Cancer Letters, 5.{1978) 1"-6
1
© Elsevier/N0rth:Ho]lland Scientific Publishers Ltd.
TISSUE-MEDIATED MUTAGENICITY O F VINYLIDENE C ~ L O R I D E IN SALMONELLA TYPHIMU~IUM TA1535
BRIAN K. JONEI~ and DAVID E. HATHWAY 1mperiaIChernicell Industries Lfmited. Central Toxicology Laboratory. Alderley Park. Cheshire. SKI O 4TJ (United Kingdom)
(Received 14 Ap, ii 1978) (Accepted 24 Aplr~l 1978)
SUMMARY Viny.lic~ene chloride is weakly posit.~ve in the Salmonella t y p h i m u r i u m T A 1 5 3 5 test, ~aediated by kidney and hvez post-mitochondrial s u p e n m t a n t (S-9 m i x ) f r o m n o r m a l mice, but strongly positive with the S-9 mix f r o m the induced animal,s. In t h e ease of mediation ~)y rat tissue, on~[y liver S-9 m i x from i n d u c e d anima!/s ~fffords a significant positive response. These findings agree with t h e greate'r availabflity in treated mice than in rats o£ reactive rinylidene chloride metabolizes, 1,1-dichloroethylene oxide and chloroacetyl chloride [~], a n d with tl~ie ~inylidene carc/nogenicity f o u n d in mice b u t n o t in rats [9]. Exploratory ti~ sue-mediated testing of vinylidene chloride involving fiver S-9 m i x ~rom mazmosets and m a n suggests a t r e n d in t h e generation of alkylating metabolites anq i their reaction w i t h bacterial D N A for t h e s e primates which resembles rats raore t h a n mice.
INTRODUC~Oh
R e c e n t work [5] emphasLzes t h a t the metabolic p a t h w a y (Fig.l) that has b e e n tentatively pro,posed for vinylidene chloride (a) (Fig. i ) in mamma~s [4, 6] does in fact operate for both mice and ra~s. However, an i m p o r t a n t difference between mice and rats con,;ists of t h e greater availability in m~ce o f 1,:[-~Jichloroethylene oxide (b) mid its rearrangement produc~, chloroacetyl chloride (c), d u e to their higher c y t o c h r o m e .P-450 activ.tty [7,1D] (Fig. 1). Reactf.ou o f this reactive metabolite 1,i[-dichloreethy~ene oxide or its rearrangem e n t pzc,duct with D N A is likely to be m o r e significant in mice t h a n rats. This 'biochmnical lesion', which raay in tumL initiate t h e carcin~genicity described [9] for v h w ~ d e n e chloride ~a mice (bu~ n o t in rats), migh~ be tested in experim e n t s w.~th m a m m a l i a n m i c m s o m e s and D N A in vitro, a n d recourse ha,; been h a d h~itia~ly to t h e ~sl~ system of Ame:~ [1, 2], as m o d e l e d to assess t h e m u t a -
,cb ½2C=CC12
P H2C"-CCl
"., ClCH2CCl
:
aSH I
~
C1CH2CO~|I
GSH
I'-C-CHCH SCI'I CC1 I II I 2 2a
HO2C-ICICH2 SCH~R 2 NH
0
Ac
(e)
-C-CHCH SC~4O~ H1 .
