in menses and was returned to her stock cage
after 72 hours. However, the 72-96 hour urine on Rhesus 96 contributed very
little to recovery of ^C-BCE£ or metabolites.
Examination of recovery data on feces in the two monkeys shows that very
little of the•compound was recovered in feces. Total recovery in samples
through 96 hours was 1.10 and 1.63% of the dose for 1 and 96, respectively.
(Table 58 shows the weight of feces samples collected at each 24-hour inter-
val for each monkey.)
Physiological Observations on Monkeys
Rhesus 1 began menstruation at 24 hours after dosing. Also within 24
hours hemorrhagic darkening occurred below her eyes. Ketones were present
in her urine at 12, 24, 36, 48 and 72 hours. She was returned to her stock
cage after 72 hours and urine collection was discontinued. An SKA 12/60
assay (Table 59) was performed on a 72-hour blood sample. The hematocrit
was 41.5% The monkey's plasma LDH was elevated slightly (LDH » 368 vs.
normal level of 271 mU/ml +_ 24.6 S.E.). Phosphorus was also slightly ele-
vated (5.2 vs. normal level of 3.6 mgms % ^ 0.21 S.E.). This monkey ate
less food and excreted much smaller urine samples than Rhesus 96.
Rhesus 96 ate very well and excreted larger volumes of urine than
Rhesus 1. In this monkey 63.5% of the dose was recovered in urine and 1.6%
of the db»e in feces in 96 hours.
Conclusions
BCEE was administered to 2 female rhesus monkeys in single oral doses
of ID mg/V.g. From 54 to 65% of the dose as l^C was excreted in the urine
and 1-1.5" in the feces. One of the monkeys showed evidence of infraorbital
irritation, a finding also observed when larger (30 mg/kg) doses of Bis(2-
chloroisopropyDether (BCIE) were administered. With 14C-BCIE recovery in
urine varied from 24 to 30% and 1-1.5% was recovered in the feces. From
these data we conclude that BCEE is probably more toxic for subhuman pri-
mates than BCIE and both compounds appear to be quite irritating to the
eyes.
Ill
-------
TABLE 57. CUMULATIVE EXCRETION OF BCEE (AS 14C) IN
MONKEYS
SINGLE ORAL DOSE OF 10 MG/KG
Percent Dose
Tine Post g
Treatment ° +
(Hours) Urine Feces U S F Urine Feces U & F
6 16.66-16.66
12 29.85 - 29.fl5 0.003 - 0.003
24 43.43 0.22 43.65 43.23 0.04 43.27
36 48.60 - 48.88 43.50 - 43.54
48 51.03 0.64 51.67 61.05 0.09 61.14
72 53.30 0.76 54.06 62.96 0.65 63.61
96 * 1.10 54.40 63.45 1.63 05.08
*
Monkey returned to stock cage.
112
-------
TABLE 58- URINE VOLUMES AND FECES WEIGHTS OF
14C BCEE-TREATED MONKEYS
SINGLE ORAL DOSE 10 MG/KG
Time Post
Treatment
(Hours)
6
12
24
36
48
72
96
1
Urine
Volume
(ml)
106.5
14.6
31.2
24.0
26.0
73.0
*
£
Feces
Weight
g
-
-
2.34
-
16.78
1.63
12.37
96
Urine
Volume
(ml)
-
26.5
400
16
320
375
561
$
Feces
Weight
g
-
-
0.47
-
1.05
13.68
55.06
*
Monkey returned to stock cage.
113
-------
TABLE 59. SMA 12/60 ASSAY ON RHESUS 1? AT 72 HRS.
FOLLOWING SINGLE ORAL 10 MG/KG/DOSE OF
BIS (2-CHLOROETKYL)ETHER
Assay
Sugar
Uric acid
Cholesterol
SCOT
LDH
P
Alk T'ase
Ca
Bilirubin (1 minute)
Bilirubin (total)
Protein (total)
Albumin
Value
95 mgm%
0.3 mgm%
135 mgtn%
36 mU/ml
368 mU/ml*
5.2 mgm%*
78 mU/ml
7.9 mgm%
0.0 mgm%
0.2 mgm%
6.6 mgiu%
2.2 mgm%
Normal
89 +_ 3.
0.61 _f
170.2 +
30.4 +_
271.2 +
3.56 +_
166.3 +
9.78 +
0.12 +_
0.26 +
8.01 +
3.77 +
Mean +_ SE**
5
0.05
6.0
2.2
24.6
0.21
17.5
0.16
0.01
0.02
0.13
0.11
*
Deviations from normal values.
