Document MMJM1Vrd2wZ0Y86ZJ9VEyb2Jk
PLAINTIFF'S EXHIBIT API-132
INTERIM REFORT API RESEARCH PROJECT MC-l
(
November 1, 1956
THE KETTERING LABORATORY
in the Department of Preventive Medicine and fndurtrial Health
( College of Medicine
UNIVERSITY OF CINCINNATI, CINCINNATI, OHIO
SC-API-2805
EXPERIMENTS INVOLVING THE APPLICATION- OP MATERIALS TO THE SKIN OP MICE LEGEND FOR DATA SHEETS ON INDIVIDUAL EXPERIMENTS
Symbols uaed In connection with average weight curves: V Time of death (from disease) of i th tumor-free mouse. l Time of death (from disease) of L th tumor-bearing mouse.
^7 r L Time mouse killed. Mouse ml-eelng f>a- a age.
Gross and microscopic pathology:
. [D
Time of appearance of first papilloma In 1th mouse.
When this symbol Is first used for a given mouse after Its death, the presence of non-invading carcinoma (Intra-epithellal) or small areas of benign neoplasm was determinable only by hlstopathology.
flj Time of appearance of papilloma which regressed later or was not confirmed by hlstopathology.
0 Time of appearance of gross changes indicative of malignancy In tumor of tth mouse. Diagnosis confirmed except In cases where no tissue section available. When this symbol is first used for a given mouse ' after its death, the Invasive malignancy of the tumor
was determinable only by hlstopathology.
X' Gross diagnosis of malignancy of tumor not confirmed by hlstopathology.
Hi ,
0i
n or 0 No tissue section available for hlstopathology because
' of extensive post-mortem decomposition or cannibalism,
X IvnaenB-net recorded, during, thla week.
* Painting discontinued for 1 week at each indicated time. Cumulative dose-response curves (summation of data from all cages unless otherwise specified)s
Experimental curve for benign papillomas.
Biperlmen4aA-^urv^-foi>-gPooflly. Malignant tuner a,
t
FIGURE I EXPERIMENT 220, SOLUTION OF METHYLCHOLANTHRENE, 1.034 PERCENT IN BENZENE
FIGURE II EXPERIMENT 268, SOLUTION OF METHYLCHOLANTHRENE, 0.287 PERCENT IN BENZENE
AVERAGE WEIGHT OP SURVIVORS (gram a)
Cage 1, 7 Mice 30-
FIGURE III - TOTAL FEED TO DELAYED COKING UNIT
/V
STARTING DATE: STRAIN OF MICE:
8/7/51 C3H
NUMBER OF APPLICATIONS EACH WEEK:2
DOSAGE OF SOLUTION PER APPLICATION: 100 mg,
i
MEAN TIME OF APPEARANCE OF TUMORS: 36.1*4.7
6s"-
4 ` tf' 12 ' 16 ' 20 ' 24 ` 2b
Cage 2, 6 Mice
CLASSIFICATION:
X
026-
V
j--i--i--i--i--i__i__i__i--i- 1
A-lEL J__1__l.-l -J__I__I---1---L
4 8 12 16 20 24 28 32 36 40 44 48 52 56
"Cage 3, 7 Mice
j--i--i----Ii--i1--iI--i1--II_lJ--lD--i--1i_1O*1Ji II it l-1. lI II 1l-*1l iI PI'Al- i1 ([5A1 > I I t._ -L
4 8 12 16 20 24 28 32 36 i|0 44 48 52 56
20 24 28 32 36
TIME AFTER FIRST PAINT ING (weeks)
40 44 48
52
FIGURE IV
EXPERIMENT 21*3, SOLUTION OF METHYLCHOLANTHRENE, 0.15 PERCENT IN BENZENE
Cage 1, 7 Mice 30
STARTING DATE: STRAIN OF MICE:
7/28/51 C3H
26 NUMBER OF APPLICATIONS EACH WEEK: 1
DOSAGE OF SOLUTION PER APPLICATION:100 mg.
26-
#0<2)
MEAN TIME OF APPEARANCE OF TUMORS: l8.11.8
CLASSIFICATION:
A
AVERAGE WEIGHT OP SURVIVORS (gram a)
8 12 16 20 21* 28 32 36 TIME AFTER FIRST PAINTING ( w e e k 8 )
lI
FIGURE V
experiments involving the application of solutions of methylcholanthrene in benzene
LEGEND FOR DATA SHEETS ON INDIVIDUAL EXPERIMENTS
Symbols used In connection with average weight curves;
^ Time of death (froifa disease) of ith tumor-free mouse.