]
HO2C~IICH2S'~2(,O2H NH2
[oHHO2CICHCII2SCH2 (02H ]
l S (CH2CO2H) 2 (D
Fzg.i Vmthdene chloridemetabolismm rodents. genici~' of gases and vapours [8]. The present paper descnbes the results obtained and ~heir possible interpretation. 1V[ATERIALSAND METHODS V~aayl chlomde and vinylldene chloride were, supplied by ]rmperi~tl ChemicA Industries Limited, Mond Division, Runcorn, Cheshtre. Vinylid~ne chloride '~as usc.d in a stabilized form, i.e., in the presence of 100 ppm ofp-meA~cx:ypher~ol, in order to minimize the risk of peroxide formal,ion and spc.ntaneou~ polymerization. There was no danger of the antioxidam~ (b.p. 250°C) c l~mmg into contact with the ,~. typhzmunum organisms, since it is involatHe m)der the experimental condltmns that were used and would have remained in -,lae~rough. Mutagenicity assays were performed using the, Salmonella/mammaLian microsome mutagemclty test of Ames [2 [, suitably modified for sereeainc' gases [8], r~ad using stratus TA1535 and TA100. Disposable plastic Petri dishes containing Vogel ~3onner ba,,lalmeditma (Difco) were overlam with 2.0 ml 'top agm,' containing 0.:l mi overnight culture of the bacterial tester strata and mammaJjan tissue post-mitochondrim supematant (S-9, diluted 1--3 with cofactor) prepared eLthez from Aroclor 1254 reduced [1] rats (Spnng--Dawley male albino) and mine (Alderley Park Swiss-derivec[ male albino) or from uninduced ral~s and mice, marmosets (Alderley Park colony, 12-month-old males) and humans (suitable autopsy samples). The seeded dishes were exposed to atmospheres of ~qDC, VCM and 8Jr
inside gasf~,ightcultareve,~sels(Quickfit F V IL jars and N[AF 2/312 hds) during incubation at 37°C for 72 h. V D C was introduced, asa liquid,through gas., tight valves (Mininel~tvalves,Pierce Chemical Products)~ id V C M was mete::ed into the vesselsthroug~ c~dibratedrotmneters to produce nominally p~:escribed
gas/air mixtures. V D C was screened at a concentration of 5 % in air since preliminar,r te~ts showed that auch a ,concentrationhad no advcr~e effect upon ~he sU.,vivalof the bacterial~esterstrain and thiswas the m a x i m u m acceptable lew.~lbe)ow the explosive concentration of V D C in air. RE,~tULTS A N D DISCUSSIONS
Vinylidene chieride has been shown 'to be weakly positive (5-fold increase in mutation frequency) in the Ames test;, mediated by liver S-9 mix from rats which had been induced by polychlorinated biphenyl (Aroclor 1254), but it h ~ not been found t;o be mutagenic (less than a 2-fold increase) in the same test system meki:iated by kidney or liver S-9 mb~ from normal (uninduced) rats. In contrast, vinylidene chloride is weakly effective in the. Ames ~est, when mediated by no~mal rrmuse-kidney (2.3-fold increase) and -liver (1.6-fold increase) S-9 mix, but it is strongly positive when mediated by mouse-kidney (2;3-fold) and -liver (1.8-fold) S-9 mix from Aroclor-induced an:~nais (Table 1). These observations svggest that the mutagenic potential of vinylidene chloride del~ends to a considerable extent upon the prevailing degree of activation o~ relevent drug-metabolizing enzymes. The foregoing results agree (i) with the greal!er availability in mice than in rats of the reactive vinylidene chloride met~,bolites, 1,1~dict,~loroethylene oxide and its rearrangement product chloroacrtyl chloride [5], Cii) with the prediction [5] (reiterated in the introductory section) that their reaction with DNA is likely to b~ more significant in mice then rats, end (iii) with the incidence of lddney adenocarcinomas ha Swiss mice which had been exposed ehl:onically by :inhalation to 2!5 and 50 ppm of vim.clidene chloride, but not in Spr$4~e--Dawley rats exposed to 200 ppm [9]. It ought to be stated that sol:ae preliminary mutagenicity data for vinylidene chlolfide had been obtained prev:busly [ 31~in S. typhimurium mediated by ~issue from mice and rats. 'The present expe~nents were conceived in an entirely different connotation (v. supra), and the results obtained augment the e~rtier information [3]. The relevarlce of the reaction with glutathione catalysed by glutathione Sepoxide transferase to 1,1.dichloroethy[ene oxide detoxifieation has been mentioned previously (v. sup.re). In fact~ twice as much'of the resulting N-acetylS-cysteinyl ace~yl derivative (e) (Fig. 1) (where R is unknown) was formed in mice as in rats, and these relative proportions parallel the activities of glutathione S-epoxide transferase in the 2 species [5]. Since liver S-9 fractions from 24-h starved animals alre used in the Ames test, the relatively low concentrations of glutathione resulting ,nig]~t mitigate aga~st the-efficient 1,1-dichleroethylene oxide (b) detoxificat:ion, ce~alysed by glutathione S-epoxide transferase.
