Document jpo1vREK6b4rg04Qnr6pLr69

FILE NAME: Oil Industry and American Petroleum Institute (API) DATE: 1956 Nov 1 DOC#: API132 DOCUMENT DESCRIPTION: API Research Project MC-1 - Interim Report Kettering Lab INTERIM REPORT API RESEARCH PROJECT MC-1 November 1, 19$6 THE KETTERING LABORATORY M B*e Department of Preventive M edian and Industriai Health College of M edicine U N IVERSITY O F C IN C IN N A T I, C IN C IN N A T I, O H IO API 06555 INTERIM REPORT - API RESEARCH PROJECT MC-1 November 1, 195& Certain factors, which were instrumental in bringing about a systematic investigation of potential hazards associated with carcinogenic materials encountered in various refining operations, were reviewed in some detail in an annual report dated October 1, 1955* This program has encompassed products from some 16 different cracking and lube refining processes. The carcinogenic potencies of the 120 samples, as gauged by the effects induced by their contact with the skin of mice, have varied over a wide range. As much as ten-fold differences have been observed between materials from similar processes, which were at least superficially comparable. These variations have demonstrated that the history of the sample, including the crude source and the operating conditions under which it was processed, were all-important in determining its final activity. The need for rapid methods for estimating potency became*pparent. Many empirical correlations between physical or chemical properties and potencies have been tested. Some of these have found limited applicability to certain classes of oils but no generally applicable analytical technique has thus far been developed. By 1901 it was evident that the complexity of the problem was such that a API 06556 - 2- details^ understanding of the relationships between the . chemical composition of the different oils and their relative potencies would be required before it would be possible to set up the desired analytical procedures to replace the lengthy biological test. At the same time, the rapid changes which had taken place in this dynamic industry, since the initiation of the project in 1946, demonstrated that this type of fundamental understanding of the problem was essential for the recognition and control of the poten tial hazards associated with the handling of carcinogenic materials. In the annual report for 1955 It was shown how the program of fractionation and biological testing of various components of selected carcinogenic oils had led to a useful hypothesis regarding the contributions of various classes of hydrocarbons to the effective potency of a given material. For the purpose of orienting the collection of experimental data these contributions ay be expressed by the following equation: PMC = M a (Ci + C. + C8 ). The summation of C 's represented the effective potency con tributed by typical polycyclic carcinogens, such as benzo pyrene. The contribution of the non-carcinogenic fraction of the oil, including the paraffins, cycloparaffins, and oat of the aromatic constituents is represented by the "hltiplication factor, The considerable importance of these non-carcinogenic components is shown by the fact that API 06557 M_ may reach values as high as 10. To determine in a syste- CL a matic manner the relative contributions of carcinogens and non-carcinogens to potency, a series of oils have been fractionated during 1956 by certain physical and chemical procedures in order to produce concentrates of the poly cyclic carcinogens as free as possible of other components of the oils. By chromatographic procedures all of the car cinogens may be collected into a concentrate suitable for biologic test to provide values for the summation (Cx + Ca + Ca ). A further subdivision of the carcinogens has been possible through the process of extraction of the oils by maleic anhydride and subsequent chromatographic concentration of the components of the extract containing four or more