L lime of death (from disease) of ith tumor-bearing mouse.
^ or l Time mouse killed.
\y Mouse missing from cage.
Gross and microscopic pathology:
[J] Time of appearance of first papilloma in ith mouse. When symbol first used for a given mouse after its death, the presence of non-invading carcinoma (intra-epithelial) or small areas of benign neoplasm was determinable only by histopathology.
[Q Time of appearance of. papilloma which later regressed or was * not confirmed by histopathology.
Time of appearance of gross changes indicative of malignancy in tumor of ith mouse. Unless otherwise indicated, diagnosis confirmed by microscopic examination of tissue sections, When symbol first used for a given mouse after its death^ the invasive malignancy of the tumor was determinable only by histopathology.
Gross diagnosis of malignancy of tumor not confirmed by histopathology.
Bor tiS No tissue section available for histopathology.
Cumulative dose-response eurves (summation of data from all cages unless
unless otherwise specified)?
'
-*----------------------- w-.--....-.*' Benign papillomas.
------- ,,.....................- - -- ---- Grossly malignant tumors.
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-201. Solution of Methvlcholonthrene. 0.0 1 Percent by Weiaht in Benzene
8 16 24 32 40 48 56 64 72 80 88 TIME AFTER FIRST PAINTING (WEEKS)
j
66 *' * TO "
Issued 2-15-54
74
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS ,TQ THE SKIN OF MICE API- 203, Solution of Methylcholonthrene, 0.086 Percent by Weight in Benzene
16 20 24 28 32 TIME AFTER FIRST PAINTING (WEEKS)
Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-204, Solution of Methylcholanthrene, 0.17 Percent by Weight in Benzene
16 20 24 28 32 36
8 12 16
TIME AFTER FIRST PAINTING (WEEKS)
24 28 Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API -204, Solution of Methylcholanthrene,O.I7 Percent by Weight in Benzene
AVERAGE WEIGHT OF SURVIVORS (GRAMS)
24 28 32 36
8 12 16
TIME AFTER FIRST PAINTING (WEEKS)
24 28 Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-204, Solution of Methylcholanthrene, 0.17 Percent by Weight in Benzene
TIME AFTER FIRST PAINTING (WEEKS)
Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-204, Solution of Methylcholanthrene, 0.17 Percent by Weight in Benzene
TIME AFTER FIRST PAINTING (WEEKS)
20 24 Issued 2-
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN Of MICt API-205, Solution of Methylcholanthrene, 0.345 Percent by Weight in Benzene
TIME AFTER FIRST PAINTING (WEEKS)
Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-205tSolution of Methylcholqnthrene, 0.345 Percent by Weight in Benzene
TIME AFTER FIRST PAINTING (WEEKS)
Issued 2-15-54
EX*P, ERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-206, Solution of Methylcholanthrene, 0.69 Percent by Weight in Benzene
16 20
32 36 TIME AFTER FIRST PAINTING (WEEKS)
Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS, TO THE SKIN OF MICE API-209, Solution of Methylcholanthrene, 0.52 Percent by Weight in Benzene
24 28 32 36
|2 16
Painting discontinued for one week at each time indicated.
TIME AFTER FIRST PAINTING (WEEKS)
24 28 Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-205,Solution of Methylcholanthrene, 0.345 Porcent by Weight in Benzene
TIME AFTER FIRST PAINTING (WEEKS)
Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-219, Solution of Methycholanthrene, O.II6 Percent by Weight in Benzene
24 28 32 36
16 20 24
TIME AFTER FIRST PAINTING (WEEKS)
28 32 36 Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-220, Solution of Methvicholonthrene. 1.034 Percent hv Weight in Ranm>
8 12 16 20 24 TIME AFTER FIRST PAINTING (WEEKS)
Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO! THE SKIN OF MICE API-238, Solution of Methylcholanthrene, 0.23 Percent by Weight in Benzene
Painting discontinued for one week at time indicated.
24 28 32 36
12 16
TIME AFTER FIRST PAINTING (WEEKS)
24 28 Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-243, Solution of Methylcholonthrene, O.l6 Percent by Weight in Benzene
8 12 16 2S
V Painting discontinued for one week at each indicated time.
