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7 cH* o fV Present d as a part of the Status Report on Vinyl Chloride, Dow Chemical of Canada, Limited, Fort Saskatachewan, Alberta to Officials of Department of Health and Environment of Alberta, Canada on September 11, 1980 by V, K. Row . Good afternoon., Ladies and Gentlemen. It has been a few years since it was my privilege to discuss with you the toxicological data on vinyl chloride. Today I am not going to burden you with a whole lot of detail but rather I want to highlight some of the observations in toxicology that I believe are pertinent to the assessment of hazard -- or safety as I prefer to think of it, I believe the most significant recent observations are those dealing with carcinogenicity recently reported by Prof. Maltoni, teratology reported by John et al. and others, and metabolism and pharmacokinetic data reported by Gehring dt al. and Butcher et al. Consequently I will focus my i attention on the recent developments in these areas. As I am sure you all know that in January of this year, OSHA submitted a series of questions to the industry about VC and PVC and asked the industry to respond. A comprehensive response to each question was developed and submitted by the industry under auspices of SPI. The Dow Chemical Company had i d major part in the preparation of the review. Sections of this submission relevant to the toxicology of VC are included in the document we are filing with you today. In this presentation, we will present a summary of the information pertinent to this meeting and not burden you with all of the detail. do 1.3^525 CONFIDENTIAL Acute Toxlcltv. Vinyl chloride, with boiling point of -13.8C is a gas at ordinary temperatures. Consequently the major route of exposure is by inhalation. Vinyl chloride was investigated as a possible anesthetic in the 1920's to 1930's. But it was considered to be unsatisfactory as an anesthetic because of the high concentrations required (10-20%) which caused circulatory and cardiac effects as well as a fire and explosion hazard. If liquid vinyl chloride were spilled on the skin, there may be severe cooling and frost bite. With respect to mutagenicity. Vinyl chloride has been shown to be mutagenic in a number of microbial test systems and In vitro Chinese hamster cell cultures, but not in neurospora. However, vinyl chloride is not mutagenic in In vivo dominant lethal assay in mice but is positive in Drosophila. That vinyl chloride is negative in any of the In vitro tests particularly where closed systems are involved or where metabolic activation is inherent in the test procedure is a mystery to me. It is also not surprising that it is negative in the D.L. test since in the whole system, defense and excretory mechanism can come into play, Chronic Toxicity. Torkelson, et al. report repeated exposures of animals for 4 hours/day, 5 days/weeks for 6 months. At 500 ppm rats showed increased liver weight and histopathological changes. At 100 ppm and 200 ppm exposures, increase in live weight was observed in rats but no changes could be 00 ^ observed In dogs or guinea pigs. All animal species showed no adverse effect at 50 ppm for 6 months. Carcinogenicity. As you are aware, investigations sponsored by the industry have been going on in Prof. Maltoni's lab in Bologna, Italy for several years. He reported his most recent data in detail at the OSHA/NIOSH/NIEHS sponsored symposium, March 20 and 21, 1980 in Bethesda, Maryland. I will summarize it here. In Maltoni's studies, laboratory animals were exposed to vinyl chloride vapour concentrations ranging from 30,000 to 1 ppm or they were fed olive oil solutions of vinyl chloride by gavage. Slide 1 Slide 2 Slide 3 Slide 4 Summarize the results of Dr. Maltoni's studies. Description. Recent inhalation work. Recent oral work. The lowest cancer effect dose by inhalation is 10 ppm which calculates to approximately 1.5 mg/ kg/day. The experimentally determined lowest cancer effect level by oral gavage is 0.3 mg/kg/ day. The differences between the two values could be due to: (1) the inexact nature of the assumptions used in the calculation; and (II) oral gavage exposures would be expected to yield higher transient levels of vinyl chloride and its activated metabolites in contact with sensitive reaction sites, DO 136527 CONFIDENTIAL -4- Metobollsm. For some time I have felt that it is easy, to criticize someone else's conclusions, but it is not always so easy to Justify one's own conclusions for personal biases are always present and they are hard to exclude from rational thinking. Consequently, if one is to criticize, he should have some scientific rationale for doing so. It was with this objective in mind that the rather extensive metabolic and Pharmacologic studies were begun in the Dow Toxicology Lab on VC and a number of other controversial materials. Other investigators have been intrigued with this approach and many contributions have been made. With respect to VC, most of our work has been published. Basically, from all the data available, it appears that VC undergoes metabolic activation via the route shown in the next few slides. Insofar as metabolites are concerned these are shown in the next slide. Why is this important? Because qlutathione and sulfhydryl compounds are biochemical scavengers of free radicals, such as are formed in the metabolism of VC. The next slide shows what happens to the sulfhydyl content of the liver following exposure to VC. This is important for its depletion reduces natural defense capability. The next slide shows the pattern of excretion of C1Z* following various oral doses of C^ labeled VC. The following slide shows the major changes in excretion of VC with dose. It is well accepted that metabolic activation rates of different species varies greatly and this has much to do with the amount of active metabolites of VC that are available to react in the tissues from given exposures. Recent studies by Butcher et al. and Gehring et al. have shown that this activation rate in rats is substantially greater than in man. These data would predict that rats are more susceptible to tumor induction by vinyl chloride than humans. This is indeed confirmed by human epidemiological studies. A series of studies have been conducted in the Dow Toxicology Research Laboratory to assess the hazard associated with exposure to vinyl chloride as well as to investigate the mechanism by which this monomer might exert its toxic effects. The purpose of the studies was to assess the embroyotoxic potential of Inhaled vinyl chloride in mice,, rats, and rabbits. These studies were supported in part by the Chemical Manufacturer's Association. Since previous studies in this laboratory suggested that the primary metabolic pathway for vinyl chloride is blocked by ethanol, it was considered possible that administration of ethanol might alter its metabolism in a manner which would enhance its toxic t)0 c C O N F ID E N T a80556,46 john TERATOLOGY STUDIES WITH VINYL CHLORIDE* Vinyl chloride, ppm Ethanol, Percent Mice Rats, rabbits Mice, rats, rabbits Mice, rats, rabbits 500 500 50 50 2500 2500 500 0 0 0 15 0 15 0 15 0 0 15** 'John. t aI, To* Appl Pharmacol 30. 