rings. In Table 1 are shown the results of a series of biological tests on such fractions which are underway or have been completed during the current year. It will be noted that only about 15 per cent of the effective potency of the heavy gjetalyt,ic cycle gas oil, API-89, was contributed by polycyclic carcinogens (API-89-3) In contrast, the entire potency of the thermal tar, API-65 can be ascribed to such polycyclic components. In both instances 2$ to 30 per cent of the basic potency due to polycyclic aromatics could be ascribed to compounds ex tracted by maleic anhydride, such as derivatives of benz(a)- anthracene. The contribution of the 0-ringed class, benzopyrenes, can be estimated from the content of this class as determined by direct chemical analysis. The third -k - major class of carcinogens, those of lowest molecular weight, are being investigated by a more direct approach, which will be discussed in a subsequent section. Measurement of the multiplication factor, MBl, cannot be made by any direct method. This contribution due to accelerators may be estimated from the ratio of the effective potency of the oil to the base potency contributed by the polycyclic fraction. Thus, for oils API-89 and API-65, the estimated values of M would be 7 and 1, respectively. The potential difficulties which preclude a direct measurement of Mfi arise from the fact that a significant part of the non-carcinogens of the oils have physical properties which are quite similar to those of the carcino gens. Hence, separation of these aromatic non-carcinogens from the carcinogens requires highly efficient application of the techniques of distillation and chromatography. Such separations are being carried out with a few selected materials, but require much more time than the simpler separations which are being applied to quite a variety of oils. It is possible, however, to obtain some useful information by direct tests of the effective accelerating properties of the non-carcinogenic components which are easily separated from the whole oil by chromatography. Thus, the fractions containing essentially all of the paraffins and cycloparaffins and monoeyeloaromaties, aa well as most of the naphthalene derivatives, are under API 06559 5 test as the vehicles for 0.05 Pr cent of methylcholanthrene. In addition, tests are being started with solutions of methylcholanthrene in a medium devised to simulate as closely as possible the combination of the non-carcinogenic components of the original oils. These media are being blended from the non-carcinogenic chromatographic fractions together with certain liquid aromatic hydrocarbons to replace the polycyclics separated in the chromatography. These liquids, tetrahydronaphthalene and diphenylmethane, satisfy the theoretical requirement for introduction into the general medium of the aromatic system, with the potentially significant influence of its Tf-orbitals on the physical characteristics of the liquid phase, without the complications of insolubility, on the one hand, or high volatility, on the other, encountered in initial efforts to employ phenanthrene or benzene for this purpose. It will be recalled from discussions in previous reports that, if the concentration of accelerators in an oil falls in the range 20 to $0 per cent (typical of catalytically cracked residua), some indication of the magnitude of their influence may be estimated by tests of the whole oils at dosage levels varying from 20 mg. to 100 mg. per application. One Is, of course, limited by the toxic nature of an oil in determining the maximum effect of such accelerators. Hence, a direct test using appropriate fractions obtained in the chemical laboratory may serve this purpose better. API 06560 Since the results of tests on simple mixtures of relatively pure hydrocarbons normally lead to a more rapid understanding of the relative contribution of different types of components, a series of parallel tests are being carried out on