**
Means established on 20 rhesus stock females of closely
matched weights.
114
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REFERENCES
1. "Drinking Water and Health," National Academy of Sciences, Washington,
D.C., 1977.
2. Arioshi, T, Ideguchi, K., Iwasaki, K., and M. Arakiki. Relationship
Between Chemical Structure and Activity. III. Dose-Response or Time
Course of Induction in Microsoroal Enzymes Following Treatment With
1,2,4-Trichlorobenzene. Chem. Pharm. Bull. 23:831, 1975
3. Carlson, G.P., and R.G. Tardiff. Effect of Chlorinated Benzenes on the
Metabolism of Foreign Organic Compounds, Tox. Appl. Pharmacol. 36:383,
1976.
4. Lingg, R.D., Kaylor, W., Pyle, S., Domino, M., Smith, C.C. and G.F.
Wolfe. Metabolism of Bis(2-Chloroethyl)Ether and Bis (2-Chloroiso-
propyDEther in the Rat, Submitted for publication, 1980.
5. Smith, Carl, C., Ihrig, J., and R. Menne. Antimalarial Activity and
Metabolism of Biguanides. 1. Metabolism of Chlorguanide and Chlorguanide
Triazine in Rhesus Monkeys and Man. Am. J. Trop. hed. Hyg. 10:694, 1961.
6. Armstrong, V. Metabolism of Chlorguanide, A Unique Mixed Function
Oxidase Substrate. Ph.D. Dissertation, University of Cincinnati, 1972.
7. Lowry, O.K., Roseb-ouqh, N.J. , Farr, A.L. , and R.J. Randall. Protein
Measurement With the Folin Phenol Reagent. J. Biol. Chen. 193:265,
1951.
8. Miller, G.L. Protein Determination for Large Numbers of Samples. Anal.
Chem. 31:964, 1959.
9. Cochin, J., ?nd J. Axelrod. Biochemical and Pharmacological Changes
in the Rat Following Chronic Administration of Morphine, lialorphine
and Normorphine. J. Pharmacol. Exp. Ther. 125:105, 1959.
10. Nash, T. The Colorinetric Estimation of Formaldehyde by Means of the
liantzsh Reaction, Biochem. J. 55:416, 1953.
11. Burns, J.J., Yu, T.F., Berger, L., and A.B. Gutman. Zoxazolamine:
Physiological Disposition, Uricosuric Properties, Amer. J. Med. 25 .•401,
1958.
12. Guarino, A.M., Gram, T.E., Schroeder, D.H., Call, J.B., and J.R.
Gillettt*. Alterations in Kinetic Constants for Hepatic Kicrosomal
Aniline Hydroxylase and Ethylmorphine N-Demethylase Associated With
Pregnancy in Rats, J. Pharmacol. Exp. Ther. 168:224, 1969.
-------
13. Guarino, A.M., Gram. T.E., Gigon, P.L., Green, F.E., and J.R. Gillette.
Changes in Michaelis and Spectral Constants for Aniline in Hepatic
Microsomes from Phenobarbital-Treated Rats. Mol. Pharroacol. 5:131,
1969.
14. Omura, T. and R. Sato. The Carbon Monoxide-Binding Pigment of Liver
Kicrosonves: II. Solubilization, Purification, and Properties, J. Biol.
Chem. 239:2379, 1964.
15. Bradley, J.V. Distribution-Free Statistical Tests. Prentice-Hall, Inc.
Inglewood Cliffs, NJ, 1968.
16. Reid, W.n., Krishna, G., Gillette, J.R. and B.D. Brodie. Biochemical
Mechanism of Hepatic Necrosis Induced by Aromatic Hydrocarbons.
Pharmacology 10:193, 1973.
17. Conney, A.H., Miller, E.G., and J.A. Miller. Unpublished Results Re-
ported in Ossch, F. Mammalian Epoxide Hydrases: Inducible Enzymes
Catalysing the Inactivation of Carcinogenic and Cytotoxic Metabolites
Derived from Aromatic and Olefinic Compounds. Xenobiotica 3:305, 1972.
18. Jollow, D. , Zampaglione, N., and J.R. Gillette. Mechanism of Protection
from Bromoben. •sne Hepatotoxicity by 3-Methylcholanthrene. Pharmacolo-
gist 13:288, 1971 (Abstract).
19. National Academy of Sciences. Nonfluorinated Halomethanes in the
Environment. Washington, D.C., 1978.