497-513 (1P77), CMA oo 'Schwcla, cl at. IctMolof], 15, 355-392 11975), M|CHS COO' t OI ' -7- or teratogenic potential. Thus, some of the vinyl-chloride exposed animals were given 15% ethanol in their drinking water during the days of exposure. The teratogenic potential of lnblbed ethanol in these three species was previously studied in our laboratory and reported at the Washington meeting. In SUMMARY, the results of these studies indicate that exposure of pregnant mice, rats, or rabbits to vinyl chloride by inhalation at concentrations sufficiently high to cause maternal toxicity did not cause a teratogenic effect In any of the three species tested. Fetal effects consisted of delayed skeletal development in mice at 500 ppm, an exposure level which was maternally toxic, and an increase in the incidence of dilated ureter in rats exposed to 2500 ppm. In mice exposed to 500 ppm of vinyl chloride, the incidence i 6f fetal resorptions was increased over concurrent, negative controls. This Incidence was at high end of the range for historical control groups in our laboratory. Ingestion of 15% ethanol in the drinking water enhanced the toxicity of Inhaled vinyl chloride, Fetal body measurements were lower among mice and rats given the combination and the ipcreases in skeletal variants, indicating delayed development, were observed in both species. The incidence of resorptions was increased in rabbits given ethanol, and maternal toxicity was enhanced by ingestion of ethanol in all three species. DO 136531 OONFTDFNTTAL -8- In 1979/ a report on teratology studies In mice and rats exposed to vinyl chloride appeared in the Hungarian literature. Their work is summarized from an English translation on this last slide. Among mice exposed to 1QQQ ppm for 4 periods of 2-hours duration each day during organogenesis an increased fetal loss was reported. I'm not sure what was meant by fetal loss. The author reports that no teratogenicity was observed in mice. Several experiments were conducted with rats. Exposure to 1500 ppm for 24 hrs/day during organogenesis was reported to have no fetal or teratogenic effects apart from an increased liver weight/ presumably in the fetuses. Exposure during the third part of pregnancy produced no deleterious effects/ whereas exposure in the first part caused increased fetal mortality and decreased fetal body weights. The exact days of gestation during which exposures were conducted were not indicated, The author reported that vinyl chloride did not potentiate the teratogenicity of trypan blue in rats, Vinyl chloride plus alcohol/ reported as given as large doses/ during the neurulation period/ which I presume to be approximately days 9 through 12 of gestation/ caused some skeletal retardation in rats, Thus/ these results as summarized in the Hungarian report are quite similar to those observed in our studies with rats and mice. C0O0NFnTI^DEN^T! AAli More recently/ a multi-generation reproduction study was sponsored by Consumer Products Safety Commission in which a8Q556,56 jo tin 1979 UNGVARY Mice . . . 1000 ppm 4 (2 hr) periods/day, organogenesis: Increased fetal loss, no teratogenicity Rots ... 1500 ppm 24 hours/day, organogenesis: No fetal or teratogenic effect apart from increased liver weight Third part of pregnancy: No effects First part of pregnancy: increased fetal mortality and decreased fetal body weight ( VCI + trypan blue: No potentiation VCI + alcohol, neurulation period: Increased skeletal retardation 00 1.36533 C O N F ID E N T !, -10- Sprague-Dawley/Wistar rats were exposed to vinyl chloride. It did not cause adverse effects in reproductive performance. Dr. Robert Hehir of the CPSC reported on these studies at the Washington meeting. We have also seen an abstract of a Bulgarian paper which was presented at the 19th International Congress on Occupational Health in which the effects of maternally inhaled VCI on embryonal, fetal, and postnatal development of rats were reported. The authors report that inhalation of 6,.15 mg/m3/day or about 2.5 ppm throughout gestation produced increased fetal mortality, decreased fetal weight and increases in a number of fetal anomalies including hemorrhages, encephalocele, hydrocephalus, and delayed skeletal ossification. Postnatal effects consisted of biochemical effects (decrease enzymatic activity of dehydrogenases, decreases in enzymatic activity of bile) and injury to the nervous system. Since this exposure level is equal to only 2.5 ppm, or 1/1000 of the levels tested in our laboratory and since details of the test methods, especially as it pertains to vapor generation and analysis are lacking in the abstracted form, we cannot fully evaluate the findings that are reported, except to say that they are not what we would have expected on the basis of any data available. 00 136S34 CONFTOENTTAl Finally^ it should be noted that the maximum dose levels tested in the study of John et al., 2500 ppm in rats and rabbits and 500 ppm in mice without teratogenic effects, provide reasonable assurance that the likelihood of VC causing teratogenic effects under present conditions of human exposure is extremely remote. To our knowledge, there are no reports which clearly relate birth defects in humans to maternal, or for that matter, paternal exposure to VCM. Two relevant studies which have been published 15, 16 in(jiCate no relationship between population exposure to VCM in the area surrounding PVC formulation plants and birth defects. Infante et al., 17 have suggested a causal relation ship between paternal exposure to VCM in the industrial setting and pregnancy outcome. The adequacy of the date supplied by Infante et al. was almost immediately questioned18. More recently, Haas and Schottenfeld19 have leveled severe criticisms at the Infante study, the following is extracted from their paper: the inferences made cannot be sustained and little light is shed on the possible association of abnormal pregnancy outcome with paternal occupational exposure to VCM." Likewise, others have raised serious doubts regarding the validity of the conclusions drawn by the Infante et al. study. For example. Downs, et al.20 completely discredited the study; their ultimate conclusion was as follows: and I quote -- "...it does not seem possible to salvage anything from study..." 00 1.36S35 OONFTDENTTAl -12- "The authors' analytical methods are invalid since the... test requires that the two rates being compared be in dependent, and this is not the case..." "...The methods used by the authors to test significance are inappropriate since pregnancies are clustered..." "...The misleading conclusions drawn by the authors were brought about through the selection and use of their control group." "...The purpose of adjusting rates is to make them comparable. Adjusting a rate r to a population A and another rateCs) to a population B, and then comparing the adjusted rates r and s is contrary to the purpose of adjusting rates, and the resulting comparison does not make any sense. "... the comparisons of rates made bv the authors ore irrelevant to the hypotheses tested by them and to their corresponding conclusions. Similarly, McMahon21 opens his review of the infante popr with the following comment: ...It is disappointing to see an article of such poor quality as this published in the Lancet. The data are subject to serious criticism on several counts. DO CONF 136536 T DENT T At. and/ after detailed point-by-point analysis of the study/ presents.the following conclusory statement: In short/ this paper deserves...no consideration whatsoever in weighing the question of whether there is or is not a genetic risk associated with exposure to VCM. The foregoing reviews/ two of which were available in 1977/ completely rebut the scientific validity of the paper by Infante/ et al. in this matter. In the absence of a scientifically sound defense of this paper by the authors or their scientific peers/ continued citation of this paper is inappropriate and misleading to the public/ especially. On a positive note/ Edmonds et al. of the U.S. CDC attempted to establish a correlation between parental occupation and congenital effects in and surrounding a PVC plant in West Virginia. i They were unable to confirm Infante's conclusions and concluded themselves that "...no relationship between infants with malformations and parent's exposure to vinyl chloride could be established." Thank you/ ladies and gentlemen/ for your attention. i |i V. K. Rowe n0 i36?