solutions of benzopyrene In blended media, Including a standard accelerator such as n-dodecane, together with cycloparaffins of high molecular weight (from white mineral oils), on the one hand, or with more volatile hydrocarbons of relatively low molecular weight, on the other. The results are shown In Table 2 and In Figure 1. It will be noted that, at the 50 mg. level of dosage, dilution of dodecane with 50 per cent of either of the non-accelerating solvents, decahydronaphthalene or beta-methylnaphthalene, did not reduce the accelerating activity of the n-paraffln . On the other hand, dilution with only 25 per cent of white mineral oil {viscosity, 3I4.O S.S.U. at 100F.) reduced the activity considerably. These results suggest that some major component of the white oils has the property of counteracting accelerating Influences to a significant extent. It has been shown previously that some of these white oils contain long-chain accelerators. The failure to obtain from them any significant adduct with urea Indicates that these are not n-paraffins, but presumably Isoparaffins and long-chain alkylcyclohexanea or cyclo pentane a, the ring being near the end of the chain (1-cyclohexyldecane is an active accelerator, whereas 7 -cyclohexyl- tridecane is almost Inactive). It has been found that, to 1 I I __ a useful extent, the monocycloparaffins can be extracted from the white oils by their formation of adducts with thiourea, the polycycloparaffins concentrating in the non-adduct. Such fractionations are being carried out on API-80 to obtain material with which to determine whether the polycycloparaffins are responsible for the inhibitory effect observed in the blends discussed above. Since it is suspected that the physiological as well as the physical properties of the liquid media are also influenced signif icantly by the presence of non-carcinogenic aromatics, suitable blends will also be tested containing such components together with the polycycloparaffins and the accelerators. The preceding paragraphs indicate the necessity for consideration of the non-carcinogenic medium as a whole in the approach to the final goal of this phase of the project, a quantitative measure of the magnitude of the factor Ma under any given set of conditions of exposure. In addition, a continuing effort has been'made to define the general characteristics of the components of this medium which are primarily responsible for its catalytic (accelerating) influence on carcinogenesis. The earlier results of tests of a variety of relatively pure hydro carbons for their ability in this respect have been summarized in a paper entitled "Carcinogenesis of the Skin. II. The Catalytic Effects of Aliphatic and Related Hydrocarbons". 'mV. - 8- A fairly general relationship has been noted between the molecular length (in the extended configuration) of the solvent and its ability to accelerate the induction of tumors by polycyclic carcinogens. As shown in Fig. 2 , activity seems to reach a maximum level at a length equivalent to that of the n-paraffins containing 12 to II4. carbon atoms. Further, the maximum effect for alkyl derivatives of benzene and naphthalene seems to be somewhat greater than that for saturated hydrocarbons. The fairly active accelerating ability of triiso propylbenzene seems to be an exception to this relationship of molecular length and activity. Aa discussed previously, the initial sample used showed in its mass spectrum evidence of an impurity having a lower molecular weight. A pure sample of 1 ,3 ,5-triisopropylbenzene, kindly supplied by Dr. E. V. Adams, has now been tested. It has shown the same accelerating activity as the original sample (Ma-- ij.). The shape of the molecule has also been considered as a potentially important characteristic in addition to its length. The saturate, 7-cyclohexyltridecane, demon strated only a relatively low activity (MR " 2.5), even though its molecular length should be optimum (see structures