Document M4LBbjL0QKZ0Vr1oL75N8xZzM

f. vi nyl Chloride K-1711-(100) fl Le ->.1 ,1 ,1-Trichloroethane K-l 71 6-(l 00) Trichloroethylene K-2520-(100) Perchloroethylene K--2521 -- (100) Butylene Oxide K-10841-(100) [mi] R&S 024312 February 1-1, 1933 TO: HSIA HEALTH ASSESSMENT GROUP RE: PPG GENOTQXICITY STUDY Gentlemen: Enclosed, for your information, find the final reocrt of tne study run for PPG by G. Williams covering the following: ,1,1-Trichloroethane 1,1,2-Tricnloroethane Tetrachloroethylene R&S 024313 EVALUATION OF SEVERAL HALOGSNATED ETH.--N'i --^NO ETHYLENE CONFOUNDS FOR GZNOTCXICITY Gary M. Williams, M.D. Tomi'<o Shi mac a , ?h.D. American Health Foundation Navlor Dana Institute for Disease Prevention One Dana Road Valhalla, N.Y. 10595 rtf*-- ~v Objectives Technical Report II. Materials and Methods A. Calculation of liquid volume to produce ^squired concentration of the compound in gas pnase. B. Salmonella/microscme mutagenesis assay 1. Mutagenesis assay 2. Cytotoxicity test 3. Interpretation of results C. nepatocyte Primary Culture (HPO/DNA repair assay 1. Initiation of HPC's 2. HPC/DNA repair assay 3. Interpretation of results III. Results A. Determination of actual concentra-ior. c.f cas chloride in chamber by Gas Chromatocrapn.- phase vinyl B. Salmonella/microscme mutagenesis assay C. HPC/DNA repair assay IV. Discussion V. References R&S 024314 W, Ara^VnffigjaSfeiiii,-.. limnruntn > R&s 024315 II. Materials and Methods A. Vapor Equivalent: Calculation of liquid volume required to produce a cesirec concentration of the compound in the gas phase: The vapor equivalent of liquids can be calculated by the following equation: V = C x VI x J2L * 111 x _L x 1x 22.4 760 d 10-2 Where VI: Chamber volume in liters MW: Molecular weight of a test compound T: Absolute temperature in chamber , ?: Atmospheric pressure in mm Hg d: Density (gram per ml.) of liquid test compound, V: Volume of the test compound to be used (in ml) * C: Target concentration (?) of test-compound m vapor m chamber. t* r7 isi Since the internal dimension of the chamber is 22.2x28.8x21.5 cm, the volume is 13.68 1. Since the exposures are performed at atmospheric U pressure P=760mm Hg and variations by temperature effects are small ffc-5 and are within sampling error range, the following is derived. '-3l -V *ShSf SP ,, C x 13.63 x MW d x 102 x 22.4 'sT l Examples of Calculated Liquid Volume used in the Chamber: Chemical Vapor Target Cone (%) 5.0 Vinyl Chloride n 1i Chemical Licuid Volume (ml) ......... 1,T,'-Tn- 1,i,2-Tri- Terracnloro- chloroethane chloroethvlene eohyler.e 3.T 2.7 3.1 33 & CO o ro co The test compounds examined in this study were vinyl chlo ride; 1,1,2-trichloroethylene, nor.-stabilized and stabilized; 1,1,1-trichloroethane, low-stabilized and stabilized; tetrachloroethylene, non-stabi1ized, stabilized, and with high purity (Table I). All test compounds were obtained from PPG Industries, Lake Charles plant. Analyses of purity were performed by the Barberton Technical Center. All test compounds were stored at -20*C at which vinyl chloride 'stayed in a liquid state._ C. Saimonella/microsone mutagenesis assay 1. Mutagenesis Assay; Modified exposure method: The assay developed by Ames (1) was modified for the study of gas phase compounds. The bacterial cultures were exposed in an aluminum chamber with internal dimensions of 22.2 cm X 28.3 cm x 21.5 cm and a plexiglass front door. Because of the size of the chamber and the possibility of compounds reacting with plastics, 60 mm glass petri dishes were substituted in place of conventional 100 mm plastic petri dishes. The internal surface area of the plexi glass door was covered with aluminum foil. Each petri dish was layered with 10 ml of minimum agar. The top agar mixture was prepared according to the conventional method by mixing 0.1 ml of the tester strain, 0.1 ml of phosphate buffer in place of a test compound, 0.5 ml of S-9 mix or phosphate buffer for activaticnfree exposures and 2 ml of molten top agar (total of 2.7ml), and 0.9 ml of this mixture was elated on top of the base acar layer R&S 024318 I .4^' **>*,*>,, j- '"''i**/ **v>' 1 .,. -** *.. f in triplicate petri dishes. Two hours was allowed for initiation of bacterial growth in soft agar. The exposure to gas phase of vinyl chloride (/C) was per- } formed as follows: calibrated volumes that woulc produce the desired gas'phase concentrations upon evaporation w-=re poured into a deep glass petri dish containing a magnetic bar. The dish containing liquid phase VC was immediately placed at t.ne bottom of the exposure chamber in which uncovered bacterial culture dishes were in place. The chamber was tightly closed and placed on a magnetic stirrer at room temperature for 20 min to- allow the evaooration of VC in the chamber. Then the cr.amber was trans ferred to a 37'C incubator and the exposure continued for 13 hrs. with continuous stirring to dissipate the gas evenly inside of the chamber. At the end of the exposure period, the gas phase compound was released from the chamber through a rear valve inside a hood with external exhaust. The bacterial cultures exposed to gas were removed from the chamber, ceverec with lids, and incubated for another 30-54 hrs. in the absence of tr.e test compound in an incubator. After a total of 43-72 hrs. incuba tion, the number of revertant colonies was counted and tne ave rage of triplicate plates was multiplied by 3 to adjust to con ventional assay conditions. The control plates used to score spontaneous revertants were prepared in similar manner ano incu bated in a regular incubator ir. the absence or test compounds. 2. Cvtotcxicicv Test: _- - --T In order to determine the cytocidal errects of caseous coi.i- pouncs, cytotoxicity tests were performed simultaneously with the 3SS?5KJ!S M mutagenicity studies. Nutrient agar plates overiayered with toe agar containing a tester strain that was diluted to the concen trations which yield a countable number of colonies and S-9 mix or phosphate buffer were placed in the chamber simultaneously with mutagenicity assay plates. Non-exposea control plates containing tester strains with or without S-9 mix were inccbazed in a 37"C incubator. The cytotoxicity of the test compounds is expressed as the percentage reduction ip. the colony number. 