>37 CONFTOf.NTtAl T2.2-183- (2) Stanford Research Inst. SRI Project No. LSC-4378-1 Submitted bv: J. M. Norris Test Strain: Bacterial-Salmonella typhimurium Yeast-Saccharomyces cerevisiae TA98, TA100, TA1535, TA1537, TA1538 D3 S-l Incomplete (-) Acrylonitrile From tox. study S-2 + D3 Vinylbenzyl chloride Lot #06174 S-3 "* Benzene Bay City Pipeline, 5/28/75 S-4 ( + ) D3 Styrene tars LUWA, Styrene finishing still tar column, 580 Bldg. S-5 ( + ) D3 Chloroacetic Acid Batch 145 (1501), 5/28/75 S-6 Styrene From inhal. tox. study (5 ppm TBC), 6/75 S-7 Chloroacetyl chloride Lot 05285-5; Batch 3105 tank, 5/28/75 S-8 -- 50/50 Polyethylbenzene/DOWFROTH* Ref. #408-3-5-1 S-9 -- Decabromodiphenyl Oxide Lot #09014-317 S-10 i ( + ) D3 Perchloroethylene (DOWPER* formulation, W. Dengler, H. Farber, 6/75) S-ll -- Divinyl Benzene Lot #05135 S-12 -- ' Pentachlorophenol (tars) 6/12/75 S-13 -- Ethyl benzene "A", 5/28/75 S-14 Pentachlorophenol new (DOWICIDE* EC-7) Lot #08024 S-15 -- #2 Fuel Oil Ref. #408-3-5-2, 5/19/75 S-16 Incomplete Pentachlorophenol Old (DOWICIDE* 7) Lot #705611 S-17 Incomplete Hydroxyethylacrylate From inhal. tox. study, 6/75 + s= = (+) ss (-) = A positive negative marginally positive marginally negative DO 1.36S38 CONFIDENT! Al. Saccharomyces Cerevisiae D3 The yeast S.. cerevisiae D3 is a diploid and is heterozygous for a mutation in an adenine-metabolizing enzyme.7 Cells homozygous for this mutation produce a red dye when grown on medium containing adenine. Adenine-requiring homozygotes can be generated from the heterozygotes by mitotic recombination. The frequency of mitotic recombination is increased by many mutagens. Mitotic recombination is indicated by the development of colonies with red pigmentation, and the degree of conversion to this pigmented colony indicates the mutagenicity of a compound or its metabolite.8 The Saccharomyces test strain from the liquid nitrogen is grown overnight at 30C with aeration in 1.0% tryptone and 0.5% yeast extract. The cells are washed twice in 0.067 M PO4 buffer (pH 7.4) and resuspended in the same buffer at a concentration of 108 cells/ml. The _in vitro yeast mitotic recombination assay in suspension consists of 5 x 107 washed, stationary-phase yeast cells in 1 ml of 0.067 M P04 buffer (pH 7.4) and 50 mg/ml of the test chemical (or a fraction of the concentration required to give 50% killing). The suspension is incubated at 30C for 4 hours. After incubation, the sample is diluted serially in sterile saline and plated on tryptoneyeast agar plates. Plates of a 10-3 dilution are incubated for 2 days at 30C, followed by 2 days at 4C to enhance the development of red pigment that is indicative of adenine-negative homozygosity. Plates are screened for red colonies or red sectors by scanning the plates with a dissecting microscope at 10 x magnification. Plates of a 10-5 dilution are incubated for 2 days at 30C for determination of the total number of colony-forming units. The in vitro yeast mitotic recombination assay in suspension with metabolic activation is carried out as above with the addition of the metabolic activation system to the incubation mixture. 4 DO 136539 CONFIDENTIAL Metabolic Activation--Aroclor 1254-Stimulated Metabolic Activation System Some carcinogenic mutagens (e.g., dimethylnitrosamine) are inactive unless they are converted to their active form by being metabolized. The metabolic activation systems we use have been described by Ames. Adult male mice are given a single intraperitoneal injection of a polychlorinated biphenyl (Aroclor 1254) at a dosage of 500 mg/kg.^ Four days after the injection, the food is removed. On the fifth day, the mice are sacrificed. The livers are removed aseptically and placed in preweighed, sterile glass beakers. The organ weight is determined, and all subsequent operations to the metabolic activation step are conducted in an ice bath. The organ is washed in an equal volume of cold, sterile 0.15M KC1 (1 ml/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 KC1, and homogenized with a Potter-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x and the supernate is removed and stored in liquid nitrogen. To the post- mitochondrial supernate are added MgClj, KC1, glucose-6-phosphate, TPN, and sodium phosphate (pH 7.4). DO 1.36540 5 CONFTDFNTTAL RESULTS AND DISCUSSION Table 1 presents the results of the microbiological assays with j3. typhimurium. None of the compounds were mutagenic in these assays. We retested S-l (acrylonitrile) several times because we had information that this compound had given a positive, mutagenic response in some assays. However, as shown in Table 2, S-l was not mutagenic with typhimurium. Table 3 presents the results of the assays for mitotic recombination in S_. cerevisiae D3. One compound--S-2--increased mitotic recombination in these assays. Compound S-2 increased mitotic recombination in three experiments at a concentration of 0.01%, although in one experiment (no. 3) the increase was not significant. Concentrations above 0.02% were toxic. A clear positive response is indicated by an increase of more than threefold in the absolute number of recombinants (mitotic recombinants per ml) as well as the relative number of mitotic recombinants (per 105 survivors). Compounds S-4, S-5, and S-10 gave increases in the number of mitotic recombinants per 105 survivors. However, none of these compounds increased the number of mitotic recombinants per ml by more than threefold. Therefore, we consider the results to be only marginally positive. None of the other compounds were mutagenic in these assays. Based on our experience with over 200 chemicals in assays with _S. cerevisiae D3, we suggest that Dow Chemical Company consider further tests in other biological assay systems--particularly iri vivo or in vitro cytogenetic assays--to measure chromosome breakage. However, because the increase in mitotic recombination induced by S-2 is small compared with the increased obtained with the positive control (1,2,3,4 diepoxybutane), it may be that this compound is of little or no hazard. This conclusion is reinforced by the absence of mutagenicity in all of the Salmonella assays with S-2. 6 DO 136541 OONFTDFNTTAL There is no indication of a dose-response in the number of mitotic recombinants per ml with S-4, S-5, or S-10. In previous studies, we have shown that the number of mitotic recombinants per 105 survivors increases as survival decreases, even when the decrease in survival is not due to a mutagen. Therefore, the apparent increase induced by these compounds may or may not be an artifact. Since none of these compounds were mutagenic in the Salmonella assays, we believe that if they are mutagens, they are only weakly so. 7 no confidential REFERENCES 1. McCann, J,, E. Choi, E. Yamasaki, and B. N. Ames. 1975. Detection of carcinogens as mutagens in the Salmonella microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA 72_, 5135-5139. 2. Simmon, V. Unpublished results. 3. Ames, B. N., E. G. Gurney, J. A. Miller, and H. Bartsch. 1972. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc. Nat. Acad. Sci. USA 69, 3128-3132. 4. Ames, B. N., F. D. Lee, and W. E. Durston. 1973. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat. Acad. Sci. USA 7Q, 782-786. 