below). In contrast, however, the corresponding 7-phenyl and 7-n-hexyl derivatives have shown a significantly more active accelerating influence (these samples were kindly supplied by API Research Project i+2 at Pennsylvania State College). CH3(CH2 )^CH (CH 2 ) 5CH3 7 -cyclohexyltridecane CH3 (CH2 ) &^CH (CH 2 )^CH3 7-phenyltridecane GR^iCBz) 7 -n-hexyltridecane API 06563 - 9- INVESTIGATION DIRECTED TOHARDS THE IDENTIFICATION OP THE CARCINOGENS OF FRACTIONS. DISTILLING IN THE RANGE 720 TO ?60*F., FROM CRACKED AND STRAIGHT RUN DISTILLATES OR RESIDUA These are the carcinogens responsible for the major part of the potency of heavy catalytic cycle gas oils, coker gas oils, and light lubricating stocks which contain little or no benzopyrene (occasionally the cracked gas oils have high enough end points to contain signif icant amounts of benzopyrene, but this seems to be the exception). In a concentrated attack on this problem of the nature of the carcinogen of lowest molecular weight in petroleum oils, the polycyclic aromatics obtained by chroma tography from a highly active residual product of Fluid Catalytic cracking, API-8, was fractionated through an 80-plate distillation column in 1955'* The' biological tests on narrow fractions distilling in the range, 650 to 760, were completed in 1956. The results are shown In Table-3. It has been demonstrated that all of these fractions contain accelerating components, although the activity in this respect apparently drops to a relatively low level by the time the concentration of carcinogen (in fraction 8-26-k or -1 ) has increased to a point capable of inducing tumors in 90 per cent of the C3H mice. API 06564 10 - The carcinogenic fractions, 8-26-1, -m, and -h, (730 to 760P.) were selected for,further fractionation. In order to reduce the complexity of the system the three fractions were first extracted with aqueous acid and base and then with maleic anhydride. The concentration of the major components of the maleic anhydride adduct from 8-26-n was estimated from its mass spectrum (ionization potential, 7 volts) as follows: n ^n^2n-l8 17 0,7% 18 5.1 19 6.1; 20 3.1 21 1.1 cnH 2n-20 0,0$ 11.1 11.8 5.6 1.8 CnH2n-22 0.3* 3.8 . 6.i| 2.3 0.8 CnH2n-2i4. - 13.3* 2.2 0.8 0.3 Suggested structures: Cycloalkylanthracenes Alkylbenzofluorenes Benz(&) anthracenes On the basis of the mass spectrum at high voltage, the cyclo- pentano substitution is considered to be the most likely structure for the CnH 2n_2o A six-membered API 06565 V % -n - cycloalkyl ring would be expected (on the basis of the ' spectrum of tetralin) to yield a strong fragment peak at m/e of 201; by loss of -CHa-CH*-. The intensity of the 201+ ion in the spectrum of the mixture was negligible. The components extracted by maleic anhydride apparently contributed about 1/6 of the potency of the original distillate, 8-26-n. About 1/5 of the accelerated potency of 0.10 shown by this fraction in the test 8-26-n-l can be ascribed to benzanthracene. It seems likely that the major contribution came from the l+-rlnged compounds having one partially saturated ring, but this cannot be stated with certainty until additional experimental evidence is available. The combined material from 8-26-1, -m, and -n, which had been freed from the components soluble in aqueous acid and base and from the anthracenlc compounds removed by maleic anhydride, was next subjected to chromatographic fractionation on activated alumina. Hass and absorption spectrometry were used to determine the distribution of the various classes of hydrocarbons through the chromatographic fractions (see Table +). The biological test on the fraction which contained almost all of the J+-rlnged chrysene and triphenylene (8-26-1J+) mixed with the more strongly adsorbed portion of the benzofluorenes, Indicated the presence of only a trace of the carcinogens of the parent distillate. The test on the most strongly adsorbed fraction (8-26-15), that which Includes nitrogen