-~ +i -N
*i
+i ÷i ÷i +,
+i
+i ÷i
÷~÷i÷i~
~4
÷i÷i
@~dd
~;
~
÷i+i÷~+i
~ d 4
~ v
o
©
oN
°~ .~
Moreovet:, disposal of "[,1-dichloroethyleneoxide (b) by epoxide hydratase s e e m e d to be u n i m p o r t a u t in rive, as t h e u~timate p r o d u c t (CO2) was forraed in very snmll a m o u n t s [ 5]. A similar Constraint to t h a t for glutathione detoxffication o f 1,1-di~:hloroethyleneox/de (b) would appear t0 apply to glutathlone detoxificafion o f chloroacetic :acid (d) (Fig. 1), which is f o r m e d by s p o n t a n e o u s rearrangement of t h e epoxide and s u b s e q u e n t hydrolysis of the ch~oroacety] chloride (c) obtained, and which leads to thiodiglycollic acid (f) (Fig. 1). Thus, in t h e case o f an epoxlCe-genarating test-substance like vinylidene chloride° the expet/m(intal conditions o f the A m e s test would appear to be weighted in favour o:~ r e a c t i o n with bacterial DNA. In addition, vinylidene chloride has also been s h o w n to be weakly positive (3-~Vold l[ncrease) in the .4xnes tes~ mediated by liver S - g m i x from a h u m a n subject, vcho hac, been receiving long-term p h e n o b a r b i t o n e medication, but it has n o t been f o u n d to be mutagenic in t h e same test system, mediated by liver S-9 either f r o m normal m a r m o s e t s or from a (as far as can be ascertained) n o r m a l h u m a n subject (Table 1). The new data suggest, b u t do n o t prove, that t h e limited n u m b e r of primates examined in this w a y respond m o r e like the ra~s t h a n t h e mice with regard to generation of alkylating metabolites of vinylidene chloride, and their reaction with bacterial DNA. Simil~,r results to those reported in Table 1 were also obtained with stcain TAIO0. O u t o f t h e asymmetrically substituted chloroethylenes, vinyl chloride is the strongest m u t a g e n in the parallel tests, b u t it is the least toxic substance, and a 50% (v/v) atmospheric concentration is n o t antibacterial to the S. Typhi. murium organisms (Table 1 ). On the o t h e r hand, o u r preliminary mad exploratoz7 tests (unpublished results) showed t h a t 1,1,2-txichloroethylene, stabilized wit'h triethylamine, does n o t appear to be m u t a g e a l c w h e n tested at t h e t% level, b u t it is by' far t h e m o s t toxic of t h e 3 substances towards the S. typhimurium organisms amd is aatibacterial at a 5% concentration. Vinylidene chloride has intermedim~e properties. Thus, its mutagenic po~tency in the test approaches t h a t o f vinyl chloride only when mediated by m e u s e S-9 re'ix from induced an~nals; vinylidene chloride .is n o t toxic to bacteria at a 5% atmospheric concentra~ti.on° ACKNOWLEDGEMENT
We t~hank o u r colleagues, Dr. E. L o n g s t a f f and Messrs P.A. Lefevre and M. P e n m a n , for their greatly app:~eciated help and advice. ]~EF~RENCES 1 Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973) Carci~ ~gens are mu:mg,ms: a silmp~.etest syster~ combiningliver homo genates for activation and bacteria for de~eetion. F~:~c. Natl. Acad Sei., USA, 70, 2281--2~185. 2 An~es,B.N., MeCann~J. and Yamaski, E. (1975) Methods for detect:ing carcinogens and mutagens with the Sv:lmonella#nammalianmicrosome muta~enicity test. Mutat. Res., 31, 347--364.