5. Ames, B. N., W. E. Durston, E. Yamasaki, and F. D. Lee. 1973. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Nat. Acad. Sci. USA 70. 6. McCann, J., N. E. Spingar, J. Kobori, and B. N. Ames. 1975. The J detection of carcinogens as mutagens: Bacterial tester strains i with R factor plasmids. Proc. Nat. Acad. Sci. USA (in press). 7. Zimmermann, F. K., and R. Schwaier. 1967. Induction of mitotic gene conversion with nitrous acid, l-methyl-3-nitro-l-nitrosoguanidine and other alkylating agents in Saccharomyces cerevisiae. Mol. Gen. Genet. 100, 63-69. 8. Brusick, D. J., and V. W. Mayer. 1973. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspectives 6^, 83-96. 9. Kier, L. D., E. Yamasaki, and B. N. Ames. 1974. Detection of mutagenic activity in cigarette smoke condensates. Proc. Nat. Acad. Sci. USA 71, 4159-4163. 8 36.543 "nfVdfnttai. Table 1 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM Metabolic Compound Activation Negative Control + pg of Compound Added per Plate Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 17 29 13 19 18 37 19 39 121 128 Positive Controls 4-o-tolylazo-o-toluidine N-methyl-N'-nitro-Nnitrosoguanidine S-l + _ - + + + + + + + + + + 25 18 19 347 306 2 0.1 0.5 1.0 5 10 50 100 500 1000 5000 0.1 0.5 1.0 5 10 50 100 500 1000 5000 3000 19 15 47 45 10 18 13 49 43 14 13 9 15 6 14 17 12 12 23 19 21 18 18 20 33 29 21 29 26 26 18 17 25 16 34 27 22 23 33 22 4000 16 16 18 15 27 23 17 13 18 19 23 22 30 22 24 19 23 21 24 17 82 89 95 64 89 97 99 86 137 74 25 24 21 20 23 22 15 17 29 27 19 24 30 20 23 20 16 19 22 24 93 96 110 95 146 111 122 133 131 121 DO 1 0 6 5 4 4 OONFTDFNTT Table 1 (Continued) Compound Metabolic Activation pg of Compound Added per Plate Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 U. 1 lb 18 49 17 98 - 0.5 14 19 25 34 145 - 1.0 12 19 28 30 162 - 5 10 22 17 39 146 - 10 9 21 19 24 155 - 50 12 19 19 27 185 - 100 5 22 29 33 158 - 500 4 Toxic Toxic Toxic Toxic + 0.1 13 20 11 15 133 + 0.5 10 17 19 30 173 + 1.0 12 14 13 30 164 + 5 13 20 11 30 154 + 10 8 18 17 26 159 + 50 10 16 15 35 203 + 100 7 23 13 14 161 + 500 6 Toxic Toxic 14 Toxic DO 1 .3 6 5 4 5 C O N F ID E N T !, Table 1 (Continued) Compound Metabolic Activation pg of Compound Added per Plate - 0.1 - 0.5 - 1.0 - 5.0 - 10 - 50 - 100 - 500 + 0.1 + 0.5 + 1.0 + 5.0 + 10 + 50 + 100 + 500 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 8 23 27 21 95 30 21 27 22 140 20 29 18 16 100 21 21 12 22 188 21 18 22 24 121 18 22 12 26 107 24 21 19 26 122 13 12 11 25 122 10 18 20 15 111 18 24 25 31 155 14 23 20 29 146 14 18 16 21 133 14 19 15 17 143 16 22 20 27 152 15 22 19 23 122 15 19 30 28 152 '-L1N3O 1JN03 00 Table 1 (Continued) NJ bo Z-n 3 arn O' z-4 --T>l ''l Compound Metabolic Activation S-4 - - - - - - - + + + + + + + + pg of Compound Added per Plate 0.1 0.5 1.0 5 10 50 100 500 0.1 0.5 1.0 5 10 50 100 500 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 20 115 22 12 19 23 120 18 10 17 20 115 19 16 11 26 127 19 16 16 38 130 25 12 15 28 115 29 10 11 23 125 17 12 6 20 99 15 144 18 20 16 20 138 18 10 17 22 103 23 15 14 27 130 18 16 20 32 96 26 12 16 28 113 14 13 14 23 155 11 11 5 6 117 Table 1 (Continued) Compound Metabolic Activation pg of Compound Added per Plate - 0.5 - 1.0 -5 - 10 - 50 - 100 - 500 - 1000 + 0.5 + 1.0 +5 + 10 + 50 + 100 + 500 + 1000 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 10 16 31 28 119 39 15 29 32 113 32 60 31 25 111 42 38 21 26 106 42 41 25 28 120 26 32 21 26 117 35 35 20 31 96 13 48 10 19 43 9 16 19 92 17 20 25 38 84 6 26 39 21 107 16 22 36 29 143 15 18 30 27 118 14 23 23 26 148 15 25 25 27 81 2 14 9 13 80 t oa 1> Table 1 (Continued) Compound S-6 Metabolic Activation gg of Compound Added per Plate Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 0.1 0.5 1.0 5 10 50 100 500 11 20 25 27 118 24 19 10 50 163 15 17 22 21 117 18 19 18 45 168 20 22 16 55 152 23 18 10 60 150 19 24 16 50 155 12 16 13 40 89 + 0.1 12 17 15 17 149 + 0.5 18 15 15 58 170 + 1.0 12 19 13 13 125 + 5 16 16 17 54 174 + 10 11 15 21 51 176 + 50 13 13 17 53 166 + 100 11 13 19 40 151 + 500 8 6 11 28 67 DO 1 3 6 5 4 9 C O N F ID E N T ! x> Table 1 (Continued) Compound Metabolic Activation + + + + + + + pg of Compound Added per Plate 1 5 10 50 100 500 0.5 1.0 5 10 50 100 500 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 10 16 31 28 119 21 14 23 24 103 9 15 42 42 101 9 35 24 24 118 10 36 19 19 95 6 40 20 15 91 8 11 15 14 86 2 13 27 28 108 11 17 17 24 104 12 19 10 16 123 8 23 18 21 122 9 19 24 23 110 6 18 26 26 110 18 20 13 13 103 00 136550 CO NFIDENT! IE- Table 1 (Continued) Compound Metabolic Activation - - - + + + + + + + U g of Compound Added per Plate Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 0.5 1.0 5 10 50 100 500 0.5 1.0 5 10 50 100 500 18 26 18 26 15 10 Toxic 5 12 9 9 12 9 15 10 12 10 10 13 13 13 23 17 21 21 16 18 17 11 14 13 18 97 14 19 102 12 21 99 15 15 90 20 16 87 18 12 99 19 13 91 22 20 92 15 21 95 12 13 104 16 20 84 18 21 92 19 25 102 Table 1 (Continued) Compound Metabolic Activation - + + + + + + + + Pg of Compound Added per Plate Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 0.5 10 11 14 16 133 1.0 27 18 14 19 114 5 13 23 16 7 121 10 22 13 19 21 87 50 23 8 16 18 83 100 20 11 18 17 90 500 28 11 10 17 87 1000 20 13 10 14 97 0.5 14 19 24 132 1.0 14 18 22 25 97 5 6 20 16 20 87 10 15 18 17 23 91 50 17 12 23 19 100 100 13 20 21 22 71 500 15 18 16 19 93 1000 16 20 14 24 90 DO 1 3 6 5 5 ? OONFTDFNTT 3> Table 1 (Continued) Compound Metabolic Activation pg of Compound Added per Plate - 0, -1 -5 - 10 - 50 - 100 - 500 +0 +1 +5 + 10 + 50 + 100 + 500 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 24 33 33 30 29 22 Toxic 9 18 17 18 13 14 8 11 16 17 16 14 4 Toxic 16 19 19 23 13 Toxic Toxic 15 16 12 12 12 12 4 12 14 18 13 19 19 Toxic 10 16 16 15 19 12 Toxic 134 107 118 110 116 92 86 17 16 24 15 11 9 Toxic 136 100 113 106 115 93 Toxic 1 X N aO ldN 00 1 Table 1 (Continued) Compound Metabolic Activation Mg of Compound Added per Plate Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 S-ll vo + + + + + + + 0.5 1 5 10 50 100 500 0.5 1 5 10 50 100 500 8 9 12 9 10 8 7 8 12 8 8 11 10 Toxic 21 19 19 19 21 Toxic Toxic 30 43 43 22 32 34 Toxic 24 40 21 36 14 17 Toxic 31 32 40 51 52 Toxic Toxic 18 20 15 28 21 16 Toxic 40 30 36 28 34 Toxic Toxic 178 137 172 147 147 96 Toxic 103 124 116 109 104 93 Toxic On 1 3 6 5 5 4 OONFTDFNTT x> Table 1 (Continued) Compound S-12 N> O Metabolic Activation pg of Compound Added per Plate - 0.5 -1 -5 - 10 - 50 - 100 + 0.5 +1 +5 + 10 + 50 + 100 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 6 7 24 17 150 12 11 28 24 171 8 4 27 17 129 8 9 32 24 151 11 11 22 8 103 Toxic Toxic 9 Toxic 88 5 3 5 5 4 Toxic 16 21 12 16 11 7 37 42 118 33 29 96 21 27 110 19 29 105 17 27 80 19 13 Toxic O' Table 1 (Continued) Compound Metabolic Activation tig of Compound Added per Plate Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 S-13 ho + + + + + + 10.5 7 20 10 20 138 11 15 20 13 122 5 13 20 20 16 131 10 9 18 21 20 128 50 15 11 12 9 161 100 10 10 10 15 87 0.5 1 5 10 50 100 3 23 21 17 118 4 22 30 18 131 2 20 27 17 148 5 24 28 18 128 2 29 19 16 117 7 19 10 12 83 `JO1' 1 Table 1 (Continued) Compound Metabolic Activation pg of Compound Added per Plate -1 - 10 - 50 - 100 - 500 +1 + 10 + 50 + 100 + 500 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 16 12 19 11 Toxic 11 11 5 9 Toxic 21 18 20 15 Toxic 46 24 37 39 Toxic 24 32 28 22 Toxic 27 25 27 22 Toxic 22 33 18 32 Toxic 143 143 111 125 Toxic 20 121 20 124 22 92 13 87 18 Toxic COHFTOFHTt Table 1 (Continued) Compound So ZO rom-t -S1' Z-t O' -* 3> T> Metabolic Activation - - + + + + + + + pg of Compound Added per Plate 1 10 50 100 500 1000 5000 1 10 50 100 500 1000 5000 