3. Interpretation of results: counted after total cf s wr.ich yielded 100 - 48 used for numerical id in this assay. Tr.e expressiuii uj. ______ _ average number of induced revertants for 3 plates after subtrac ting the number of spontaneous revertants is expressed as the number of revertants per dose per plate. Cytotoxicity of the compound at each dose is expressed as the reduction In numbers of colonies relative to that of the nonexposed set. The compound is reported negative in this assay if the number of induced revertants did not exceed twice the sponta neous revertants (1) at the highest doses that did not exceed 20% toxicity. r D. Kecatocyte Primary Culture (H?C)/CNA reoair assay '<. Initiation c-f Hepatocyte Cultures: Hat hepatocyte primary cultures (KPC's) were initiated according to the modified procedures of Williams (2). . .1 JO So CO foo -fe CO4 CD Adult male fischer strain 344 rats were anesthetized with' 50 mg/kg body weight Nembutal Sodium Solution (Abbott Laborato ries). Perfusion of liver was performed with a peristalic pump which was sterilized by circulation of 70 % ethanol followed by sterile watr. A ventral raidline incision was made from the xipnisternum to the pubic bone and the liver was exposed. A 2) gauge butterfly needle was inserted in the portal vein and clamped in place with a serrafine forceps. Perfusion was then commenced with 37*C ste rile Solution I which consisted, of 0.5 mM ethylene glycol-bis(3aminoethyl ether) N-N'tetracetic acid (EGTA) (Sicma) in Ca++, Mg++ free Hanks' balanced salt solution buffered with IQmM N-2hvdroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) (Calbiochem), cK Adjusted to 7.35 with IN NaOK. A perfusion rate of S ml/roin. is maintained for 1.5 min. Immediately following the start of perfusion with Solution I, ligation of the infrahepatic inferior vena cava was completed and the vein was severed distally to permit the perfusate to run o waste.i Whi1e this first perfusion was bei nc performe .horacic inferior vena cava was car.nulated by puncturing right atrium and the perfusate was collected via this return can nula. At this point, the proximal segment of the subhepatic inferior vena cava was clamped to close the system and the pump speed was then increased to 40 ml/min for 2.5 min. Completion of perfusion with Solution I was followed by per fusion with 250 ml of a sterile Solution II, containing 100 unit/ml type 1 coilacenase (Sigma) in williams medium E (WME) R&S 024320 1 I t buffered with 10 mM Hepes, pH adjusted to 7,35. This solution at 37*C was perfused for 12 min at 20 ml/min without recirculation of the return perfusate. The liver was covered with sterile, gaute and a 40W bulb positioned 6 cm above the liver for warming. Following perfusion, the liver was removed to a sterile petri-dish containing warm WMS and, in a sterile hood, was trimmed of extraneous fat and connective tissues. The liver was then removed to a second petri'-dish containing 50 ml of Solution II. The portis hepatis was grasped with forceps and the capsule of the liver was opened at numerous points on the inferior sur face with small scissors and removed. Cells were detached by gentle combing with a stainless steel comb, leaving a fibrcus plug of hepatic connective tissue to be discarded. Using a wide bore pipet, 25 ml aliquots of the suspended nepatocytes were pippetted into 50 ml centrifuge tubes, and the volume brought to 50 ml per tube with WMS supplemented with calf serum at 10% (V.'ME-CS i o) Cells were sedimented at 50xG for 25 min, suspended in W'ME-CS i q and gently mixed by inverting each tube several times. A 20-fold dilution of this cell suspension is prepared and 0.5 ml cf the diluted hepatocytes was added to 0.1 ml of 0.4% trvsar. blue stain (Gibco) for determination cf viability using a hemocytometer. f The perfusions performed in this study yielded 0.8-2.0x10 cells ter ICOg body weight and only those preparations with a cell viability higher than 90% were used. R&S 024321 2. HFC/DNA repair assay: a) Modified long term vapor phase exposure: The HPC/DNA repair assay developed by Williams (3) and modified (4) was fur ther adapted to the present studies as fellows: One million cells were seeded on 4.9 so cm glass coversiigs placed in 60, mm glass oetri dishes with 4 ml of WME-cSio- After 3 hrs of attachment, the cells were washed with L-15 medium 'phosphate buffered for use in non CO2 incubator) containing a total of 40uCi [3;-']Td.A {[^HjTdR L-15) for incorporation during ON'A. repair synthesis. The dishes were placed in the chamber and the test concentrations of liquid phase compound:; were introduced in the same manner as described in C.l. After 13 hrs exposure to test compounds, coverslips were processed according to the conventional method (3) for preparing autoradiographs. b) Modified short term vapor phase exposure: In order to assess repair of damaged DNA at reduced cytotoxic levels of test compounds, the exposure time was shortened. The cell preparation and exposure method remained the same as described in 2.a) except that exposures were reduced to 3 hrs. After this, exposed cul tures were removed from the chamber and washed with two chances of serum free Leibovich's L-15 medium. Then the cultures were fed with 4mi of [^HjTcR L-13 and incubated at 37*C for another 15 hrs to allow DNA repair synthesis. 3. Interpretation cf results: The scoring method routinely used was designed to avoid false positives by choosing she r.ighest cytoplasmic count of each cell as a background and by adjusting number of ceils than were -G scored to meet statistical significance, depending cr. the nuc lear/cytoplasmic grain count ratio. Cr.lv those cells which were viable at tr.e time or fixation, ^ indicated by swollen nuclei (as a result or mcuoaticr. in the hypotonic sodium citrate), and those evenly coatee wicn emulsion were scored. 