3 Barf~ch, H., Malaveine, C., Montesano, R. ~md T0nmtis, L. (] 975)Tis,..me-mediated mutagenieity ofvinyIidene chloride and 2-cholorbut~tdiene in Salmonella typb~tzurlum. Natnre (London), 255, 6414-643. 4 Hathway , D.E. ( 1977) Comparative r~Lammalian mel;aboUsm.of vinyl and :vinylidene chlorides ifi relation to oncogenic potential. EnVirOn. He.~iltl~,Perspect., 21, in press. 5 Jones, BIK. and Hathway, D.E (1978) Species differ~mees in the metabolism of vlnylidene chloride between ~mice and rats. Br~'J Cancer, 37~ 411--416. '~' "~ .... 6 Jones, B.K. and Hathway, D.E. (1978) The biologic~l fate of vinylidene :chloride in rats.
Chem.-Biol. Interact., 20, 27--4].. 7
L~tterst,CL., Mimnaugh, E.C., Reagala, P~.L. m~d G~.am, I'.I~L(1975)Comparisc,n of in vitro dru~ mei~boHsm by lung,liver and Iddney of several c o m m o n laboratory sp~Jclies. Drug. Metah. Dispos., 3,259--265. 8 Longstaff. E. and McGregor, D.B. (1978) M~ntagenicity o~ a halocarbon re:~gerant, monochlorodifluorome~hane (R-.22) in Salmonella typhimuriam. Toxicol. Lett.,in press. 9 Maltoni, C., Cotti, G., Morisi, L. and Chieco, P. (1977) Carcinogeniclty bio-assays of vinylidene chloride; r~search plan and early r ~ mlts. )/Jed.Lay., 68,241--262. ~0 Yllner, S. (1971) Metabolism of chloracetat ;.1-~4C in the mouse. Acta Pha;:maeol. Toxic~L, 30, 69--60.
1
© Elsevier/N0rth:Ho]lland Scientific Publishers Ltd.
TISSUE-MEDIATED MUTAGENICITY O F VINYLIDENE C ~ L O R I D E IN SALMONELLA TYPHIMU~IUM TA1535
BRIAN K. JONEI~ and DAVID E. HATHWAY 1mperiaIChernicell Industries Lfmited. Central Toxicology Laboratory. Alderley Park. Cheshire. SKI O 4TJ (United Kingdom)
(Received 14 Ap, ii 1978) (Accepted 24 Aplr~l 1978)
SUMMARY Viny.lic~ene chloride is weakly posit.~ve in the Salmonella t y p h i m u r i u m T A 1 5 3 5 test, ~aediated by kidney and hvez post-mitochondrial s u p e n m t a n t (S-9 m i x ) f r o m n o r m a l mice, but strongly positive with the S-9 mix f r o m the induced animal,s. In t h e ease of mediation ~)y rat tissue, on~[y liver S-9 m i x from i n d u c e d anima!/s ~fffords a significant positive response. These findings agree with t h e greate'r availabflity in treated mice than in rats o£ reactive rinylidene chloride metabolizes, 1,1-dichloroethylene oxide and chloroacetyl chloride [~], a n d with tl~ie ~inylidene carc/nogenicity f o u n d in mice b u t n o t in rats [9]. Exploratory ti~ sue-mediated testing of vinylidene chloride involving fiver S-9 m i x ~rom mazmosets and m a n suggests a t r e n d in t h e generation of alkylating metabolites anq i their reaction w i t h bacterial D N A for t h e s e primates which resembles rats raore t h a n mice.