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 42 25 25 85 28 67 46 80 41 61 41 63 32 33 16 20 14 36 8 35 11 30 7 39 9 36 11 33 43 37 103 37 41 94 36 43 112 30 42 109 22 32 81 14 32 85 18 22 97 22 20 98 31 21 125 38 43 90 48 32 121 54 38 102 53 43 91 46 48 86 Table 1 (Continued) Compound Metabolic Activation - - + + + + + + + pg of Compound Added per Plate 0.25 0.5 1.0 10 50 100 500 0.25 0.5 1 10 50 100 500 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 13 9 14 6 8 1 Toxic 7 6 9 10 6 5 Toxic 21 24 25 31 20 21 Toxic 22 15 17 21 22 25 Toxic 31 20 24 28 19 13 Toxic 33 26 23 38 9 13 Toxic 23 36 32 26 25 24 Toxic 20 17 29 22 25 34 Toxic 102 101 106 90 90 71 Toxic 103 103 124 105 82 91 Toxic oo ,:A^ O00-1'1a CDOON F ID E N T ! Table 1 (Concluded) Compound S-17 Metabolic Activation - + + + + + + Mg of Compound Added per PI 0.5 1.0 5 10 50 100 0.5 1.0 5 10 50 100 Average Histidine-Positive Revertants per Plate TA1535 TA1537 TA1538 TA98 TA100 20 22 10 9 12 Toxic 17 12 10 14 12 Toxic 14 10 19 15 Toxic Toxic 11 19 19 12 Toxic Toxic 13 10 19 13 12 Toxic 12 17 22 15 Toxic Toxic 14 8 9 7 Toxic Toxic 19 10 14 19 Toxic Toxic 83 66 76 77 Toxic Toxic 79 79 96 57 55 Toxic Table 2 IN VITRO ASSAYS WITH SALMONELLA TYPHIMURIUM--STRAIN TA100 Compound Negative Control S-l i ' Metabolic Activation - + + + + + + + ug of Compound Added per Plate Average TA100 Revertants per Plate 1000 1500 2000 2500 3000 3500 4000 1000 1500 2000 2500 3000 3500 4000 138 145 134 117 125 109 95 109 124 148 166 144 153 169 153 148 N-a> oO O -nZ o 4J-- om O' Z--A 'CT g Tab Le 3 IN VrTRO ASSAYS WITH SACCHAROMYCES CEREVISIAE D3 Exper No. 1 2 3 Compound Metabolic Activation Percent Concentration (w/v or v/v) Surviving Cells per ml (x 10"'7 ) Percent Survivors Mitotic Recombinants per ml (x 10~3) Mitotic Recombinants per 105 Survivors Negative Control S-2 Negative Control Positive Control 1,2,3,4 Dlepoxybutane S-2 Negative Control Positive Control 1,2,3,4 Diepoxybutane S-2 -- + _ - + __ + _ + + __ + + + + + 0.01 0.01 0.1 0.1 0.05 0.05 0.01 0.01 .04 .04 .01 .01 .02 .02 .03 .03 6.7 4.5 6.5 4.0 Toxic Toxic 7.8 6.0 4.1 5.2 6.5 6.7 5.3 5.8 5.9 4.5 4.1 4.9 2.2 2.5 Toxic Toxic 100 100 97 89 100 100 53 87 83 112 100 100 111 78 77 84 42 43 6 5 15 13 10 4 1478 1286 22 24 3 5 761 663 9 4 15 20 9.0 11.1 23.1 32.5 12.8 6.7 3605 2473 33.8 35.8 5.7 8.6 1290 1473 22.0 8.2 68.2 80.0 Table 3 (Continued) 1 Exper. No. Compound Metabolic Activation Percent Concentration (w/v or v/v) 1 Negative Control + S-3 Negative Control - + - + - + - + 0.01 0.01 0.1 0.1 0.5 0.5 Positive Control 1,2,3,4 Diepoxy butane S-3 3 Negative Control _ + _ + 0.05 0.05 0.1 0.1 S-3 0.2 + 0.2 viving s per ml : 10~7) 6.7 4.5 7.0 6.7 7.8 5.4 Toxic Toxic 7.8 6.0 4.1 5.2 Percent Survivors 100 100 104 149 116 120 100 100 53 87 Mitotic Recombinants per ml (x 10"3) 6 5 13 15 8 15 10 4 1478 1286 Mitotic Recombinants per 10lj Survivors 9.0 11.1 18.6 22.4 10.3 27.8 12.8 6.7 3605 2473 8.0 103 7.8 130 8.5 100 7.2 100 6.6 78 1.5 21 7 9 7 5 0 4 8.8 11.5 8.2 6.9 26.7 00 1.36563 C O N F ID E N T ! > Table 3 (Continued) Expe r. No. Compound_____ Metabolic Activation Pe rcen t Concent rat ion (w/v or v/v) 1 Negative Control Positive Control 1,2,314 Diepoxybutane + NJ 2 3 O o o o 2- -7 -a OP'P Negative Control S-4 Netagive Control Positive Control 1,2,3,4 Diepoxybutane S-4 + + + + + + + 0.05 0.05 0.01 0.01 0.1 0.1 0.1 0.1 .04 .04 .02 .02 .04 .04 .06 .06 .08 .08 .10 .10 1 'iving ; per ml 10-7) Percent Survivors Mitotic Recombinants per ml (x 10-3) Mitotic Recombinants per 10Ij Survivors 7.8 100 6.0 100 10 4 4.1 53 1478 5.2 87 1286 12.8 6.7 3605 2473 5.5 71 5 5.3 88 8 3.9 50 10 3.8 63 12 8.5 100 7.2 100 7 5 0.5 6 2.0 27 5 7 5.3 100 5.8 100 3 5 5.9 111 761 4.5 78 663 9.1 15.1 25.6 31.6 8.2 6.9 100 35.0 5.7 8.6 1290 1473 2.6 49 1 3.8 5.9 102 5 8.5 5.6 106 5 8.9 4.8 83 6 12.5 3.9 74 9 23.0 4.5 78 Contaminated 3.6 68 4 11.1 3.6 62 4 11.1 2.2 42 5 22.7 3.1 53 5 16.1 ) Table 3 (Continued) Exper. No. 1 2 uj o 3 z: o -1T-it om --t0*>"s Z-t 2Otn' p _____ Compound___ Me t abo Lie Ac t iv.it ion Percent Concentration (w/v or v/v) Negative Control + S-5 - 0.01 + 0.01 - 0.1 + 0.1 - 0.5 + 0.5 Negative Control + S-5 - 0.01 + 0.01 - 0.1 + 0.1 Negative Control + Positive Control 1,2,3,4 Diepoxybutane + 04 04 S-5 - 0.1 + 0.1 - 0.2 + 0.2 - 0.3 + 0.3 - 0.4 + 0.4 - 0.5 + 0.5 Surviving Cells per ml (x 10-?) 5.7 4.4 6.7 7.4 6.6 3.7 Toxic Toxic 8.5 7.2 6.8 7.5 7.2 7.0 5.3 5.8 5.9 4.5 Percent Survivors 100 100 118 168 116 84 100 100 80 104 85 97 100 100 111 78 Mi tot ic Recombinants per ml (x 10-3) 4 3 3 5 0 5 7 5 1 2 6 2 3 5 761 663 Mi tot ic Recombinants per 10'J Surv Ivors 7.0 6.8 4.5 6.8 13.5 8.2 6.9 1.5 2.7 8.3 2.9 5.7 8.6 1290 1473 5.5 4.8 4.5 5.7 2.7 3.8 4.5 4.3 .3 2.9 104 83 85 98 51 66 85 74 6 50 7 5 3 2 2 2 4 7 0 6 12.7 10.4 6.7 3.5 7.4 5.3 8.9 16.3 -- 20.7 Table 3 (Continued) I Exper. No. Compound Metabolic Activation Percent Concent rat ion (w/v or v/v) Surviving Cells per ml (x 10-7) Percent Survivors Mitotic Recombinants per ml (x 10`3) Mitotic Recombinants per 10^ Survivo rs 1 Negative Control - + 7.8 100 6.0 100 10 4 12.8 6.7 Positive Control 1,2,3,4 Diepoxybutane + 0.05 0.05 4.1 53 1478 3605 5.2 87 1286 2473 S-6 - 0.1 .9 12 1 11.1 + 0.1 2.3 38 1 4.3 2 Negative Control w S-6 + + 0.05 0.05 8.5 100 7.2 100 .2 2 .4 6 7 5 3 3 8.2 6.9 3 Negative Control - + 5.4 100 4.6 100 2 4 3.7 8.7 S-6 0.03 1.2 22 5 41.7 + 0.03 2.4 52 3 12.5 4 Negative Control + 10.1 9.75 100 100 8 15 7.9 15.4 Positive Control 1,2,3,4 Diepoxybutane + 0.04 0.04 9.0 90 1712 1902 8.2 84 1071 1306 o o Jn O' Z -a O' =a O' S-6 - 0.01 + 0.01 - 0.02 + 0.02 - 0.03 + 0.03 - 0.04 + 0.04 9.2 11.0 12.5 14.3 15.5 12.6 6.6 1.6 91 112 124 147 153 129 65 16 7 12 15 12 12 16 8 3 7.6 10.9 12.0 8.4 9.7 12.7 12.1 18.8 Table 3 (Continued) ) Exper. No. 1 2 3 01 ^ -n o-n VOi' ZM <O' '"1 Compound Metabolic Activat ion Negative Control S-7 Negative Control S-7 Negative Control Positive Control 1,2,3,4 Diepoxy butane S-7 + -- + + + + + + + + + + + + Percent Concent rat ion (w/v or v/v) 0.01 0.01 0.1 0.1 0.5 0.5 0.01 0.01 0.1 0.1 0.04 0.04 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 Surviving Cells per ml (x 10-7) 5.7 4.4 6.6 3.7 7.1 6.6 Toxic Toxic 8.5 7.2 11.8 8.4 9.6 7.4 10.1 9.75 9.0 8.2 8.5 9.8 8.4 11.5 Toxic 10.8 Toxic Toxic Percent Survivors 100 100 116 84 125 150 100 100 139 117 113 102 100 100 90 84 84 101 83 118 110 Mitotic Recombinants per ml (x 10~3) 4 3 8 8 3 3 7 5 4 4 5 5 8 15 1712 1071 5 8 4 13 16 Mitotic Recombinants per 10"' Survivors 7.0 6.8 12.1 21.6 4.2 4.5 8.2 6.9 3.4 4.8 5.2 6.8 7.9 15.4 1902 1306 5.9 8.2 4.8 11.3 14.8 Table 3 (Continued) Exper No, 1 2 OwJ 3 Compound Me tabu]ic Actjvat ion Negative Control - + Positive Control 1,2,3,4 Diepoxybutane - + S-8 - + Negative Control - + S-8 - + Negative Control - + Positive Control 1,2,3,4 Diepoxybutane - + S-8 - + - + - + - + Percen t Conrentration (w/v or v/v) 0.05 0.05 0.1 0.1 0.2 0.2 0.04 0.04 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 Surviving Cells per ml (x 10'7) 7.8 6.0 4.1 5.2 7.0 6.3 8.5 7.2 5.1 4.2 10.1 9.75 9.0 8.2 9.9 5.7 11.9 11.4 10.0 7.9 8.8 6.3 Percent Survivors 100 100 53 87 90 105 100 100 60 58 100 100 90 84 98 58 118 117 100 81 87 65 Mitotic Recombinants per ml (x 10-3) 10 4 1478 1286 8 3 7 5 11 7 8 15 1712 1071 6 6 6 8 8 12 10 5 Mitotic Recombinants per 10s Survivors 12.8 6.7 3605 2473 11.4 4.8 8.2 6.9 21.6 16.7 7.9 15.4 1902 1306 6.1 10.5 5.0 7.0 8.0 15.2 11.4 7.9 Table 3 (Continued) Exper. No. 1 2 L4>J 3 o ^z-Tt So Ow Zrn S'- -a O' M vO Compound Metabolic Activation Negative Control S-9 + + + Negative Control S-9 + + + Negative Control Positive Control 1,2,3,4 Diepoxy butane S-9 + + + + + + + Percent Concentration (w/v or