3etween 5 to 20 cells randomly selected from each quadrant of the coversiip were counted. The number ot cells to be scored decer.cs on the r.uclear/cytopiasmic ratio ot grams required to meet statistical significance. Counts were obtained for each nucleus; background was determined by counting 3 nuclear sized areas adjacent to the nucleus. Net nuclear grain counts were calculated by subtracting the highest cytoplasmic count from the nuclear count. Results are reported as the mean standard deviation of net grain counts. The test compound is reported positive when the minimum net grain count exceeded 5 per nuclei ana was co.nsistantly above controls in 2 experiments. Cytotoxicity of the test compound was identified by the absence cf S-?hase cells in the autcr ad iocrapr. and by general morphology and scored according to the scale - (25% of total cells showing toxic effects), 2 + (50%'', 3 - (7d%) and 4 - (near ly 10C%). When there were not enough cells with intact nuclei and cytcclasma because of extensive cytotoxicity, tne sample was desicnatec as "not countable." R&S 024323 t III. Results A. Concentration of gas phase compounds Gas chromatographic analysis of gas phase VC in the chafr.ber (Table II) verified the reliability of the gas equivalent liquid calibration,- the transfer technique of liquid into the chamber anc the seal of chamber. Furthermore, in order to detect any possible reaction of. 3J 99 (0 o to CtoO 1,1,1-trichloroethane or 1,1,2-trichloroethylene with the alumi num of the exposure chamber, the concen.tr at ions of these gases in air in chamber were analyzed. As shown in Table III, gas chroma tographic analysis of these gases at 17 hours were within the range of sampling error indicating retention of these gases in the chamber and no reaction with aluminum. 3. Salmonella/microsome mutacenesis assay As shown in Tables IV-a and Fig. 1, vinyl chloride (VC) induced reversion mutation at the histidine locus of the strains TA100 and TA1535 in the presence of the activation system in a dose related manner without cytotoxicity. In the absence of the S' activation system, the recovery of revertants of TA1C-0 did not ,'fe exceed 2-roid the background, whereas with TA1 535 , mucat ions were induced. The greater increase in the number of revertants in the presence cf the Aroclor-mduced rat S-9 mix indicates metabolic activation of this compound. However, vc did not induce rever tants in the other strains examined (TAS8, TA1 533 and TA1537 ) ever, in the presence of an activation system (Table v) indicating TA100, TA93, TA1535, TA1533 and TA 1 537 . The only taster strains in which a near two-fold increase in revertants occurred were TA1GC and TA1535; therefore the assays were only repeated cn these two tester strains. 1, 1,1-trichioroethane with low concentration of stabilizer produced no increase in revertants in either TA100 or TA1535 at up to 7.5% vapor concentrations which killed more than 90% of the organisms. At 10%, which had greater than 95% toxicity/ rever tants were recovered at above 2-fold increases in strains TA7 00 and TA153S- The addition of an -9 preparation had no effect. 1.1.1-trichloroethane with stabilizer induced revertan: colonies of TAI00 at the concentrations of 5% and 7.5% only in the presence of activation systems (2.6-fold anc 2.9-fold increa ses respectively). These mutagenic effects oceured at levels of toxicity of 30% or less. The tester strain TA1535 was more sen sitive to this material by demonstrating significantly increased revertant induction even at the concentration of 2.5% (a 12.4fold increase in the absence of S-9 mix and an 3.3-fold increase in the presence of 5-9 mix)(Table IV-a). Non-stabi1ized 1,1,2-trichloroethylene showed extensive cytotoxicity without revertant induction at any dose in either TA100 or TA1535. 1.1.2- trichloroethylene with stabilizer killed both tester strains completely at the concentrations above 7.5%, but induced significantly high number of revertant colonies at 5% both in TA100 (greater than 4-fold increase) and TA1535 (greater than 37-fold increase) regardless cf the presence of S-9 mix. At this 3a1 o TO -U CO IcOn cose, toxicity exceeded 95%. With TA1535, revertant induction was also observed at the doses of 2.5% (4-fold increase) and 1% (2-fold increase) even in the absence of S-9 mix. Toxicity was moderate at these coses (Table iv-a). Non-srabi1ized tetrachloroethylene was cytotoxic at the doses of 5% and greater. An increased recovery of revertant colonies (5-fold increase) occurred only in TA1535 and at greater than 93% toxicity (Table IV-b). The presence of an S-9 fraction made no contribution. Tetrachloroethylene with stabiliser showed total cell kil ling at concentrations cf 3% and greater. At 2.5%, whicn was greater than 97% toxic, induced revertant colonies were observed in both tester strains TA1Q0 (2-fold increase) and TA1535 (10fold increase). In both strains, the presence of activation sys tems did not make a significant contribution. Tetrachloroethylene of high purity demonstrated total ceil killing effects at a concentration of 2.5%, but did not induce revertants in TA100 or TA1535 at lower doses either in the pre sence or absence of S-9. C. KPC/DNA repair assay As shown in Table VI exposure of hepatocytes to vinyl chlo ride in the gas phase induced unscheduled DMA synthesis indica ting the production of DNA damage to the hepatocytes. The incuction of DMA repair by vinyl chloride was observed at 5% without cell killing effect and was dose related within the coses we exa mined, 5%, 2.5%, 0%, bv modified exposure method (Table VI). Of six case materials examined in this assay, neitner 4 * 33 i fio (/) o to -C* CO ro o> 1i 1,1,1-trichloroethane with low or regular concentrations of sta bilizer, nor 1,1,2-trichloroethylene without or with stabilizer induced DNA repair even at doses that exhibited extensive cell killing either by the modifie-. gas phase exposure (Table VI) or by the conventional liquid phase exposure method (Tables vil-a and Vll-b). Tetrachloroethylene in the non-stabilized rorm eli cited DNA repair synthesis when hepatocytes were exposed wO 0.1% of this compound by the modified method and in the stabilized material at 0.1% (Table VI). Decrease in time of exposure to these compounds did not reduce their cytotoxic effect on hepato cytes nor did it induce DNA repair (Tables VI, VH-a and Vll-b). R&S 024327 --'i-*i--Urtj y^^AfV^.'