INTRODUC~Oh
R e c e n t work [5] emphasLzes t h a t the metabolic p a t h w a y (Fig.l) that has b e e n tentatively pro,posed for vinylidene chloride (a) (Fig. i ) in mamma~s [4, 6] does in fact operate for both mice and ra~s. However, an i m p o r t a n t difference between mice and rats con,;ists of t h e greater availability in m~ce o f 1,:[-~Jichloroethylene oxide (b) mid its rearrangement produc~, chloroacetyl chloride (c), d u e to their higher c y t o c h r o m e .P-450 activ.tty [7,1D] (Fig. 1). Reactf.ou o f this reactive metabolite 1,i[-dichloreethy~ene oxide or its rearrangem e n t pzc,duct with D N A is likely to be m o r e significant in mice t h a n rats. This 'biochmnical lesion', which raay in tumL initiate t h e carcin~genicity described [9] for v h w ~ d e n e chloride ~a mice (bu~ n o t in rats), migh~ be tested in experim e n t s w.~th m a m m a l i a n m i c m s o m e s and D N A in vitro, a n d recourse ha,; been h a d h~itia~ly to t h e ~sl~ system of Ame:~ [1, 2], as m o d e l e d to assess t h e m u t a -
,cb ½2C=CC12
P H2C"-CCl
"., ClCH2CCl
:
aSH I
~
C1CH2CO~|I
GSH
I'-C-CHCH SCI'I CC1 I II I 2 2a
HO2C-ICICH2 SCH~R 2 NH
0
Ac
(e)
-C-CHCH SC~4O~ H1 .
]
HO2C~IICH2S'~2(,O2H NH2
[oHHO2CICHCII2SCH2 (02H ]
l S (CH2CO2H) 2 (D
Fzg.i Vmthdene chloridemetabolismm rodents. genici~' of gases and vapours [8]. The present paper descnbes the results obtained and ~heir possible interpretation. 1V[ATERIALSAND METHODS V~aayl chlomde and vinylldene chloride were, supplied by ]rmperi~tl ChemicA Industries Limited, Mond Division, Runcorn, Cheshtre. Vinylid~ne chloride '~as usc.d in a stabilized form, i.e., in the presence of 100 ppm ofp-meA~cx:ypher~ol, in order to minimize the risk of peroxide formal,ion and spc.ntaneou~ polymerization. There was no danger of the antioxidam~ (b.p. 250°C) c l~mmg into contact with the ,~. typhzmunum organisms, since it is involatHe m)der the experimental condltmns that were used and would have remained in -,lae~rough. Mutagenicity assays were performed using the, Salmonella/mammaLian microsome mutagemclty test of Ames [2 [, suitably modified for sereeainc' gases [8], r~ad using stratus TA1535 and TA100. Disposable plastic Petri dishes containing Vogel ~3onner ba,,lalmeditma (Difco) were overlam with 2.0 ml 'top agm,' containing 0.:l mi overnight culture of the bacterial tester strata and mammaJjan tissue post-mitochondrim supematant (S-9, diluted 1--3 with cofactor) prepared eLthez from Aroclor 1254 reduced [1] rats (Spnng--Dawley male albino) and mine (Alderley Park Swiss-derivec[ male albino) or from uninduced ral~s and mice, marmosets (Alderley Park colony, 12-month-old males) and humans (suitable autopsy samples). The seeded dishes were exposed to atmospheres of ~qDC, VCM and 8Jr
inside gasf~,ightcultareve,~sels(Quickfit F V IL jars and N[AF 2/312 hds) during incubation at 37°C for 72 h. V D C was introduced, asa liquid,through gas., tight valves (Mininel~tvalves,Pierce Chemical Products)~ id V C M was mete::ed into the vesselsthroug~ c~dibratedrotmneters to produce nominally p~:escribed
gas/air mixtures. V D C was screened at a concentration of 5 % in air since preliminar,r te~ts showed that auch a ,concentrationhad no advcr~e effect upon ~he sU.,vivalof the bacterial~esterstrain and thiswas the m a x i m u m acceptable lew.~lbe)ow the explosive concentration of V D C in air. RE,~tULTS A N D DISCUSSIONS