2B 32 36
|5 20
TIME AFTER FIRST PAINTING (WEEKS)
J
30 35
40
Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS ,TO I HE SKIN OF MICE API-243, Solution of Methylcholanthrene, 0.16 ' Percent by Weight in Benzene
12 16 20 24 28 32 36
|2 16
TIME AFTER FIRST PAINTING (WEEKS)
24 28 32 Issued 2-15-54
EXPERIMENTS INVOLVING THE APPLICATION OF MATERIALS TO THE SKIN OF MICE API-268, Solution of Methcholcmthrene, 0.29 Percent by Weight in Benzene
TIME AFTER FIRST PAINTING (WEEKS)
Issued 2-15-54
INTERIM REPORT - API RESEARCH PROJECT MC-1 November 1, 1956
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 apparent. 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 1951 it was evident that the complexity of the problem was such that a
-2 -
detailed 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 understending 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 195 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 may be expressed by the following equation:
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 most of the aromatic constituents is represented by the multiplication factor, M . The considerable importance of these non-carcinogenic components is shown by the fact that
-3 -
M. may reach values as high as 10. To determine in a syste 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 (Ca + Ca + Cs). 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 10 per cent of the effective potency of the heavy catalytic 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 20 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 5-ringed class, benzopyrenes, can be estimated from the content of this class as determined by direct chemical analysis. The third
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, Ma . 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
would be 7 and 1, respectively.
The potential difficulties which preclude a direct
measurement of
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 monocycloaromatics, as
well as most of the naphthalene derivatives, are under
5
test as the vehicles for 0.05 per 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.
6
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 $0 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-paraffin . On the other hand, dilution with only 25 per cent of white mineral oil (viscosity, 3^.0 S.8.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 alkylcyclohexanes or cyclo pentanes, the ring being near the end of the chain (1-cyclohexyldecane is an active accelerator, whereas 7-cyclohexyltridecane is almost inactive). It has been found that, to
(
. I'
(
7
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 AFI-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.
fhe 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, fhe 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. fhe Catalytic Effects of Aliphatic and Related Hydrocarbons",
6
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 Pig. 2, activity seems to reach a maximum level at a length equivalent to that of the n-paraffins containing 12 to 14 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 triisopropylbenzene seems to be an exception to this relationship of molecular length and activity. As 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. W. Adams, has now been tested. It has shown the same accelerating activity as the original sample (Ma-- 4)
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 (Mo* 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 42 at Pennsylvania State College).
7-cyclohexyltridecane Ma =* 2.5
7-phenyltride cane Ma~ 9
7-n-hexyltridecane
9
INVESTIGATION DIRECTED TOWARDS THE IDENTIFICATION OP THE CARCINOGENS OF FRACTIONS, PISTOLLING IN THE RANGE 720 TO 760P. 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.
10 -
The carcinogenic fractions, 8-26-1, -m, and -n, (730 to ?60F.) 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
^n2n-l8
0nH2n-20
17 0.7$
0.8$
18 5.1
11.1
19 6.4
11.8
20 3.1
5.6
21 1.1
1.8
cnH2n-22 0.3$ 3.8 6.4 2.3 0.8
cnH2n-24 -
13.3$ 2.2 0.8 0.3
Suggested structures:
Cycloalkylranthracenes
Alkylbenzofluorenes
Benz(&)anthracenes.
On the basis of the mass spectrum at high voltage, the cyelopentano substitution is considered to be the most likely structure for the Cxfi2n-20 hydrocarbons. A six-membered
cycloalkyl ring would be expected (on the basis of the spectrum of tetralin) to yield a strong fragment peak at m/e of 20k by loss of -CHa-CH*-. The intensity of the 20k 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, 9-26-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|.-ringed 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 anthracenic compounds removed by maleic anhydride, was next subjected to chromatographic fractionation on activated alumina. Mass and absorption spectrometry were used to determine the distribution of the various classes of hydrocarbons through the chromatographic fractions (see Table 4). The biological test on the fraction which contained almost all of the 4-ringed chrysene and triphenylene (8-26-Hj.) 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
12
of carbazole, has shown mainly that this fraction is exceed
ingly toxic to the mice. No tumors have been produced
in 3J4. weeks. One of the intermediate fractions (8-26-11),
that containing cyclopentanophenanthrenes, fluoranthenes,
and benzofluorenea, but essentially free of compounds
having 4 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 4-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,
and for a more direct
comparison with the potencies of the parent distillates,
which were tested as $0 per cent solutions in benzene.
These new tests, 8-26-7, -8, -9, -10, and -12, have been
under way about four months. The results of these tests
should Indicate the particular classes of polycyclic hydro
carbons responsible for the major part of the potency in
this distillation range, 720 to 760P. According to the
physical and chemical behavior of the classes indicated,
a further concentration of the carcinogens will be carried
out until their certain identification has been accomplished.