-Vv.^ *vJ^V'^ iv. discussion In this study, the genotoxicitv of one chlorinated ethane and two chlorinated ethylene compounds was compared to that of vinyl chloride by measurement of mutation at the histidine locus in Salmonella tvphimurium and induction of unscheduled DNA syn thesis in hepatocytes. Compounds were tested by vapor phase exposure in both tests as well as liquid phase exposure in the HPC/DNA repair test. With respect to Salmonella/microsome mutagenicity test, mutagenic activity cf vinyl chi-oride, a positive control used .in this study, was in accord with a previous report (S). ' Exposure to 1, 1, l-trichloroethar.e with stabilizer increased the recovery of revertants of TA1535 and TA100 with little influ ence of the S-9 mix (Table IV-a, Fig. 3); however, 1,1,1-trichloroethane with low concentrations of stabilizer did not induce revertants (Table IV-a, Fig.2) within the dose range of 1,1,1trichloroethane with stabilizer that produced an increased revertant recovery. This suggests that the compound used to stabilize trichloroethane may be responsible for the induction of revertant colonies. The pH cf 1,1,1-trichloroethane was neutral at the time of testing and therefore acidic decomposition during storage did not occur. A potential role of the stabilizer in the induction of reverse mutation was also seen in the case of 1 , ,2-rrichloroethylene (TCE, Figs. 4 and 5). Trichloroethylene without stabilizer did not induce reverse mutations even at a dose that <illed 57% cf the cells (Table IV-a), whereas TCE with stabilizer R&S 024328 ; induced both high cytotoxicity (95%), and revertant colonies (above 4-fold in TA100 and 37-fold in TA98; Table IV-a). These data suggest that the induction of revertant colonies may be attributable to the stabilizer for this solvent but that cytotoxicity may be an effect of TCE itself. Atconcentrations of tetrachloroethvlene which were highly cytotoxic (97%), a greater'reversion response was produced by the stabilized material than the non-stabilizea, while no enhancement of revertants were seen following exposure to high purity tetrachloroethvlene. The induction of revertants by the stabilized products of these halogenated ethane and ethylene compounds demonstrates the need for consideration of the role of stabilizers in the mutagenicity of test materials that represent mixtures. The data are consistent with stabilizer involvement since there was no enhancement of revertants associated with the presence of the S-9 i fraction. Typical stabilizers, including those found in these solvents, are biologically active without enzymatic activation. Additionally, since the chlorinated ethane/ethvler.es did net exhibit greater reversion activity in the presence of S-9 (as did vinyl chloride, a true mutagen), this increased response at high cytotoxic levels does not lixely reflect true mutagenic activity. A previous report by Henschler et al. (9) on technical trichloroethylene also demonstrated the important role of stabilizers. They found that eoichlorohydrin and 1,2-epoxy butane, which were present as stabilizers, were highly mutagenic against Salmonella tester strains and suggested that they could be respensiole for carcinogenic effects in mice (9). 16 R&S 024329 7 " | | | I | ` The ocher important factor in the interpretation of results is the degree of cytotoxicity. The increase in revertant colonies at the coses which kill more than 951 of the ceils (Tables IV-a and IV-b) is of unknown mutagenic significance 1 because mechanisms other than these normally thought to be responsible for causing bacterial mutations (DNA-chemical interactions) may be operative. The different results in the mutagenesis assay between the benefice mutagen vinyl chloride (high reversion rate at low cytotoxicity, dose-response relationship) and the three sol-vents (an increase in recovery of revertents only at high cytotoxicity and a lack of cose-responsiveness) further support the interpretation that the latter materials possess no mutagenic activity. In testing the haloge.nated ethanes and ethylenes in the modified vapor phase HPC/DNA repair test, vinyl chloride was weakly active at the concentrations tested, and in the absence of cytotoxicity. Reduction in exposure time did not reduce the degree of cell killing but resulted in more extensive cell killing effects of some of these compounds, suggesting that maximum intracellular concen.tr at ions may be achieved very rapidly. Neither 1 ,1,1-trichloroethane or 1, i ,2-trichloro ethylene with, without or with low stabilizers, induced repair synthesis. However, increased grain counts were obtained with both nor.-stabilized and stabilized tetrachloroethyler.e at concen trations exhibiting cytotoxic activity. As shown in the bacterial studies, extraneous materials in the samples of tetrachloroethylene may be responsible for their L'TS activity. 17 R&S 024330 I 024331 In the conventional liquid phase H?C/DNn repair tes^( >-he test compounds produced no repair synthesis, although whe cos tive control, 2-acetylaminof luorer.e, was highly active. Never theless, the compounds were cytotoxic as witr. vapor phase exposure. The absence of positive results with tetrachloroethylene, as opposed to those obtained with vapor phase exposure, may indicate that the impurities postulated to be responsible tor this effect are either poorly soluble or highly volatile. In summary, based on the'present results, we find no evidence that 1,1,1-crichloroethane, 1,1,2-trichloroethylene and tetrachloroethylens P6r s6 sre cenowoxic* (/> w0 _ Ames, 3., McCar.n, J. and Yamasaki, E. ( 1 975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-micrcsome mutagenicity test. Mutstio: Res., 31:347-264. Williams, G.K., Bermudez, E., and Scaramuzzino, D. (1977). Rat hepatocyte primary cell cultures. Ill Improved dissociation and attachment techniques and the ennancement of survival by culture medium. In Vitro 13:509-317. Williams, G.M. (1977). Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary ceil cultures. Cancer Res. 37: 1845-1351. 4. Williams, G.M. and Laspia, M.F. (1979). The detection of various nitrosamines in the hepatocyte primary culture/DNA repair test. Cancer Letters 6:199-205. 5. Baden, J.M., Kelley, M.J., Ma2ze, R.I. and Simmon, 7.F. (1979). Mutagenicity of inhalation anesthetics; nitrons oxide, cyclopropane, trichloroethylene and divinyl ether. Br. J. Anaesth. 51:417-421. 6. Cerna, M. and Kvpenova (1977). Mutagenic activity of chloroethylenes*analysed by screening system tests. Mutation Res. 46:214-216. 7. Nestmann, E.R., Otson, R., Kowbel, D.J., Sothwell, P.D. and Harrington, T.R. (1932). Mutagenicity of fabric-protecting oroducts containing 1,1,1-trichloroethane m a modified Salmonella assay. Environ. Mutagen. 4:412-413. 3. Simmon, V.F., Kauhanen, K. and Tardiff, R.G. (1977). I Mutagenic activity of chemicals identified in drinking water. Prog. Genet. Toxicol. (Proc. Symp.) 2:245-2:3. 4 9. Her.schler, D. , Eder, E. , Neudecker, T. and Meczler, M. (1977). Carcinogenicity of Trichloroethylene: Fact or 1 Artifact? Arch. Toxicol. 37:233-236. > ! \ 1 9- R&S 024332 I \ f i TABLE I LIST OF TEST COMPOUNDS Chemical Vinyl Chloride (VC) 1>1, 2-Trichloroethylene iTCE) Stabilized (Trichlor 136) Son-stabilized (Trichlor 119) Container Stainless Steel ' Cylinder Assav 99.99% P.T.ber Glass Bottle 99.90% 1i1>1-Trichioroethane Stabilized (Triethane 324A) Lew-stabilized (Triethane 321) 99.30% Tetrachloroethylene (Perchloroethvlene) Stabilized (Perchlor 230) Non-stabilizea (Perchlor 200) Kiqh purity More retailed assay information on these materials will be maintained only by PPG for proprietary reasons. 20 - * Calculated Concentration3 Actual Concentration*3 0 Tiir.e TT~HrsT 10% 17.6% 15.9% 5% 1% control (blank) 7.24% 6.03% 0.59% 0.99% Not Detected a Vapor equivalent of liquid was calculated accorcir.c to the equation described in Materials and Methods. Gas samples were obtained at time 0, the immedia ely after the dissipation of gas phase vinyl chloride inside o the chamber and 17 hrs after the incubation at 37*c. 30 99 <n ro w W i * Compound 1.1.1-Trichloroethane low-stabilized 1.1.2- Trichloroethylene non-stabilized Actual Concentration3 0 Time 17 hours 5.0% 5.0% 4.2 % 4.5 % Vapor equivalent of liquid was calculated according to the equation described in Materials and Methods. The concentra tion of gas in air in chamber was analysed by gas chromato graphy at 0 time (immediately after the dissipation of cas inside the chamber), and at 17 hours. I 33 fie to 8 CO CO 07 t TAHLE IV-a ffAf.MONEhl.A/MICHOSOME MUTAGENESIS ASSAYS ON 11AI.OGEN ATEl) ETHANES AND KTHYI.ENKS BY MODI ELKO EXPOSURE MUTHODa 1,1,1 Tr ich toroeUmne low-stabiIized Cone {%) 10.0 7. 5.0 2.5 1.0 0. 1 10.0 7.5 5.0 2. 5 No S-9 No. of i nduced revertants: per plate^ TA TOO TA 15 35 With S-9 No Si-9 With S-9 192 { 100*)C 494 (100%) 190 (100%) 606 { 74%) 209 ( 75%) 4 13 ( (16%) 69 ( 00%) 200 ( 05%) 20 (100%) 120 ( 100%) 14 ( 100%) 112 ( 100%) 167 ( 100%) 20 1 ( 100%) 77 ( 04%) 1 1 3 ( 100%) 79 ( 77%) 54 ( 100%) 27 1 ( mo%) 9 55 ( 7(1%) 217 ( 90%) 170 ( 100%) 1 10 ( 90%) 37 ( 90%) 370 (1.4%) 2(1 (1.0%) 0 < 79%) 0 ( 0 6%) 423 (0.4%) 93 (2.2%) 2 { 091.) 14 ( 100%) 7 3 (4.5%) 5 (4.1%) 12 ( 69%) 6 < 90%) 90 (1-3%) 2 ( 1.7%) 0 ( 70%) 1 ( 94%) 1, 1,2 Trichloroethylene non-stabi1ized 10.0 7.5 5.0 2.5 1.0 10.0 7.5 5.0 2.5 1.0 92 (2.5%) 3H (t-2%) 3 02 ( 71%) 105 ( 93%) 69 (100%) 0 (2.6%) 0 ( 21%) 7 ( 95%) 1 13 (1.5%) 4 16 (0.9%) 461 ( 74%) 315 ( 07%) 146 ( 7 1%) 25 (1.3%) 24 7 (7.4%) 338 ( 09%) 248 (100%) 12 ( 811) Mn Growth No Growth 0 (2.7%) 0 (4.4%) 0 ( 14%) 57 ( 95%) 7 ( 41%) 31 ( 91%) 20 (2.2%) 234 (4.0%) 366 ( 60%) 190 ( 95%) 0 ( 97%) 0 (2.6%) 0 ( 21%) 17 ( 95%) stabilized 10.0 7.5 5.0 2.5 1.0 722 23 17 to til e (2.0%) ( 10%) ( 41%) method No Growth . No Growth 7 69 (3.0%) 36 ( 17%) 42 { 46%) described by Amo s 939 116 43 ( 1 ) with (4.7%) ( 33%) ( 08%) s\ ight 904 (3.6%) 109 1 41%) 25 { 00%) mudiiicyt: i ijus as described in Materials and Methods. Tester stains with S-9 mix or with buffer solution were | mured onto basal agar medium and incubated at 37*C for 2 1/2 hrs. to allow initiation of cell growth, then exposed to gas phase of each compound in tightly sealed chamber for 10 hrs. Average of 2 experiments each consisting of 3 replicate plates. Figures represent total reveitaut *; minus spontaneous rovertants. Surviving fraction of cells as determined by simultaneous cytotoxicity test. "No growth" indicate no surviving cells on cytotoxicity test plate. - 23 - a,.... I mm TABLE MHMkanai SALMONELLA/M ICRO.50MB `MUTAGEN L5S IS ASSAYS ON HALOGBNATED ETHANES AND BTHYl.BNBS I3Y MODIFIED EXPOSURE METHOD3 Compound Cone (%} No S-9 No. of induced revertant per plate** TA 100 With S-9 No S-9 JZ with S-9 Te trachloroethy 1 one non-stabi1ized 10.0 75 5.0 2.5 1.0 0. 1 No Growth No Growth No Growth 243 ( 1.91) 269 . (1.11) 11 ( 20%) 34 ( 31%) 0 ( 71%) 0 ( 02%) 1 35 ( 1.5%) 16 ( 24%) 10 ( 77%) 14 0 ( 1 . 1%) 9 ( 26% ) 3 ( 0 3%) stabilized 10 0 7.5 50 2.5 1 .0 0.1 No Growth No Growth ______________ __ No Growth 3 15 (2.3%) 356 ( 1-7%) 199 (1.0%) 240 (2 .2%) 0 ( 20%) 0 ( 33%) 0 ( 41%) 0 ( ?.R%) 0 ( 71%) 0 ( 00%) 7 ( 90%) 0 ( 04%) Tetracliloroethy lene High Purity 2.5 1.0 0.1 No Growth 0 (0.6%) 0 (4.0%) 0 (4.0%) 19 ( 10%) 11 ( 2 4 ( 22%) 19 ( 39%) 24 ( 55%) Spontantous Revertant! 177 157 20 24 aThe assays were perfotmed according to the method described by Ames (1) with slight modifications as described in Materials and Methods. Tester stains mixed with S-9 mix or with buffer solution wen; poured onto basal agar medium and incubated at 37*C Cor 2 1/2 hrs. to allow initiation ol: cell growth, ='tlien exposed to gas phase of each compound in tightly sealed chamber Cor 18 hours. Average of 2 experiments, each consisting of 3 replicate plates. Figures represent total revertants minus spontaneous revertants. f cSurviving fraction of cells as determined by simultaneous cytotoxicity test. "No growth" indicate!; 't&Z no surviving cells on cytotoxicity test plate. LPZ0 SSU - 24 - ij^Ti^iff*^i >,M,,<.,,,,||. ii ijl'hQii f **suyw^*'i'l''.'