v/v) 0.01 0.01 0.1 0.1 0.3 0.5 0.01 0.01 0.1 0.1 0.04 0.04 0.2 0.2 0.4 0.4 0.6 0.6 0.8 0.8 1.0 1.0 Su rviving Celts per ml (x 10~7) 5.7 4.4 7.0 6.6 4.9 4.8 7.3 5.5 8.5 7.2 8.4 7.0 5.0 6.3 8.2 7.3 9.3 5.5 6.5 4.7 7.5 6.5 5.7 5.2 7.5 7.4 8.7 6.1 Percent Survivors 100 100 123 150 86 109 128 125 100 100 98 97 59 88 100 100 113 75 79 64 91 89 70 71 91 101 106 84 Mitotic Recombinants per ml (x 10-3) 4 3 1 13 18 5 8 1 7 5 1 2 3 3 5 5 950 1056 8 8 3 3 3 8 7 8 10 7 Mitotic Recombinants per lO1* Survivors 7.0 6.8 1.4 19.7 36.7 10.4 11.0 1.8 8.2 6.9 1.2 2.9 6.0 5.0 6.1 6.8 1021 1920 12.3 17.0 4.0 4.6 5.3 15.4 9.3 10.8 11.5 11.5 Table 3 (Continued) Exper. No. 1 Compound Negative Control S-10 2 Negative Control S-10 3 Negative Control Metabolic Activat Lon _ + - + - + - + - + - + - Pe rcent Concent ration (w/v or v/v) 0.01 0.01 0.1 0.1 .01 .01 0.1 0.1 Positive Control 1,2,3,4 Diepoxybutane S-10 + - + + - + - + + 0.04 0.04 0.01 0.01 0.02 0.02 0.03 0.03 0.04 0.04 0.05 0.05 rviving Is per ml x 10-7) 5.7 4.4 4.8 3.2 Toxic Toxic 8.5 7.2 6.0 7.2 Toxic Toxic 8.2 7.3 9.3 5.5 Percent Survivors 100 100 84 73 100 100 71 100 100 100 113 75 Mitotic Recombinants per ml (x 10"3) 4 3 5 3 7 5 6 7 5 5 950 1056 Mitotic Recombinants per 10s Survivors 7.0 6.8 10.4 9.4 8.2 6.9 10 .0 9.7 6.1 6.8 1021 1920 7.0 85 4 5.7 7.7 105 1 1.3 3.0 37 8 26.7 4.0 55 9 22.5 .9 11 4 44.4 3.1 42 3 9.7 .6 7 8 133.0 1.3 18 5 38.5 Toxic Toxic Table 3 (Continued) ) Exper. No. 1 2 Compound Negative Control S-ll Negative Control S-ll Metabolic Activation - + - + - + + -- + Percent Concentration (w/v or v/v) 0.01 0.01 0.1 0.1 0.01 0.01 rviving Is per ml x 10-7) 5.7 4.4 2.7 1.2 Toxic Toxic 8.5 7.2 2.7 2.7 Percent Survivors 100 100 47 27 100 100 32 38 Mitotic Recombinants per ml (x 10~3) 4 3 3 1 7 5 4 2 Mitotic Recombinants per 10^ Survivors 7.0 6.8 11.1 8.3 8.2 6.9 14.8 7.4 00 1.36571 OONFTDFNTTAl Table 3 (Continued) Exper No. Compound Metabolic Activat ion Negative Control S-12 Negative Control S-12 Negative Control Positive Control 1,2,3,A Diepoxy butane S-12 + + + - + + - + + + + - + + - + - + Percent Concentration (w/v or v/v) 0.01 0.01 0.1 0.1 0.01 0.01 0.1 0.1 0.04 0.04 0.002 0.002 0.004 0.004 0.006 0.006 0.008 0.008 0.01 0.01 ' viv ing .s per ml : 10~7) 5.7 4.4 1.7 1.3 Toxic Toxic 8.5 7.2 2.4 4.1 .2 .7 8.2 7.3 9.3 5.5 Percent Survivors 100 100 30 30 100 100 28 57 2 10 100 100 113 75 Mitotic Recombinants per ml (x 10"3) 4 3 13 5 7 5 2 4 Toxic Toxic 5 5 950 1056 Mitotic Recombinants per 10^ Survivors 7.0 6.8 76.5 38.5 8.2 6.9 8.3 9.8 6.1 6.8 1021 1920 8.6 105 8.5 116 7.7 94 5.6 77 5.5 67 6.4 88 5.1 62 5.0 68 4.9 60 6.8 93 7 10 8 5 7 6 2 6 5 10 8.1 1.2 10.4 8.9 12.7 9.4 3.9 12.0 10.2 14.7 DO 1.365 7 2 CONFTDFNTTA!,. Table 3 (Continued) Exper. No. 1 2 3 4 Compound Negative Control S-13 Negative Control S-13 Negative Control S-13 Metabolic Activat ion + - + - + + - + + + - Negative Control Positive Control 1,2,3,4 Diepoxybutane S-13 + + + + Percent Coneent rat ion (w/v or v/v) 0.01 0.01 0.1 0.1 0.01 0.01 0.1 0.1 0.02 0.02 0.04 0.04 0.01 0.01 0.02 0.02 viving s per ml : 10-7) 5.7 4.4 6.9 4.4 Toxic Toxic 8.5 7.2 6.3 4.9 Toxic Toxic 5.4 4.6 1.9 1.9 8.2 7.3 9.3 5.5 Percent Survivors 100 100 121 100 100 100 74 68 100 100 35 41 100 100 113 75 Mitotic Recombinants per ml (x 10"3) 4 3 5 3 7 5 3 3 2 4 2 3 5 5 950 1056 Mitotic Recombinants per 10<) Survivors 7.0 6.8 7.2 6.8 8.2 6.9 4.8 6.1 3.7 8.7 10.5 15.8 6.1 6.8 1021 1920 7.8 95 7.3 100 8.8 107 7.2 99 2 2 4 2 2.6 2.7 4.5 2.8 oo 136^73 r.ONFTDENTT Table 3 (Concluded) ) Exper. No. 1 2 3 4 Compound Negative Control Metabolie Activat ion - + S-14 - + - + Negative Control + S-14 Negative Control S-14 Negative Control Positive Control 1,2,3,4 Diepoxybutane S-14 - + - + + + + + + + Percent Cone ent rat ion (w/v or v/v) 0.01 0.01 0.1 0.1 .01 .01 .1 .1 .005 .005 0.04 0.04 0.01 0.01 0.05 0.05 rviving Is per mL x 10-7) 5.7 4.4 1.1 1.1 Toxic Toxic 8.5 7.2 4.5 4.1 .5 .8 5.4 4.6 1.3 1.8 8.2 7.3 9.3 5.5 Percent Survivors 100 100 19 25 100 100 53 57 6 11 100 100 24 39 100 100 113 75 Mitotic Recombinants per ml (x 10-3) 4 3 3 3 7 5 8 3 2 2 2 4 2 1 5 5 950 1056 Mitotic Recombinants per 10*` Survivors 7.0 6.8 27.3 27.3 8.2 6.9 17.8 7.3 3.7 8.7 15.4 5.6 6.1 6.8 1021 1920 8.2 100 5.5 75 2.5 30 3.4 47 9 5 4 7 11.0 9.1 16.0 20.6 DO 1 3 6 5 7 4 C O N F ID E N T ! Final Report IN VITRO MICROBIOLOGICAL MUTAGENICITY STUDIES OF DOW CHEMICAL COMPANY COMPOUNDS Prepared for: DOW CHEMICAL COMPANY Midland, Michigan Attention: L. W. Rampy Submitted by: Vincent F. Simmon, Ph.D. Manager, Microbial Genetics Program Denis C. Poole Microbiologist SRI Project No. LSC-4378 Approved: CT. "iv. Newell,'--Director Department of Toxicology W. A. Skinner, Executive Director Life Sciences Division 26 April 1976 r>.> - ^ DO OOHF toF.NT4AL UNO 55 8 8 2 SUMMARY SRI examined 17 Dow Chemical Company compounds for mutagenicity in five strains of Salmonella typhimurium and for mitotic recombina tion in Saccharomyces cerevisiae D3. The results of the Salmonella tests for all compounds and the cerevisiae tests for 14 compounds were presented in an interim report submitted in January. Four compounds required further testing: S-l, S-15, S-16, and S-17 were retested in the >. cerevisiae assay procedure, and the results are reported here. S-l and S-15 only marginally increased mitotic recombination, and S-16 and S-17 did not increase mitotic recombination at all. 1 00 1.36576 CONFTDFNTTAl INTRODUCTION SRI examined 17 Dow Chemical Company compounds for mutagenicity in five strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98, TA100) and for mitotic recombination in Saccharomyces cerevisiae D3. The results of all the Salmonella tests and of 14 of the 17 Sac charomyces tests were reported in January 1976. SRI retested four compounds for mitotic recombination with S. cerevisiae D3. An Aroclor 1254-stimulated, rat-liver-homogenate metabolic activation system was included in this assay procedure to provide metabolic steps that the yeast are either incapable of con ducting or that they do not carry out under the assay conditions. The purpose of this study was to determine whether the compounds caused genetic mutation in microorganisms. The assay procedure with S, typhimurium has been proven to be 85 to 90% accurate in detecting carcinogens as mutagens, and it has about the same accuracy in identifying chemicals that are not car cinogenic. 1 The assay procedure with S. cerevisiae is about 60% accurate in detecting carcinogens as agents that increase mitotic recombination.2 The combination of these two assay procedures significantly enhances the probability of detecting potentially hazardous chemicals. However, because the test systems are not 100% accurate, neither a positive nor a negative response proves that a chemical is hazardous or nonhazardous to man. 2 .36^77 CONFt'D^NT'C^- METHODS Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 The S. typhimurium strains used at SRI were obtained from Dr. Bruce Ames of the University of California at Berkeley.3-5 All are histidine auzotrophs (his") by virtue of mutations in the histidine operon. In addition to the mutations in the histidine operon, five of the indicator strains have mutations in the lipopolysaccharide coat (rfa") and deletions that cover a gene involved in the repair of uv damage (uvrB~). The rfa" mutation makes the strains more permeable to large molecules, thereby increasing their sensitivity to these molecules. The uvrB mutation decreases repair of some types of chemically damaged DNA and thereby enhances sensitivity to some mutagenic chemicals. Strain TA1535 is reverted to histidine prototrophy (his +) by many mutagens that cause base-pair substitu tions. Strains TA1537 and TA1538 are reverted by many frameshift mutagens. TA1537 is more sensitive than TA1538 to mutation by some acridines and benzanthracenes, but the difference is quantitative rather than qualitative. TA100 is derived from TA1535 by the intro duction of the R factor plasmid pKMIOl.6 The introduction of this plasmid, which confers ampicillin resistance to the strain, greatly enhances the sensitivity of the strain to some base-pair substitution mutagens. We have shown that mutagens such as benzyl chloride and 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (known as AF2) can be detected in plate assays by TA100 but not by TA1535. The presence of this plasmid also makes strain TA100 sensitive to some frameshift mutagens--e.g., ICR-191, benzo(a)pyrene, alfatoxin B^, and 7,12dimethylbenz a)-anthracene. TA98 is derived from TA1538 by the addition of the same plasmid, and the plasmid makes this strain more sensitive to some mutagens. 