Analytical techniques for the direct determination of this
class in complex oils may then be developed.
( 13 -
API
EXPERI MENT
NUMBER
DESCRIPTION
FRACTION
DISTIL LING BENZO
GRAVITY <750f. PYRENE (API) (per cent) (per cent)
RELATIVE CARCINO
GENIC POTENCY,
?MC (at 100 mg.;
89 Catalytically
27.0
cracked sidestream
(F.C.C. heavy cycle
gas oil)
100
0.001
oa
33 Coker gas oil
16.8 60 0.008 Q.4
63 Thermal tar
6.6 43.8 0.04
0.11X
00
O0ra
1
120 Straight run dis 19.5 -- 52 tillate (naphthenic )
105 Straight run dis 28.9 tillate (waxy)
115 Straight run distil - 23.2 late (naphthenic)
72.9 50
0.0003 .002
0.11 0,03
(
- 13 TABLE I RELATIVE CONTRIBUTIONS OP CARCINOGENS AND NON-CARCINOGENS TO THE EFFECTIVE POTENCY OP VARIOUS REFINERY STREAMS
CHROMA TOGiyLPHIC FRACTIONAL'ION
FRACTION EXTRACTED BY MALEIC ANHYDRIDE
Saturates
Thru Alkylnaphthalenes
API PMC
EXPERI MENT
with
0.05$f
NUMBER Yield MC
Poly
BASIC
cyclic CONTRI
Aromatics BUTION
and OF
Non-hydro POLY
carbons CYCLIC S
(yield) TO Pm1
ESTI
Yields
MATED
ACCELER
ATING
FACTOR 2-3 4+
a rings rings
Contri bution
of
4+ Ring Fractiox
to PMCl
89-1 89-2
1.64$ 0.2$
0.00 0.012
89-3 89-4 89-6 33-3 33-4
8l2$ 81 11
19# 33
0.04 2
inc.
(2.1) 2.2
inc.
33-5 65-1 65-2
6? Inc.
60
0.12
1
(0.9) 1.1 2*01
65-3 120-1
40 inc .
(1.2) 0.4 /v 0.004
120-2
33 Inc
120-3
67 inc.
105-1 84 inc.
115-1
91 inc.
1. Determined by biological test on solution of polycyclic fraction in benzene at appropriate concentration, three 20 mg. appli-
ons sflcli 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
DILUENT
CONCEN
TRATION
SCHEDULE
OF OF
BENZOPYRENE
APPLICATIONS
{$ by
RELATIVE (no./week -
weight) SPREAD1
ng.)
MEAN TIME
OF APPEARANCE
OF TUMORS
RELATIVE CARCINO
GENIC POTENCY,
(weeks)^*3 .. PrMC ^
66$ 100$ Dodecane None
0.04
2.9
2 - 50
23.7
0.14
666 100$> Dodecane None
0.08
2.9
2 - 50
20.4
0.18
295 100$ Dodecane None
0.20
2.9
3 - 50
14.7
0.21
658 None
100$ Decahydronaphthalene
0.08
1.8 est, 2 - 50
0^43
n^0.05
659 50$ Dodecane 50$ Decahydronaphthalene
0.08
2.4 est , 2 - 50
20.0
0.19
660 50$ Dodecane 505? Beta-methylnaphthalene
0.08
2 - 50
19.1
0.20
613 None
85$ Beta-methylnaphthalene ,
15$ Benzene
0.20
2 - 50
30.61
= 0.10
614 75$ Dodecane ' 25$ white oil (340 SSU/1000)
0.20
2.65 est . 3 - 50
20.4
0.12
297 50$ Dodecane 50$ white oil
0.20
2.4 est
3 - 50
20.2
0.12
296 None
100$ white oil
0.20
1.8
3 - 50
27.9
0.07
1. Based upon relative spread of 1.0 for solutions of benzopyrene in benzene. 2.. Experience indicates an experimental variation of 15$.
3* In these tests, essentially all of the C3H mice develop squamous cell papillomas and carcinomas if they survive.
- 15
TABLE 3
TUMOR-INDUCING ACTIVITY OP PRODUCTS OP FRACTIONAL DISTILLATION OP THE
POLYCYCLIC AROMATICS OP A CATALYTICALLY CRACKED OIL
API EXPERI
MENT NUMBER
8-26-a1
FRACTION DISTILLED
28.5 - 30.8#
ESTIMATED BOILING RANGE
(in 0 F. at 760 mm.)
641 - 646F.