.">^ afl*w. TABLE V SALMONKLLA/M ICHOSOMH MUTAGENESIS ASSAYS ON IIAl.OGENATED ETHANES AND ET1IYLENEB BY MODIFIED EXPOSURE METHOD'1 Compound Vinylchloride Cone.{*) 7.5 1,1,1-Trich loroothane low-stabi l ized 10.0 stabi1ized 5.0 1,1,2-Trichloroethylene non-stabi1ized 2.5 stabilized 2.5 Tetrachloroothylone non-stabi1ized 1.0 stabi1ized 1 .0 No. o induced revcrtimts p<*r pi fite*3 TA 90 No S-9 With S-9 to ( 1 001) c 17 ( 100%) TA 1530 No S-9 With S-9 0 ( 100%) 10 ( 100%) TA 15 37 No S-9 With :. 9 4 (00%) 1 ( 100%) r (17%) 3 (04%) 12 ( 00% ) 9 (94%) 19 (17%) 0 ( 100%) 10 ( 10%) 0 (100%) 0 (10%) 0 (91%) 0 (7.8%) 0 ( 7 6% ", 0 ( 30%) 2 ( 34%) 0 (23%) 2 ( 22%) 0 (40%) 0 ( 23%) 1 (46%) 0 (23%) 0 ( 50% ) 0 ( 11%) 0 { 571.) 1 (H%) 10 (47%) r> (49%) 9 (43%) 12 (35%) 3 ( 39%) 0 (40%) 3 ( 39%) 4 (39%) 8 (39%) 1 (36%) 4 (45%) 1 ( 40% ) Spontaneous Revertants 47 47 20 19 8 11 a The assays wore performed according to the method described by Ames (1) with slight mod i f i cut i nun as described in Materials and Methods. Tester stains with S-9 mix or with buffer solution were pouted onto basal agar medium and incubated at 37*C for 2 1/2 hrs. to allow initiation of cell growth, then exposed to gas phase of each compound in tightly sealed chamber Cor 18 hrs. ^ Average of 2 experiments each consisting of 3 replicate plates. Figures represent total roverI.anl ; minus spontaneous revertants. c Surviving fraction of cells as determined by simultaneous cytotoxicity test. "No growth" indicate *i no surviving cells on cytotoxicity test plate. 8Efr20 SSfcl - 23 - ta(UjK vr 1IPC/DNA UK PM It assay;? on iim/xiknated rtiianrs and rtiiylenks oy MOD t Kl Kl) RXl'OiJHRE MKTtlOD*1 Compound Vinyl Chloride 1,1,1 Tr ichloroethane low-stab! 1 izcd Cone. (% l*1 s.O 2. r> 2. 5 1.0 0. 1 lixpo;rure__TLmo _ -J~Inrs. T0'"li'rs. An torad Lograph Autoradiograph Crains/Nucleus Cytotoxicity Grains/Nucleus Cytotox: Not Done Not Done 11.341.9 c 4.943.5 0 0 Not Countable 0.G 1.7 0.042.2 4+ 4+ 34 2.142.0 4.63.0 3. 140.9 11 n 0 stabiiized 1,1,2 Trichloroethylene non-stabilized 5.0 2.5 1.0 0. 1 2.5 1.0 0. 1 1.345.0 1. 142.0 0 0 0 0 .9241.0 44 Not Done 34 Not Countable 2.4 .844.7 11 0 .764.4 11 24 .204.2 4 1- 24 3.242.0 44 2K 2.54.9 4 i stabilized Tetrachloroethylene non-stabilizcd 2.5 0.240.4 1.0 1.041.8 0. 1 1.042.2 2.5 Not Countable 1.0 Not Countable 0. 1 10.846.1 2+ 24 24 44 44 34 Not Countable 1 . 243.3 2.841.3 Not Countable Not Countable 2.54.9 1 I 0 0 11 4t 14 stabilized 2.5 Not Countable l .0 Not Countable 0. 1 6.244.9 4+ 44 24 Not Countable Not Countable 15.941.6 44 44 1f Cells attached on glass covers lips in reduced volume of culture medium were exposed to gas phase of each compound in tightly sealed chamber for designated period of time. Modification of the meI hod developed by Williams (3). k Rach compound was introduced into the chamber in liquid phase at the volume that produce desired concentration in gas phase according to the equation described iri Materials and Methods. c Average of 2-3 coverslips of each, 20 nucleus were counted. - 26 - eeefrzo ssh TAULK VU-a IM'C/ONA REPAIR ASSAYS ON IIALOCKNATRI) KTIIANRS AND ETIIYT.ENKS I1Y CONVENTIONAL MRTHOI)a Ji ltiecs. Exposure Time - _ "IB1 (1 fIIirIsh.,' Compound Cone. ( ?.) Autoradiograph Grains/Nucleus Cytotoxicity Autoradiograph Grains/Nuclcus Cyiotox 1,1 Trichloroethane low-stabiiized stabi1ized 1.0 0. 1 0.01 0.001 0.0 00 t 1.0 0. 1 0.0 1 0.0 0 t Not Countable Not Countable Not Countable 0 0 Not Countable 0.t 0.2 0 0 4t 41 2+ 2t 0 4I 24 2I 0 Not Countable Not Countable .5 t.4 .0 t.2 0 Not Countable Not Countable .3 J .r> .7 *. 7 4) 11 1i (1 0 4t 4t 4l 0 1,2 Trichloroethylene non-stabi1ized stabi1ized 1.0 0. 1 0.01 0.00 1 1.0 0.1 0.01 0.001 Not Countable Not Countable 0 0 Not Countable Not Countable 0 0 4t 4+ 4+ 2t 4+ 4+ 2+ 0 Not Done Not Countable .2 .9 Not Countable Not Countable .6 * .0 .7 *1.0 4t 11 H 4* 1t 0 a The an says were performed according to the method described by Williams (3) with slight modifications as described in Materials and Methods. .Vinyl chloride was not tested by this method, k Rach compound was- introduced into the petri dish in liquid phase to have desired final concentration. c Positive control, d Negative control. - 27 - omzo S9u TABLE Vtl-b lll'C/DNA RKPAIU ARRAYS ON IIAl.OGENATED ETHANES AND KTUYbKNER MY CONVENTLONAI. METHODn Compound Cone.(H) Exposure Time XlTrs. ' f if'fir s~. Autorad iograpfi A u to rad fog r a pi i Grains/Nucleus Cytotoxicity Grains/Nucleus CyInto*icily Tetrachloroethylene non-stabilized 1.0 0. 1 0.01 0.00 1 0.0001 Not Countab)e Not Countable Not Countable 0 1.0*2.23 Ar 4+ <1 + 3+ 3+ Not Done Not. Countable Not Countable .2 *. ! .4 .2 4t 4i 11 1l stabilized 1.0 0. 1 0.01 0.001 Not Countable Not Countable Not Coun tab)e 0 4r 4t 4i 2+ 2 Acetylaininof luorenec io_5m 160 .0*74.9 0 Ptuorcne^ 10-5m Not Done ! Pyrene 10-^M 0 0 Cell Control 0 0 0 U1M.0 . 1* .1 170.2*20.6 1.6* .6 2.75*2.76 Not Done Not Done Not Done 4y 11 t) 0 0 0 The assays were performed according to the method described by Williams (3) with slight modifications as described in Materials and Methods. Vinyl chloride was not tested by this method. ^ Each compound were introduced into the petri dish in liquid phase to have desired fLnal concent.rat. ion, c Positive control. ^ Negative control. - 20 - ttetzo svu ;~iiJ ' - A* ' ->* 'Vf* " R&S 024342 VINYL CHLORIDE F ic Dcse-response curves for vinyl cnloride with Saiir.one 1 ia tvo'nimurium tester strains with (--o--) and without {--. activation system containing 150ul of oclcr induced ra 5-9. Each point represents the geomet ic mean of triplicate plates from one experiment, Spontaneous revertants are indicated by arrows. 2? ; f f i? i ?ia*2. 1,1,l-TRICHLOSOETHANE, Low-stabilized Cose-resoonse curves for 1, 1 ,1-trichlcroechane witn low concentrations of stabilizer with Salmonella typhiir.urturn strains with (--o--) and vitncut (-----) sc^ivation svstem containing 150ul or Rrocacr induced raw i.-9. Each point represents * the geometric mean of triplicate dates frcrr. one experiment* Spontaneous revertants ate indicated by arrows. - 30 - R&S 024343 i !'U. :!o 2.: 5.3 ::.a \ R&S 024344 * l VVe**, " ,,'"* $ J*'.