Vinylidene chieride has been shown 'to be weakly positive (5-fold increase in mutation frequency) in the Ames test;, mediated by liver S-9 mix from rats which had been induced by polychlorinated biphenyl (Aroclor 1254), but it h ~ not been found t;o be mutagenic (less than a 2-fold increase) in the same test system meki:iated by kidney or liver S-9 mb~ from normal (uninduced) rats. In contrast, vinylidene chloride is weakly effective in the. Ames ~est, when mediated by no~mal rrmuse-kidney (2.3-fold increase) and -liver (1.6-fold increase) S-9 mix, but it is strongly positive when mediated by mouse-kidney (2;3-fold) and -liver (1.8-fold) S-9 mix from Aroclor-induced an:~nais (Table 1). These observations svggest that the mutagenic potential of vinylidene chloride del~ends to a considerable extent upon the prevailing degree of activation o~ relevent drug-metabolizing enzymes. The foregoing results agree (i) with the greal!er availability in mice than in rats of the reactive vinylidene chloride met~,bolites, 1,1~dict,~loroethylene oxide and its rearrangement product chloroacrtyl chloride [5], Cii) with the prediction [5] (reiterated in the introductory section) that their reaction with DNA is likely to b~ more significant in mice then rats, end (iii) with the incidence of lddney adenocarcinomas ha Swiss mice which had been exposed ehl:onically by :inhalation to 2!5 and 50 ppm of vim.clidene chloride, but not in Spr$4~e--Dawley rats exposed to 200 ppm [9]. It ought to be stated that sol:ae preliminary mutagenicity data for vinylidene chlolfide had been obtained prev:busly [ 31~in S. typhimurium mediated by ~issue from mice and rats. 'The present expe~nents were conceived in an entirely different connotation (v. supra), and the results obtained augment the e~rtier information [3]. The relevarlce of the reaction with glutathione catalysed by glutathione Sepoxide transferase to 1,1.dichloroethy[ene oxide detoxifieation has been mentioned previously (v. sup.re). In fact~ twice as much'of the resulting N-acetylS-cysteinyl ace~yl derivative (e) (Fig. 1) (where R is unknown) was formed in mice as in rats, and these relative proportions parallel the activities of glutathione S-epoxide transferase in the 2 species [5]. Since liver S-9 fractions from 24-h starved animals alre used in the Ames test, the relatively low concentrations of glutathione resulting ,nig]~t mitigate aga~st the-efficient 1,1-dichleroethylene oxide (b) detoxificat:ion, ce~alysed by glutathione S-epoxide transferase.
-~ +i -N
*i
+i ÷i ÷i +,
+i
+i ÷i
÷~÷i÷i~
~4
÷i÷i
@~dd
~;
~
÷i+i÷~+i
~ d 4
~ v
o
©
oN
°~ .~
Moreovet:, disposal of "[,1-dichloroethyleneoxide (b) by epoxide hydratase s e e m e d to be u n i m p o r t a u t in rive, as t h e u~timate p r o d u c t (CO2) was forraed in very snmll a m o u n t s [ 5]. A similar Constraint to t h a t for glutathione detoxffication o f 1,1-di~:hloroethyleneox/de (b) would appear t0 apply to glutathlone detoxificafion o f chloroacetic :acid (d) (Fig. 1), which is f o r m e d by s p o n t a n e o u s rearrangement of t h e epoxide and s u b s e q u e n t hydrolysis of the ch~oroacety] chloride (c) obtained, and which leads to thiodiglycollic acid (f) (Fig. 1). Thus, in t h e case o f an epoxlCe-genarating test-substance like vinylidene chloride° the expet/m(intal conditions o f the A m e s test would appear to be weighted in favour o:~ r e a c t i o n with bacterial DNA. In addition, vinylidene chloride has also been s h o w n to be weakly positive (3-~Vold l[ncrease) in the .4xnes tes~ mediated by liver S - g m i x from a h u m a n