INCIDENCE OP
TUMORS (# - weeks)
0#- 80
RELATIVE CARCINO
GENIC POTENCY,
PMC
-
8-26-b
30.8 - 32.0
646 - 651
0-80
-
8-26-c
32 - 34-1.
651 - 656
0-80
-
8-26-d 8-26-e
34.1 - 36 36 - 38
656 - 662 662 - 668
0-80 0-80
-
8-26-f
38 - 40
668 - 674
7-80
-
8-26-g
40 - 43.9
674 - 686
28 - 80
-
8-26-h
43.9 - 46
686 - 693
12 - 80-
-.
8-26-1
46 - 50.3
693 - 709
36 - 80
/'v'O.Ol
8-26-j 8-26-k1
50.3 - 52.5 52.5 - 54*8
709 - 717 717 - 727
71 - 73 83 - 28
0.03*.01 S0.211
8-26-1 8-26-m1
54.8 - 57.3 57.3 - 60.4
727 - 736 736 - 747
90 - 58 87 - 45
n.no9"*-"*.o25 0.291
8-26-n
6o4 - 62.4
747 - 759
100 - 46
0O,1id5+-.*0072
Contributions of Accelerators to Potencies of Fractions above
8-26-2
Solution of Benzopyrene in fraction2 of ' 8-26-g, -h, and -i; plus 20# benzene
Acceler ating
Factor
5
8-26-4
Solution of Benzopyrene in fraction2 of 8-26-1, -m, and -n; plus 30# benzene
2
1. All fractions tested as 50/S solutions In benzene except 8-26-k and -m, which were accelerated with $0% dodecylbenzene.
2. Chromatographic fractions including saturates through naphth
alenes, 27# of 8-26-g, -h, and -i; 23# of 8-26-1, -m, and -n.
TABLE 1+
RELATIVE POTENCIES OF CHROMATOGRAPHIC FRACTIONS OF CARCINOGENIC FRACTIONS OF LOWEST MOLECULAR WEIGHT FROM CATALYTICALLY CRACKED RESIDUUM, API-8
CHROMATOGRAPHY
BIOLOGICAL TESTS
PERCENT ELUTED
APPARENT COMPOSITION
API
EXPERI
MENT. NUMBER
PERCENT ELUTED.
CONCEN TRATION
IN. SOLUTION
TESTED
SOLVENT
SCHEDULE
OF APPLI INCIDENCE
CATIONS . OF (no./week TUMORS
- mg.) (^-weeks)
rH
CT-
1
O
CO
0 - 22% Saturates 21-27 Naphthalenes 2^ - 90 Phenanthrenes 50 - 8? Pyrenes
Fluoranthenes m - 94 Benzofluorenes
91 - 94 Chrysene, etc.
94 - loo Non-hydrocarbons
8-26-7 0-26-8 8-26-9 8-26-10 8-26-11 8-26-12 8-26-14 8-26-15
26 - 6k%
64 - 60 ' 80 - 85 85 - 88 08 - 91 91 * 93 93 - 94 94 - ioo
334# 32,6
9.6 6.2 4.1 3.8
0.9 10.4
Benzene Benzene Benzene Benzene Dodecane Benzene Dodecane Dodecane
3-15 0% - 19 3-15 18 - 19 3 - 15 0-19 3-15 0 - 19 3-50 70 - 30 3-15 0-19 3-50 10 - 35 3-50 0-35
FIGURE 1 - INFLUENCE OF CYCLOPARAFFINIC DILUENTS ON THE ACCELERATING ACTIVITY OF n-DODECANE
MEAN TIME OP APPEARANCE OP PAPILLOMAS (w eeks)
0.08
0.12
0.16
0.20
0.24 0.28
0.32
0.36
. DOSAGE OF BENZOPYRENE USED (mg. BP/standard Area*/Week)
Area covered by 100 mf. BP Jn benzene.
FIGURE 2 - MOLECULAR LENGTH OF SOLVENT vs. ACCELERATING ACTIVITY
I H*
03
I
I
( - 19
Prom The Kettering Laboratory in the Department of Preventive Medicine and Industrial Health, 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 P. Heyroth, Ph. D., M. D. A. Wesley Horton, Ph. D. John J # Phair, M. D. Fred Shaffer, M. 5. Klaus L. Stemmer, M. D. Russell Tye, M. S. Waldo J. Younker, M. S. Patricia Clapsaddle Lenore Hull Irvin Raplen Effie West
Report
A. Wesley Horton, Ph. D
Date: November 1, 1956
Director
(