`Vf - ">* ,.* - -----"- *-*-< zfxpilfiarv' f`* .. 'W^**v*<fv* ' ^ *v:.-nwv *<* rx :5J5 i I 310 150 - 1.3 2.5 5.0 a loo '.0.0 ' Fig.4. o;o 1C.0 1,1,2-TRICHLOROETHYLENE, Non-stabiIized Dcse-resoonse curves for 1 ,1 , 1-trichioroechylene without stabilizer with Salntor.e 11 a typhiir.urlut teste, strains with --o--) and without (___) activation evsrayt containing IcOul or iroclor inctceo ra. s-.. Each ooir.t reoresents the geometric near, or rri?lic=-s elates f r on one experiment. Spontaneous reverta.nts a.*' indicated bv arrows* - 32 - 31 * W o N> b c*n R&S 024346 ? R&S 024347 t.-, isis Trt 100 i } f 'er ttrachictoat'nyl*ne without SiilSir with Salmonella tuoh^uti^ t . e s..;-na ' r* ( -- -- c -- i d ontaininc :5 0ul presents the r.e experiment. trows. L '- - w ' ,* of Acoclor i-^ueea saoi.t-o-jw --c 0 -SC** CO ... ^flon ;re indicated oy - !. i. !: i' i);. *' "A 152* TA IDO 30 B CO o to -u CO GO Sic . 7 J.*. .-V 5-0 D.j lo.o TETRACH LC RO STHYL EM E, Stabilized Dose-response curves for tetrachioroechylene with stabilizer with Salmonella tvphimurium tester strains with (--o--) and without (----) activation system containing 153 ul of Arcelor induced rat S-S. Each point represents the geometric mean of triplicate plates from one experiment. Spontaneous revertants are indicated bv arrows,. - 35 - APPEND 1 X t-a: SUMMARY OF FIRST SET OF KXPER1 MliNTS SALMON ELI tA/M ICROSOMK MUTAGENESIS ASSAYS ON ItAlOGENATED ETHANES AND ETHYLENES*1 " Number of. Total Revertants per Plate Compound Cone. (%) Vinyl Chloride 1,1, 1-T'r i chi or oo thane low-stabilized 10. 0 7.,5 5.0 2. 5 1.,0 0. 1 10..0 7..5 5..0 2..5 TA-100 _______________________ ________ TA-15.15 without S-9 with S-91 w 1 thout. S-9 with S- 9 297, 37 5, <100 , 222 , 213, 210, JJ0, 354 , 4 60 , 2 67 , 17 1 , 2 37, 369b 37 2 4 14 249 207 106 730, 013, 6 B 4, 354, 306, 279, 670, 0 37 , 690, 369, 264, 252, 762 079 435 372 205 276 174 , 225 , 93, 120, 90, 75, 159, 2 10, 105, 156, 1 14, 66, 130 2 20 93 123 63 01 250 , 300, 369 094, 1014, 1029 225, 315, 10 I 195, 192, 195 126, U, 167 63, 60, 60 5 34 , 135, 100, 160 , 474 , M7, 147, 135, 53 1 234 1 56 159 409, 190 , 105, 195, 591, 200, 126, 162, 555 135 160 204 70, 66, 72 21, 33, 30 30, 39, 39 39, 27, 2 1 111, 90, 96 12, 21, 24 24, 15, 1 ) 33, 15, 2 f stabilized , 1,2-Trichloroethylene non-stabi1ized 10..0 7 .. 5 5..0 2..5 1,.0 10..0 7..5 5..0 2.. 5 1..0 294 , 520 , 4 7.9 , 342 , 200 , 249 , 4 32 , 456, 32 t , 237 , 159 513 4 47 340 306 0, 0, 0, 100, 246 , 0, 0, 0, 150, 190, 0 0 0 132 129 2 50 , 477 , 555, 402, 354 , 200 , 6 00, 591 , 495 , 360, 240 642 612 307 297 0, 0, 0, 135, 267 , 0, 0, 0, 96, 190, 0 0 0 165 231 42, 237, 310, 252, 27, 54, 3 24 , 3 36 , 300, 30, 45 240 342 24 3 33 0, 0, 0 0, n, 0 0, o, 0 24, 36, .18 72, 70, 75 33, 192, 313, 252, 30, 10, 261 , 304 , 24 6, 10, 60 3111 264 2 37 27 0, 0, 0 0, '0, 0 0, 0, 0 24, 30, 15 60, 40, 48 stabi1ized 10,.0 7,.5 5,.0 2,.5 1,.0 Spontaneous Revertants 0, 0, 0 102, 70, 90 690, 1020, 1014 153, 129, 144 177, 1110, 240 0, 75, 1023, 130, 106, 0, 39, 861, 171, 165, 0 10 9 12 117 237 10 3, 213, 1 71c . 17 1, 150, 150 0, 90, 1042, 138, 70, 0, 0 48, 30 037, 1002 14 1, 1 17 75, 63 24, 21, 24 0, 0, 120, 30, 805, 11(11, 90, 120, 39, 0 0 36 375 1)4 75 10, 10, 24 a The assays were performed according to the. method described by Ames (1) wilh slight modi 1 icaliour. <i described in Materials and Methods. Tester stains with S-9 mix or with buffer solution w? e ponied onto basal agar medium and incubated at 37'C for 2 1/2 hrs. to allow initiation of cell growih, the exposed to gas phase of each compound in tightly sealed chamber Cor 10 hrs. b Total number of reverfants for each triplicate. c Spontaneous revertants for each triplicate from vinyl chloride 2.5% experiment. 6WZ0 SSH APPENDIX I-h: SUMMARY OF FIRST SET OF EXPERIMENTS SALMON ELI, A/M I CROSOME MUTAGENESIS ASSAYS ON UAI-OGENATED ETHANES AND ETIIY DENES'* Number of Total Itcvcrtants per Plate Compound __ TA-100 Conc.(%) without S-9 with S-9 ___ TA-1535 without S-9 wftii S-9 Tetrachloroethyleno non-stab L1ized 7.5 0, 0, 0 0, 0, 0 5.0 0, 0, 0 0, 0, 0 2.5 135, 4 0 3 , 399b 405, 4 35 , 423 1.0 20 1 , 147, 24 3 171, 225, 1119 0. 1 159, 111, 1 47 100, 163, 17 1 0, 0, 147, 45, 24, 0, 0, 156, 51 , 36, 0 0 126 21 40 0, Or 162, 30, 27, 0, 0, IlH, 3 1, 42, 0 0 18 1 36 28 r.tabi lized Tetrachloroethyleno high purity 10.0 7.5 5.0 2.5 t.o 0.1 2.5 t.o 0. 1 0, 0, 0 Not Done 0, 0, 0 460 , 5 6 1, 534 57, 36 , 33 129, 153, 144 0, 0, 0 99, 84, 87 123, 1 38, 156 0, 0, 0 Not Done 0, 0, 0 60 6 , 538 , 53 1 12, 30, 54 144 , 147, 156 0, 0, 0 0 1, 75, 96 153, 144, 106 0, 0, 0 Not Done 0, 0, 0 291 , 2 50 , 2 64 10, 30, 24 24, 33, 45 0, 0, 0 24, 36, 21 27, d 2, 33 0, 0, 0 Not Done 0, 0, 0 321 , 36 3, 3 15 18, 12, 111 12, 12, 18 0, 0, 0 4 0 , 36, 51 30, 42, 48 Spontaneous Revertants 182, 2 13, 174 171, 150, 150 24, 24, 24 18, M, 21 a The assays were performed according to the method described by Arnes (1) with slight modifications as described in Materials and Methods. Tester stains with S-9 mix or with buffer solution were poured onto basal agar medium atul incubated at 37*C for 2 1/2 hrs. to allow initiation of cell growth, then exposed to gas phase of each compound in tightly sealed chamber for 18 hrs. b Total number of revertants Eor each triplicate. c Spontaneous revertants for each triplicate from vinyl chloride 2.5% experiment. osetzo S9u MMgfJ -'i^tl Compound APPENDIX 11-a: SUMMARY OF SECOND SET OF EX PEE 1MENTS S A [.MONET, LA/H IC110 SOME MUTAGENESIS ASSAYS ON MALOGENATED lO'H ANES AND HTI1YLENES*"1 Number of Total Uovertants per Plate Cone.