subject, vcho hac, been receiving long-term p h e n o b a r b i t o n e medication, but it has n o t been f o u n d to be mutagenic in t h e same test system, mediated by liver S-9 either f r o m normal m a r m o s e t s or from a (as far as can be ascertained) n o r m a l h u m a n subject (Table 1). The new data suggest, b u t do n o t prove, that t h e limited n u m b e r of primates examined in this w a y respond m o r e like the ra~s t h a n t h e mice with regard to generation of alkylating metabolites of vinylidene chloride, and their reaction with bacterial DNA. Simil~,r results to those reported in Table 1 were also obtained with stcain TAIO0. O u t o f t h e asymmetrically substituted chloroethylenes, vinyl chloride is the strongest m u t a g e n in the parallel tests, b u t it is the least toxic substance, and a 50% (v/v) atmospheric concentration is n o t antibacterial to the S. Typhi. murium organisms (Table 1 ). On the o t h e r hand, o u r preliminary mad exploratoz7 tests (unpublished results) showed t h a t 1,1,2-txichloroethylene, stabilized wit'h triethylamine, does n o t appear to be m u t a g e a l c w h e n tested at t h e t% level, b u t it is by' far t h e m o s t toxic of t h e 3 substances towards the S. typhimurium organisms amd is aatibacterial at a 5% concentration. Vinylidene chloride has intermedim~e properties. Thus, its mutagenic po~tency in the test approaches t h a t o f vinyl chloride only when mediated by m e u s e S-9 re'ix from induced an~nals; vinylidene chloride .is n o t toxic to bacteria at a 5% atmospheric concentra~ti.on° ACKNOWLEDGEMENT
We t~hank o u r colleagues, Dr. E. L o n g s t a f f and Messrs P.A. Lefevre and M. P e n m a n , for their greatly app:~eciated help and advice. ]~EF~RENCES 1 Ames, B.N., Durston, W.E., Yamasaki, E. and Lee, F.D. (1973) Carci~ ~gens are mu:mg,ms: a silmp~.etest syster~ combiningliver homo genates for activation and bacteria for de~eetion. F~:~c. Natl. Acad Sei., USA, 70, 2281--2~185. 2 An~es,B.N., MeCann~J. and Yamaski, E. (1975) Methods for detect:ing carcinogens and mutagens with the Sv:lmonella#nammalianmicrosome muta~enicity test. Mutat. Res., 31, 347--364.
3 Barf~ch, H., Malaveine, C., Montesano, R. ~md T0nmtis, L. (] 975)Tis,..me-mediated mutagenieity ofvinyIidene chloride and 2-cholorbut~tdiene in Salmonella typb~tzurlum. Natnre (London), 255, 6414-643. 4 Hathway , D.E. ( 1977) Comparative r~Lammalian mel;aboUsm.of vinyl and :vinylidene chlorides ifi relation to oncogenic potential. EnVirOn. He.~iltl~,Perspect., 21, in press. 5 Jones, BIK. and Hathway, D.E (1978) Species differ~mees in the metabolism of vlnylidene chloride between ~mice and rats. Br~'J Cancer, 37~ 411--416. '~' "~ .... 6 Jones, B.K. and Hathway, D.E. (1978) The biologic~l fate of vinylidene :chloride in rats.
Chem.-Biol. Interact., 20, 27--4].. 7
L~tterst,CL., Mimnaugh, E.C., Reagala, P~.L. m~d G~.am, I'.I~L(1975)Comparisc,n of in vitro dru~ mei~boHsm by lung,liver and Iddney of several c o m m o n laboratory sp~Jclies. Drug. Metah. Dispos., 3,259--265. 8 Longstaff. E. and McGregor, D.B. (1978) M~ntagenicity o~ a halocarbon re:~gerant, monochlorodifluorome~hane (R-.22) in Salmonella typhimuriam. Toxicol. Lett.,in press. 9 Maltoni, C., Cotti, G., Morisi, L. and Chieco, P. (1977) Carcinogeniclty bio-assays of vinylidene chloride; r~search plan and early r ~ mlts. )/Jed.Lay., 68,241--262. ~0 Yllner, S. (1971) Metabolism of chloracetat ;.1-~4C in the mouse. Acta Pha;:maeol. Toxic~L, 30, 69--60.