compounds such as derivatives API 06566 12 of carbazole, has shown mainly that this fraction is exceed ingly toxic to the mice. No tumors have been produced in 3U weeks. One of the intermediate fractions (8-26-11), that containing cyclopentanophenanthrenes, fluoranthenes, and benzofluorenes, but essentially free of compounds having ^ fully aromatic rings, has shown some carcinogenic activity, but was not responsible for more than about 10 per cent of the potency of the original distillates. The foregoing tests on fractions derived from 8-26-1, -m, and -n were accelerated by the use of dodecane as a solvent. This permitted a relatively rapid elimination of a possible [(.-ringed aromatic carcinogen from consideration. Tests of the remaining fractions are being carried out at their original concentration (or at a doubled concentration) in benzene as a solvent in order to obtain a better measure of their relative potencies, P TM , and for a more direct comparison with the potencies of the parent distillates, *-*- .v which were tested as 50 per cent solutions in benzene. These new tests, 8-26-7, -8 , -9, -10, and -12, have been under way about four months. The results of these tests should indicate the particular classes of polycyclic hydro carbons responsible for the major part of the potency in this distillation range, 720 to 760P. According to the physical and chemical behavior of the classes indicated, a further concentration of the carcinogens will be carried out until their certain identification has been accomplished. Analytical techniques for the direct determination of this class in complex oils may then be developed. API 06567 ! RELATIVE CARCINO FRACTION GENIC API DISTIL POTENCY, EXPERI LING BENZO *MC MENT GRAVITY <750F. PYRENE (at NUMBER DESCRIPTION (API) (per cent) (per cent' 100 mg.] I 89 Catalytically 27.0 100 0.001 2il cracked sidestream (F.C.C. heavy cycle gas oil) 33 Coker gas oil 16.8 60 0.008 o.U 65 Thermal tar 6.6 U3.8 o.ou 0.11X 120 Straight run dis 19.5 ~52 tillate (naphthenic ) o o Mil > 0.07 (50 mg.) 105 Straight run dis 28.9 tillate (waxy) 72.9 115 Straight run distil - 23.2 ^ 5 0 late (naphthenic) 0.0003 .002 0.11 0.03 API 06568 - 13 TABLE I r e l a t i v e c o n t r i b u t i o n s o p c a r c i n o g e n s a n d n o n -c a r c i n o g e n s to THE EFFECTIVE POTENCY OF VARIOUS REFINERY STREAMS CHROMATOGRAPHIC FRACTIONATION FRACTION EX'IRACTED BY MALEIC A ^HYDRIDE Saturates Thru Alkylnaphthalenes API PMC EXPERI with MENT 0.0 NUMBER Yield MC Poly BASIC cyclic CONTRI Aromatics BUTION and OF Non-hydro POLY carbons CYCLIC S (yield) TO Pm c 1 ESTI Yields MATED ACCELER ATING FACTOR 2-3 k* "a rings rings Contri bution of I4.+ Ring Fractior t0 pox 89-1 89-2 89-3 19# 0^ 89-1+ 8l2* 89-6 81 *0.11 I.64.* 0,00 0 .2JC 0.012 I 33-3 (2 .1 ) 2 .2 ine. 33-1+ 33 ine. 33-5 67 ine. 65-1 60 65-2 0.12 1 (0.9) 1 .1 0.03 65-3 120-1 120-2 120-3 105-1 i+O inc. 33 67 inc. 81; inc. inc. (1 .2 ) 0 .1+ a/ O.OOli 115-1 91 inc. 1. Determined by biological teat on solution of polycyclic fraction in benzene at appropriate concentration, three 20 mg. appli cations each week. 2. A direct test of this fraction has produced 1 benign papilloma in 36 weeks. TABLE 2 INFLUENCE OF VARIOUS DILUENTS ON THE ACCELERATING ACTIVITY OF n-DODECANE API EXPERI MENT NUMBER ACCELERATOR 665 100ji Dodecane DILUENT None CONCEtfTRATION OF BENZOPYRENE by weight) 0 .01*. SCHEDULE OF APPLICATIONS RELATIVE (no./week - SPREAD1 mg.) 2.9 2-50 MEAN TIME OF APPEARANCE OF TUMORS (weeks)^3 23.7 RELATIVE CARCINO GENIC POTENCY, PrMC ^ O.II4. 666 100j6 Dodecane None 0.08 2.9 2-50 2 0 .14. 0.18 295 lOOjf Dodecane None 0.20 2.9 3-50 lii.7 0.21 650 None lOOjf Decahydronaphthalene 0.08 1 .6 est, 2 - 5 0 rj I4.3 a ^ 0 .0 5 659 50j6 Dodecane 50jf Decahydro- naphthalene 660 5 0 Dodecane 50j6 Beta-methyi- naphthalene 0.08 0.08 2 .1| est . 