20. Ames, B.N., McCann, J., and E. Yamasaki. Methods for Detecting Carcin-
ogens and Mutagens With the Salmonella/Mammalian Microsome Mutagenicity
Test. Mut. Res. 31:347, 1975.
21. Smith, C.C., Lingg, R.D., and P.G. Tardiff. Comparative Metabolism of
Haloethers. N.Y. Acad. Sci, Conference on Aquatic Pollutants and
Biological Effects With Emphasis on Neoplasia. Ann. N.Y. Acad. Sci.
298:111, 1977.
22. Smith, C.C. and R.D. Lingg. Metabolism of 8-Haloethers in Rats and
Monkeys. International Symposium on Analysis of Hydrocarbons and Halo-
genated Hydrocarbons in the Aquatic Environment. May 23, 1978, Hamil-
ton, Ontario, Canada.
116
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APPENDIX
METABOLISM OF B-HALOETHERS IN RATS AND MONKEYS
Carl C. Smith, Ph.D., Department of Environmental Health
University of Cincinnati College of Medicine
Cincinnati, Ohio 45267
Robert D. Lingg, Ph.D., Health Effects Research Laboratory
United States Environmental Protection Agency
Cincinnati. Ohio 45268
Bis(2-chloroethyl)ether f^CEE) and bis(2-chloroisopropyl)ether (BCIE)
have been identified as contaminants in some drinking water supplies.
These compounds labeled with ^C, havo been administered orally to rats and
monkeys. As reported by Lingg ejt aJU (1978) most of the 14C following an
oral dose of labeled BCEE in rats appeared in the urine. Some 10% was re-
covered in the expired air as ^CO^, ancj almost none was exhaled unchanged.
Using a purging technique no volatile metabolites were detected. The urine
was separated by standard techniques into acid, neutral and basic fractions.
The acid fraction, containing essentially all the ''c activity, was deriva-
tized with diazomethane and a trimcthylsilylating agent (BSTFA) and analyzed
by GC-MS. Two metabolites, thiodiglycolic acid and 2-chloroethyl-B-D-
glucosiduronic acid, were identified in this acid fraction.
14
In the case of BCIE the rat excrotes C-containing metabolites pri-
marily in the urine and very little BCIE is excreted unchanged. Using the
same purging technique, three volatile urinary metabolites were detected.
These consisted of propylene oxide, l-chloro-2-propanol and 8-chloroisopropyl
isopropyl ether. No sex-related differences in the rodent pattern of
volatile metabolites was detected, Following isolation of the urinary acid
fraction and appropriate derivatization, two metabolites were identified by
GC-MS. These consisted of 2 (l-mcthyl-2-chloroethoxy) propionic acid and the
glucuronide of l-chloro-2-propanol.
In the monkey, metabolism of labeled BCIE leads to the same 5 volatile
and non-volatile urinary metabolites as were identified in rodent urine.
However, much less "C is recovered in urine and feces suggesting that more
BCIE or 14C metabolites are exhaled (Smith, 1977). No additional metabolites
were detected by these procedures. The sinian species appears to form the
glucuronide of l-chloro-2-propanol more extensively than does the rodent.
Our current information on metabolic fate of these compounds is
summarized as follows:
117
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3CEE
glucuronyl
transferase
Cl-CH2-CH2-0-glucuronide
(minor metabolite)
Reaction with
glutathione transferase
followed by several
steps
I
HOOC-CH -S-CH -COOH
(major metabolite)
BCIE
Cl-CH-CH-0-CH-CH,
2 I I 3
Cl-CH-CH-0-CH-COOH
2 I I
(minor metabolite)
(major metabolite)
reductive
dochlorination
dechlorination and
terminal oxidation
Ci-CH -CH-0-CH-C1
C'H3 «3
H
I
Glu.
currently unidentified
major metabolite
ether
cleavage
-HC1
Cl-CH0-C-0-Glu < C1-CH..-C-OH-
2 i \.. _ 2 |
at.
-XCH.-CH-CH
-9 3 2
minor metabolite
minor metabolite
minor metabolite
118
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References
1. Lingg, R.D., et al. (1978) Society of Toxicology - 17th Annual Meeting,
San Francisco, California, March 12, 1978 - Abstract No. 69 on page 59.
2. Smith, C.C. and R.D. Lingg. Metabolism of 6-Haloethers in Rats and
Monkeys presented at the International Symposium on the Analysis of
Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment,
May 23-25, 1978, Hamilton, Ontario.
119
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