(%) TA-100 without S-9 with S -- 9 TA-1535 without S -9 w t tii ;; - 9 Vinyl chloride 1,1,lTrichlorocthane low-stabi1ized 20.0 10.0 5.0 10.0 7.5 5.0 2.5 453, 429 , 420b 4 30 , 301, 396 312, 3 60, 35 1 f> 0 3, 231 , 15 1 , 102 , 561, 30 3 , 10 6, 1.15, 579 2 10 2 19 2 07 stabiiized 10.0 279 , 3 12, 321 7.5 52(1, 432, 513 5.0 4 60, 4U9, 502 2.5 .17 2, 35 1 , 135 t .0 ir,5, 252, 220 1,1,2-Trichloroethylene non-stabi1ized 7.5 ' o, 0, 0 5.0 o. 0, 0 2.5 165, 103, 174 1.0 100, 177, 174 stab L1ized 7.5 93, 70, 99 5.0 894 , 861 , 915 2.5 250, 234, 202 1.0 210, 147 , 204 Spontaneous Revertants 103, 144c 550 , 525, 505 610, 633, 594 573, 505, 450 621, 333, 7.13, 111, 6 15, 294 , 174, 153, 606 252 160 195 276 , 477, 657 , 47 1 , 202, 20 0 , 600, 627 , 534 , 26 I , 267 642 663 543 264 0, 0, 130, 106, 0, 0, 174, 100, 0 0 171 213 72, 1026, 174, 109, 90, 825, 202, 210, 26 906 273 207 150, 1 20 207 , 240, 204 17 1 , 10 3, 190 01, 102, CK) 102, 77, 33, 17, 173, 15, 77, 30, 1 14 24 21 21 40, 237 , 399 , 252, 21, 54, 374 , 363, 20 7 , 36 , 27 210 30/ 276 42 0, 0, 0 0, o. 0 30, 33, 21 24, 10, 36 63, 93 3, 177, 39, 51, 963, 132, 54, 40 970 111 66 15, 15 309, 260 , 7 7H 2 7 6 , 276 , 12 1 210, 10 1, 19(1 177, 30, 30, 24, 120, 30, 21, 21, 1 35 39 15 33 4 2, 192, 471 , 162 , 27, 40, 264 , 165 , 225 , 12, 30 3 10 44 1 207 10 0, 0, 0 0, 0, (1 10, 30, 24 2 1 , 27, 4 2 45, 36, 6 36 , 1041, 147, 17 1, . 27, 36, 60 9 27 15 1 54 30, 21 n The assays were performed according to tlie method described by Ames. (1) with slight modifications as described in Materials and Methods. Tester stains with S-9 mix, or, with buffer solution w*ve poured ontobasal agar medium and incubated at 37*C for 2 1/2 hrs. to allow initiation of cell growth, then exposed gas phase of each compound in tightly sealed chamber for 1R hrs. 1 Total number of revertants Cor each triplicate. c Spontaneous revertnnts for each duplicate from vinyl chloride 5$ experiment. % Compound APPENDIX U-b: SUMMARY OP SECOND SET OE EXPERIMENTS SALMON EM. A/M I CKO SO ME MUTAGENESIS ASSAYS ON HALOGEN ATEN ETHANES AND ETHYLENE,S'1 Number of Total Revertants per Plate ic.m TA- 100 w 11 hlOUt S-9 with S- 9 TA- 1535 -- without S -9 with S-9 Te trichloroethylene non-stabi1ized stabilized Te trichloroethylene high purity Spontaneous Revertants 2.5 1 .(1 0. 1 2.5 1.0 0. 1 2.5 1.0 0. 1 304 , 396, 4 2 3l} 20 1 , 159, 177 Ml, 150, 165 444 , 4 77, 460 225 , 1 JO, 103 114, 1 30, 1 7 1 0, 0, 0 69 , 105, 54 14 1 , 130, 150 103, l 44C 417, 4 60, 400 177, 195, 186 144 , 153, 1 17 4f 1, 4 60 , 525 103, 109, 195 14 1, 1 56, 100 0, 0, 0 102, 90, 75 165, 2 13, 192 150, 120 183, 144, 174 36, 39, 24 24, 21, 24 165, 144, 106 36, 36, 2 l 21, 10, 10 0, 0, 0 42, 2 1 , 42 54, 4 2, 36 15, 15 130, 17 1 , 144 24, 30, 4 2 10, 21, 24 mi. 109, 10 6 30, 4 2, 24 24, 24, 24 0, Or 07, 0 1, 0 40, 5 4, (irt 30, 24 a The assays were performed according to the method described by Ames (1) wi t!i slight modifications a described in Materials and Methods. Tester stains with S-9 mix or with buffer solution won. ponm) onto basal agar medium and incubated at 37*C for 2 1/2 hrs. to allow initiation of cell giowth, I he exposed to gas phase of each compound in tightly sealed chamber for 10 hrs. Total number of revertants for each triplicate. c Spontaneous revertants Cor each duplicate from vinyl chlroide 5% experiment. s> ZSSfrZO ssu Compound APPENDIX HI: SUMMARY OF FRAME SHIFT MUTATION 'IT.STS S AI .MON E L4,A/M IC ROSO M B MUTAG EN F.S IS ASSAYS OH ITAIAGENATEO ETHANES AN ETIlYliRNRS'"* Number of Total nevertantr, jier Plate TA-100 TA-1535 TA-I5.W Cone.(%) w L thont S-9 with S -9 without S-9 with S -9 without S-9 with S- 9 Vinyl chloride 7.5 1,1,1-Trichloroethane low-stabi1ized 10.(1 stabi11 zed 5.0 1,1,2-Trichloroethylone non-stabi1ized 2.5 stabilized 2.5 Tetrachloroethylene non-stabi1ized 1.0 stabilized 1.0 Spontaneous Revertants 57, HI, 54*> 40, 72, 51 21 , 15, 15 60, '15, 4 0 27, 30, 3 3 45, 42, 54 22, 42, 42 51, 30, 3 6 10, 36, 2 1 :io, 57, .16 *1 2 , 54, 36 42, 45, 33 45, 45, 42 15, 24, 27 21 , 27, 2 4 ds, 30, 42 42, 36, 36 57, 4 2, 48 45, 40, 51 33, 60, 4 0C 54, 42, 45 33, 15, 24 24, 12, 111 21, 15, 2 1 27, 36, 27 36, 27, Id 15, 21, 15 27, 21, 10 15, 12, 21 15, 10, 30 30, 15, 27 15, 15, 30 3, 9, 12 6, 9 f 9 9, 3, 15, 12, 6 3 6, 12, 6, 12, 0 r, 3, 3, 9 9, 12, 15 3, 0, 6 6, 12, 17 15, to, 15 12, 15, 6 12, 6, 9 6, 111, 12 9, 9, 9 12, 12, 1,1 a The assays were performed according to the method described by Ames (1) with slight modiFicalinns as described in Materials arid Methods- Tester stains with S-9 mix or with bufTer solution were injured unlit basal agar medium and incubated at J7*C for 2 1/2 hrs. to allow initiation ot cell growth, then exposed i gas phase of each compound in tightly sealed chamber for 1R hrs. 1 Total number of revertants Cor each triplicate. c Spontaneous revertants for each triplicate from vinyl chloride experiment. ZSZtZQ S9U Conpouixl AI'PMJMX III: SUMMARY OF FRAME SHUT HUI'ATION TISTS SAiAOJi-;r.iA/Miao<OMi- wpaoeni-sis assays un tiAUXiiwATU) biuanes wo irmYf.iKiW1 Cone. {1) TA-90 witlKMlt S-'l Number of 'Ibtal Revertants per Plate TA-1530 with S-9 wiU*out S-9 with S-9 TA-1537 witliout S-9 with S-9 ,;s. Srf*7n Vinyl chloride 7.5 1, t, 1-TVichlorocthanc lew-stahi 1 ized 10.0 stabilized 5.0 1,1,2-'tV ichloroothy 1 one non-stain li zed 2.5 stabilized 2.5 Porchloroothy1one non-slobilized 1.0 stabi1ized 1.0 57,01 ,54lj( UU1%)C 40,72,51(120%) 21,15,15(120%) 27,36,27(103%) 3, 9,12 (00%) 6, 9, 9 (109%) 60,15,-10 (17%) 27,30,33 (HU) 15,12,54 (60V) 22,42,42 (94%) 51,30,36 (17%) ia,36,21{100%) 36,27,10 (10%) 15,21,15(100%) 9, 3, 6 ( 10%) 15,12, 3 (91%) 6, 12, 0 (7.0%) 6,12, 6 ( /6V.) 30,57,36 (50V) 42,51,36 (34V) 42,45,33 (52%) 15,24,27 (40%) 27,21,10 (16%) 45,45,42 (22%) 21,27,24 (23%) 15,12,21 (11%) 3, 3, 9 (30%) 9,12, 15 (11%) i. 0, 6 (23%) 6,12, 12 (11%) 45,30,12 (41%) 57,42,40 (43V) 42,36,36 {49%) 33,15,24 (39%) 15, 10,30 (39%) 15,10,15 (39%) 45,40,51 (35%) 24,12,10 (40%) 30,15,27 (39%) 12,15, 6 (36%) 6, 10, 12 (45%) 9. 9, 9 (40%) Spontaneous llevertant s 33,60,H?1 54,42,45 21,15,21 15,15,30 12, 6, 9 12,12, 12 The assays were jvr formed accordtng to Uie method descrilxx! Ijy Ames (1) with slight modifiealions as describe! in Material.*! and Methods. Tester stains with S-9 mix or with buffer solution were poured onto hasal agar medium and inculwl.ed at .1/ t h 2 1/2 hrs. to allow initiation of cell growth, then ex(x>sed to gas phase of each compound in tightly sealel chamUr for 10 hrs. ** 'Itotai number of rovertnnts for ead) triplicate. The surviving fraction of cells at that concentration. " Spontaneous rovertnnts for eadi triplicate fran vinyl chloride experiment. tszpzo s'sa