2 - 5 0 2-50 20.0 19.1 0.19 0.20 613 None 85?f Beta-methyl- naphthalene, I5jt Benzene 0.20 2-50 30.6X * 0.10 6ll* 297 296 75?6 Dodecane 5 0 Dodecane None 2556 white oil (31*0 SSU/1000) 5056 white oil lOOJo white oil 0.20 0.20 0.20 2.65 est 3 - 5 0 2 .I4. est 1.8 3 - $0 3-50 2 0 .k 20.2 27.9 0.12 0.12 0.07 1. Based upon relative spread of 1.0 for solutions of benzopyrene in benzene. 2. Experience indicates an experimental variation of 3 . In these tests, essentially all of the C3H mice develop squamous cell papillomas and carcinomas if they survive. - 15 - TABLE 3 TUMOR-INDUC ING ACTIVITY OF PRODUCTS OF FRACTIONAL DISTILLATION OF THE POLYCYCLIC AROMATICS OF A CATALYTICALLY CRACKED OIL API experi ment NUMBER 8-26-a1 8-26-b 8-26-c 8-26-d 8-26-e 8-26-f 8-26-g 8-26-h 8-26-i 8-26-j 8-26-k1 FRACTION DISTILLED 28.5 - 308% 30.8 - 32.0 32 - 34.1 34.1 - 36 36 - 38 38 - 40 40 - 43.9 43.9 - 46 46 - 50.3 50.3 - 52.5 52.5 - 54.8 ESTIMATED BOILING RANGE (in * F. at 760 mm.) 641 - 646F, 646 - 651 651 - 656 656 - 662 662 - 668 668 - 674 674 - 686 686 - 693 693 - 709 709 - 717 717 - 72J INCIDENCE OF TUMORS (% - weeks) 0%- 80 0-80 0-80 0-80 0-80 7 -8 0 28 - 80 12 - 80 '36 - 80 71 - 73 83 - 28 RELATIVE CARCINO GENIC POTENCY, PMC - - - - - - - A^O.01 0 .03.01 S 0.211 8-26-1 54.8 - 57.3 727 - 736 90 - 58 0.09-.05 8-26-m1 8-26-n 57.3 - 60.4 60.4 - 62.4 736 - 74? 747 - 759 87 - 45 100 - 46 0 .291 0 i+* ^ u,15-.07 Contributions of Accelerators to Potencies of Fractions 8-26-2 8-26-4 Solution 8-26-g, Solution 8-26-1, 2 of Benzopyrene in fraction of -h, and -ij plus 20% benzene 2 of Benzopyrene in fraction of -m, and -n; plus 30% benzene above Acceler ating Factor 5 2 1. All fractions tested as 50% solutions in benzene except 8-2'OT and -m, which were accelerated with $0% dodecylbenzene. 2. Chromatographic fractions including saturates through naphth alenes, 27% of 8-26-g, -h, and -I; 3% of 8-26-1, -m, and -n. TABLE IR RELATIVE POTENCIES OP CHROMATOGRAPHIC FRACTIONS OP CARCINOGENIC FRACTIONS OF LOWEST MOLECULAR WEIGHT FROM CATALYTICALLY CRACKED RESIDUUM, API-8 CHROMATOGRAPHY BIOLOGIC/IL TESTS PERCENT ELUTED APPARENT COMPOSITION API EXPEBI- MENT NUMBER PERCENT ELUTED. CONCENTRATION IN. SOLUTION TESTED SOLVENT SCHEDULE OF APPLI- INCIDENCE CATIONS . OF (no./week TUMORS - mg.) (J-weeks) 's*. ro ro 1 0 Saturates 21 - 27 Naphthalenes 25 - 90 Phenanthrenes 8-26-7 26 - 61+J6 33.1+ Benzene 3 - 1 5 0% - 19 50 - 07 Pyrenes 8-26-8 61+ - 80 3 2 .6 Benzene 3 - 1 5 18 - 19 80 - 91 Fluoranthenes 8-26-9 80 - 85 9.6 Benzene 3-15 0 - 1 9 O' 1 P* Benzofluorenes 8-26-1 85 - 88 6.2 Benzene 3 - 1 5 o - 19 91 - 9U- Chrysene, etc. 8-26-11 8-26-12 88 - 91 91 - 93 l+.l Dodecane 3.8 Benzene 3-50 3-15 70 - 30 0-19 8-26-11+ 93 - 91+ 0.9 Dodecane 3 - 5 0 10 - 35 91+ - loo Non-hydrocarbons 8-26-15 91+ - 100 1 0 .1+ Dodecane 3-50 0 - 3 5 CD API 06572 ME OF APPEARANCE OF PAPILLOMAS (weeks) ) FIGURE 1 - INFLUENCE OF CYCLOPARAFFINIC DILUENTS ON THE ACCELERATING ACTIVITY OF n-DODECANE LEGEND Composition of Solvent IOO56 dodecane 75/6 dodecane, 2556 white oil (3U0 S.S.K 5056 dodecane, Q 5056 white oil O IOO56 white oil y dodecane, 50% deca- hydronaphthalena IOO56 decahydronaphthalene All tests at %0 mg./applica ->1 tion except where indicated. 1 > o a\ Ln 0.08 0.12 0.16 0.20 0.2ij. 0.28 0.32 O .36 DOSAGE OF BENZOPYRENE USED (mg. BP/standard Area*/Week) * Area covered by 100 mg. BP in benzene. m i i i i i______ ..j____________ i______i-- i-- i-- i-- i-- i-- l 14 16 18 20 22 2k 26 28 30 > MOLECULAR LENGTH IN EXTENDED CONFIGURATION (A) COT> *^J prom The Kettering Laboratory in the Department of Preventive Medicine and Industrial HeaiLth, College of Medicine, University of Cincinnati, Cincinnati, Ohio. Investigative Team; Bernard H. Braun, Ph. D. Prank P. Cleveland, M. D. Ralph T. Denham, B. S. Mary Jane Graf, B. S. Francis F. Heyroth, Ph. D., M. D. A. Wesley Horton, Ph. D. John J. Phair, M. D. Fred Shaffer, M. S. Klaus L. Stemmer, M. D. Russell Tye, M. S. Waldo J. Younker, M. S. Patricia Clapsaddle Lenore Hull Irvin Rapien Effie West Reports A. Wesley Horton, Ph. D. Approved: Director Date; November 1, 1956