3 DO 136578 CONFIDENTIAL All the indicator strains are stored at -80C. For each experi ment, an inoculum from frozen stock cultures is grown overnight at 37c in a nutrient broth consisting of 1# tryptone and 0.5# yeast extract. After stationary overnight growth, the cultures are shaken for 3 to 4 hours to ensure optimal growth. Each culture is checked for sensitivity to crystal violet. The presence of the rfa~ mutation makes the indicator strains sensitive to this dye, whereas the parent strain, rf a~*~, is not sensitive to the dye. However, the mutation is reversible, leading to the accumulation of rfa* cells in the culture. Therefore, the cells must be tested routinely to ensure their sensi tivity to crystal violet. Each culture also is tested by specific mutagens known to revert each test strain (positive controls). To a sterile 13 x 100 mm test tube placed in a 43C heating block, we add in the following order: Assays in agar (1) 2 ml of 0.6# agar (2) 0.1 ml of indicator organisms ! (3) 0.5 ml of metabolic activation mixture (optional) (4) Up to 100 p.1 of a solution of the test chemical. For negative controls, we use steps (l), (2), and (3) (optional) and 100 p,l of the solvent used for the test chemical. This mixture is stirred gently and then poured onto minimal agar plates. After the soft agar has set, the plates are incubated at 37c for 2 days. The number of his+ revertants (colonies that grow on plates lacking a sufficient amount of histidine to support colony formation) are counted and recorded. Some of the revertants are j^ rountinely tested to confirm that they are his , require biotin, and are sensitive to crystal violet (rfa ). 0.6# agar contains 0.05 mM histidine and 0.05 mM biotin. Minimal agar plates consist of 15 g of agar, 20 g of glucose, 0.2 g of MgS04.7 HgO, 2 g of citric acid monohydrate, 10 g of K2HP04, and 3.5 g of NaHNH^PO^. HgO per liter. 4 00 136579 r.ONFTDENTTAl Saccharomyces cerevisiae D3 The yeast . cerevisiae D3 is a diploid heterozygous for a mutation in an adenine-metaboli2ing enzyme.7 Cells homozygous for this mutation produce a red dye when grown on medium containing adenine. Adeninerequiring homozygotes can be generated from the heterozygotes by mitotic recombination. Many mutagens increase the frequency of mitotic recombi nation. Mitotic recombination is indicated by the development of colonies with red pigmentation, and the degree of conversion to this pigmented colony indicates the mutagenicity of a compound or its metabolite.8 The Saccharomyces test strain from the liquid nitrogen is grown over night at 30C with aeration of 1.0% tryptone and 0.5% yeast extract. The cells are washed twice in 0.067M P04 buffer (pH 7.4) and resuspended in the same buffer at a concentration of 10s cells/ml. The i_n vitro yeast mitotic recombination assay in suspension consists of 5 x 107 washed, stationary-phase yeast cells in 1 ml of 0.067M PO^ buffer (pH 7.4) and 50 mg/ml of the test chemical (or a fraction of the concentration required to give 50% killing). The suspension is incubated at 30 for 4 hours. After incubation, the sample is diluted serially in sterile saline and plated on tryptone-yeast agar plates. Plates of a 10-3 dilution are incubated for 2 days at 30c, followed by 2 days at 4c to enhance the development of the red pigment indicative of adenine-negative homozygosity. To detect red colonies or red sectors, we scan the plates with a dissecting microscope at 10 x magnification. Plates of a 10~5 dilution are incubated for 2 days at 30C for determination of the total number of colony-forming units. The i_n vitro yeast mitotic recombination assay in suspension with metabolic activation is conducted as above with the addition of the meta bolic activation system to the incubation mixture. Metabolic Activation--Aroclor 1254-Stimulated Metabolic Activation System Some carcinogenic mutagens (e.g., dimethylnitrosamine) are inactive unless they are converted to their active form by being metabolized. Ames 5 00 136580 CONFIDENTIAL has described the metabolic activation systems we use,10 Adult male mice are given a single 500-mg/kg intraperitoneal injection of a polychlorinated biphenyl (Aroclor 1254).10 Four days after the injection, the animals' food is removed. On the fifth day, the mice are killed. The livers are removed aseptically and placed in preweighed, sterile glass beakers. The organ weight is determined, and all subsequent operations to the metabolic activation step are conducted in an ice bath. The organ is washed in an equal volume of cold, sterile 0.15 M KC1 (1 ml/g of wet organ), minced with sterile surgical scissors in three volumes of 0.15 M KC1 and homogenized with a potter-elvehjem apparatus. The homogenate is centri fuged for 10 minutes at 9000 x g, and the supernatant is removed and stored in liquid nitrogen. To the postmitochondrial supernate are added MgCl2, KC1, glucose-6-phosphate, TPN, and sodium phosphate (pH 7.4). 6 DO 136&01 C0NF AL RESULTS AND DISCUSSION Tables 1 through 4 present the results of the microbiological assays of each compound for mitotic recombination in S. cerevisiae D3. We tested each compound once to determine the maximum dose tolerable to the microorganism and at least twice more to accurately determine the effects of the chemical on mitotic recombination. A positive response is indicated by a more than threefold increase in the absolute number of mitotic recombinants per ml as well as in the relative number of mitotic recombinants per 105 survivors. Compounds S-l and S-15 caused an increase in the mitotic recom bination frequency that was twofold greater than the normal, expected frequency. However, in repeated testing, neither compound gave a strong positive response and neither compound was mutagenic in the previous Salmonella assay. Therefore, we do not believe S-l and S-15 are mutagens by these procedures. Because compounds S-16 and S-17 did not increase mitotic recombination in any of these assays, we do not believe these compounds are mutagens by these procedures. 7 00 136^82 oonftdfnttai. REFERENCES 1. J. McCann, E. Choi, E. Yamasaki, and B. N. Ames. Detection of carcinogens as mutagens in the Salmonella microsome test: Assay of 300 chemicals. Proc. Nat. Acad. Sci. USA _72> 5135-5139. 2. V. Simmon. Unpublished results. 3. B. N. Ames, E. G. Gurney, J. A. Miller, and H. Bartsch. Carcinogens as frameshift mutagens: Metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc. Nat. Acad. Sci. USA 69, 3128-3132 (1972). 4. B. N. Ames, F. D. Lee, and W. E. Durston. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat. Acad. Sci. USA 7(), 782-786 (1973). 5. B. N. Ames, E. W. Durston, E. Yamasaki, and F. D. Lee. Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection. Proc. Nat. Acad. Sci. USA 70, (1973). 6. J. McCann, N. E. Spingar, J. Kobori, and B. N. Ames. The detection of carcinogens as mutagens: Bacterial tester strains with F factor plasmids. Proc. Nat. Acad. Sci. USA (in press, 1975). 7. F. K. Zimmermann and R. Schwaier. Induction of mitotic gene conversion with nitrous acid, l-methyl-3-nitro-l-nitrosoguanidine and other alkylating agents in Saccharomyces cerevisiae'. Mol. Gen. Genet. 100, 63-69 (1967). 8. D. J. Brusick and V. W. Mayer. New developments in mutagenicity screening techniques with yeast. Environ. Health Perspectives 6^ 83-96 (1973). 9. L. D. Kier, E. Yamasaki, and B. N. Ames. Detection of mutagenic activity in cigarette smoke condensates. Proc. Nat, Acad. Sci. USA 71, 4159-4163 (1974).10 10. B. N. Ames, J. McCann, and E. Yamasaki. Methods for Detecting Carcino gens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364 (1975). 8 Table 1 IN VITRO ASSAYS OF COMPOUND S-l WITH SACCHAROMYCES CEREVISIAE D3 Exper. No. Compound Metabolic Activa tion Percen t Concentration (x/v or v/v) Surviving Cells/ml (x 10"7) Percent Survivors Recombinants per ml (x 10"3) Recombinants per 10 s Survivors 1 Negative control 4- S-l _ + + + 2 Negative control _ + Positive control 1,2,3? 4-Diepoxybutane -- _U S-l - + -U 0.01 0.01 0. 1 0. 1 0. 5 0.5 0.05 0.05 0.1 0.1 0.5 0.5 6.7 4.5 100 100 6 5 7.6 6.4 7. 1 5. 3 4.8 6. 2 97 123 106 118 72 138 10 8 13 15 8 18 7.8 6.0 100 100 10 4 4.1 5.2 53 1478 87 1286 8.3 7. 1 Toxic Toxic 106 118 15 16 9.0 11.1 13.1 12.5 18.3 28.3 16.7 29.0 12.8 6.7 3605 2473 18.1 22.5 DO 1 .3 6 5 8 4 CONFTDFNTT Table 1 (concluded) 1 Exper. No. Compound Metabolic Ac tiva tion Percent Concentration (x/v or v/v) Surviving CelIs/ml (x 10"7) Percent Survivors Recombinants per ml (x 10"3) Recombinants per 10s Survivors 3 >- o 4 bo zo -n a-T\ 'j3>s ZM ^05 ^ O' > Negative control Positive control 1,2,3,4-Diepoxybutane S-l Negative control Positive control 1,2,3,4-Diepoxybutane S-l - -L -- + + - 4- -U + _ + _ 4. _ 4- + - - f 0.04 0.04 0. 1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 0.5 0.5 0.04 0.04 0.1 0. 1 0.2 0.2 0.3 0.3 0.4 0.4 0.5 0.5 5.3 3.6 3.4 4.4 4.9 3.9 5.3 3.2 4.0 3.4 3.8 2.9 2.6 3.4 6.3 6.0 4.5 4.3 6.0 5. 1 7.0 5.3 6.0 4.9 4.5 5.3 3.6 4.2 100 100 64 122 92 108 100 89 75 94 72 81 49 94 100 100 71 72 95 85 111 88 95 82 71 88 57 72 4 2 236 225 1 1 4 2 2 1 4 2 5 5 10 8 1125 818 7 4 4 6 7 10 14 10 12 12 7.5 5.6 694 511 2.0 2.6 7.5 6.3 5.0 2.9 10. 5 6.9 19.2 14.7 15.9 13.3 2500 1902 11.7 7.8 5.7 11.3 11.7 20.4 31. 1 18.9 33.3 28.6 ) Table 2 TN VITRO ASSAYS OF COMPOUND S-15 WITH SACCHAROMYCES CEREVISIAE 33 Ex per. No. Compound Negative control Metabolic Activation Percent Concentration (x/v or v/v) Surviving Cells/ml (x 10~7) Percent Survivors Recombinants per ml (x 10 3) Recombinants per 10s Survivors 5.7 4.4 100 100 4 3 7.0 6.8 Positive control 1,2,3,4-Diepoxybutane S-15 0.05 0.05 0.01 0.01 0.1 0. 1 .8 .3 4.7 6.4 Toxic Toxic 14 3 82 145 514 94 3 10 6.4 15.6 Negative control 8.5 7.2 100 100 7 5 8.2 6.9 Positive control 1,2,3,4-Diepoxybutane 0.05 0.05 .3 4 152 1.6 22 566 3537 S-15 O O w CP . IP t oa 4 O' 0.01 0.01 0.1 0. 1 6.2 6.8 3.3 3.7 73 94 38 51 4 2 13 10 6.5 2.9 39.4 27.0 1 Table 2 (concluded) Exper. No. Compound________ 3 Negative control Metabolic Activation Positive control 1,2,3,4-Diepoxybutane S-15 4 O o U) i : 'J' 4 3) 4 Vl Negative control Positive control 1,2,3,4-Diepoxybutane S-15 4- tercent :entrat ion > or v/v) Surviving Cells per ml (x 10~7) Percent Survivors Recombinants per ml fx 10'*3)i4* Recombinants per 105 Survivors 0.04 0.04 0.02 0.02 0.06 0.06 0.08 0.08 0.10 0.10 0.04 0.04 0.02 0.02 0.06 0.06 0.08 0.08 0.10 0. 10 5.3 3.6 3.4 4.4 4.8 5.3 5.7 5.3 4.2 4.9 4.9 4.3 4.8 4.6 6.6 5. 5 6.6 4.6 5.6 4.8 5.4 5.7 5.7 4.5 100 100 64 122 91 147 108 147 79 136 92 119 100 100 138 120 138 100 117 104 113 124 119 98 4 2 236 225 4 1 3 4 1 1 4 4 3 6 583 1198 4 2 3 7 6 5 2 12 7.5 5.6 694 511 8.3 1.9 5.3 7.5 2.4 2.0 8.2 9.3 6.3 13.0 883 2178 6.1 4.3 5.4 14.6 11.1 8.3 3.5 26.7 1 Table 3 IN VITRO ASSAYS OF COMPOUND S-16 WITH SACCHAROMYCES CEREVISIAE D3 Expe r. No. Compound Me tabolic Ac tiva tion Percent Concentration (x/v or v/v) Surviving Cells/ml (x 10-7) Percent Survivors Recombinants per ml (x lO"3) Recombinants per 10s Survivors 1 Negative control 4- Positive control 1,2,3,4-Diepoxybutane S-16 "b _ 4- 0.05 0.05 0.01 0.01 0. 1 o.l 5.7 4.4 0.8 0.3 1.2 2.3 Toxic Toxic 100 100 14 3 21 52 4 3 514 94 10 3 7.0 6.8 83 13.0 2 O O' Z 3! -4 00 r-t 3) 3> Negative control Positive control 1,2,3,4-Diepoxybutane S-16 + _ 4- _ -I_ 0.05 0.05 0.01 0.01 0. 1 0. 1 3.5 7.2 0.3 1.6 3.5 3.8 Toxic Toxic 100 100 4 22 41 53 7 5 152 566 10 10 8.2 6.9 3537 28.6 26.3 1 Table 3 (concluded) Ex per No. Compound Me tabolie Activation Percen t Concentration (x/v or v/v) Surviving Cells/ml (x 10-7) Percent Survivors Recombinants per ml (x 10-3) Recombinants per 105 Survivors 3 Negative control 4- Positive control 1,2,3^4-Diepoxybutane S-16 - + _ 4- - + - + - + 0.04 0.04 0.004 0.004 0.008 0.008 0.01 0.01 0.02 0.02 5.3 3.6 3.4 4.4 5.3 4.5 3.9 3.4 3.3 2.5 3.8 3.0 100 100 64 122 100 125 74 64 92 47 72 83 4 2 236 225 2 1 3 3 3 1 2 4 7.5 5.6 694 511 3.8 2.2 7.7 8.8 9.0 4.0 5.3 13.3 I Table 4 IN VITRO ASSAYS OF COMPOUND S-17 WITH SACCHAROMYCES CEREVIS1AE D3 Exper. No. Compound Metabolic Activa tion Concentra tion (x/v or v/v) Surviving Cells per ml (x 10-7) Recombinants Percent per ml Survivors (x 10"3) Recombinants per 10s Survivors 1 Negative control + Positive control 112, 3,4-Diepoxybutane -U S-17 4- - -L 2 Negative control Positive control 1 j 2;3,4-Diepoxybu tane S-17 _ + _ + - 0.05 0.05 0.01 0.01 0. 1 0.1 0.05 0.05 0.01 0.01 5.7 4.4 0.8 0.3 3.6 1.7 Toxic Toxic 8.5 7.2 0.3 1.6 5.9 5. 5 100 100 14 7 63 38 100 100 4 22 69 76 4 7.0 3 6.8 514 94 1 2.8 8 47.1 7 8.2 5 6.9 152 566 3537 IS 30.5 4 7.3 DO 1 3 6 5 9 0 CO NFIDENT! Table 4 (concluded) Exper. No. Compound Metabolic Activation Concen tra tion (x/v or v/v) Surviving Cells per ml Percent (x lO"7) Survivors Recombinants per ml (x 10 3) Recombinants per 10s Survivors 3 Ol Negative control Positive control 1,2,3,4-Diepoxybutane S-17 -1_ _ + -- + -- 4- 0.04 0.04 0.006 0.006 0.010 0.010 0.020 0.020 0.03 0.03 5.3 3.6 3.4 4.4 4.8 4.2 4.6 3.5 6.3 4.7 4.7 4.0 100 100 64 122 91 117 87 97 119 131 89 111 4 2 236 225 5 1 1 1 3 5 3 5 7.5 5.6 694 511 10.4 2.4 2.2 2.9 4.8 10.6 6.4 12.5 . T2.2-183-(2) lord Research Inst. 3$eoj ct N . LSC-4278-1 fB n System: Aroclor tisulated mouse liver by: L. W. Rampy Test Strain: Bacterial-Salmonella typhimurium Yeast-Saccharomyces cerevisiae TA98,TA100, TA1535, TA1537, TA1538 03 ctnon-act. Bact- Yeastact. non-act. Yeastact. S-l (+) (+) Acrylonitrile From tox. study 1 ,iv) S-2 + + vinylbenzyl chloride Lot 106174 / S-3 - Benzene -t > t- - / Bay City Pipeline, 5/28/75 S-4 - - (+) (+) Styrene tars * * ?Lf - ' LUWA, Styrene finishing still tar column, 580 Bldg S-5 - - (+) (+) Chloroacetic Acid A' ^ < Batch 145 (1501), 5/28/75 '-6 Styrene * / i From inhal. tox. study (5 ppm TBC), 6/75 S-7 Chloroacetyl chloride Lot 05285-5; Batch 3105 tank, 5/28/75 S-8 50/50 Polyethvlben2ene/ DOWFROTH* c,/1 Ref. 1408-3-5-1 S-9 Decabromodiphenyl Oxide Lot #09014-317 K ^ - S-10 (+) (+) Perchloroethylene (DOWPER* formulation, W. Dengler, H. Farber, 6/75) : S-ll S- A8-12 '. - . ------------------6/12/75 itvi'ii - (' - c' Divinyl Benzene Lot #05135 ^ to*i v ^1 Pentachlorophenol (tars) ^ + * positiv (all strains unless otherwise indicat d) - *;cnegativ (all strains unless otherwise indicat d) (4-) ^Imargiijally positive (all strains unless otherwise indicated) (*) " '`margiiiiilly negative (all strains unless otherwis indicated) 5-4-76