- Email: [email protected]
Skin tumorigenesis by an extract of amber petrolatum
TOXICOLOGY
Skin
AND
APPLIED
Tumorigenesis WILLIAM
8,
PHARMACOLOGY
by
LIJINSKY,
(1966)
113-ll’i
an
USIBERTO
Extract
of Amber
Petrolatum
SAFFIOTTI,
AND
SHUBIK
Division of Oncology, The Chicago Medical
Institute School,
Received
February
PHILIPPE
for Medical Research, Chicago, Illinois 60612
10, 1965
An extensive study of refined petroleum waxes (Shubik et aE., 1962) showed complete absenceof carcinogenicity when they were tested biologically. Simultaneous chemical studies revealed no carcinogenic polynuclear hydrocarbons in them, although somenoncarcinogenic polynuclear hydrocarbons were present in concentrations ranging up to 0.6 ppm. Since someamber or yellow petrolatums were found to contain total polynuclear hydrocarbons at concentrations of the order of ten times as much as in refined paraffin waxes, a study of the biological effects of petrolatum was also undertaken. The results of chemical analysis of ten samplesof petrolatum have already been reported (Lijinsky et al., 1963) and no known carcinogenic compoundswere detected. One samplewas selected for detailed biological study, fractionated into aromatic and nonaromatic portions and the fractions, as well as the petrolatum itself, were tested by topical application to mouseskin. These tests were analogousto those carried out with petroleum waxes, the results of which were entirely negative (Shubik et al., 1962). METHODS
Chemical
A sampleof petrolatum (N.F. grade; amber) was obtained,l and the concentration of polynuclear hydrocarbons determined spectrophotometrically (Sample 10, Table 1, Lijinsky et aZ., 1963). Forty kg of this petrolatum were chromatographed on silica gel. Five-kg portions were mixed with 5 liters of hot isooctaneand filtered through a 28 X S cm column of silica gel (100-200 mesh), the waxy material was washed into the filtrate with l-2 liters of hot isooctane, and the adsorbedaromatic material eluted with 1.5 liters of benzene. All solvents were freed of polynuclear compounds by chromatography before use by the method of Lijinsky and Raha (1961). All the benzene eluates were combined, and the solvent was removed by distillation under reduced pressure. The oily residue weighed 483 g ( 1.2% of the starting material). The polynuclear compoundswere further concentrated by partition between cyclohexane (500 ml) and nitromethane (4 X 500 ml) in which the polynuclear compounds passedinto the nitromethane (Lijinsky et al., 1963). The nitromethane was removed by distillation on a steambath under nitrogen in WZCUO, leaving a pale yellow oily residue (58 g or 0.15% of the starting material). The cyclohexane was likewise distilled, leaving a dark brown oil. Both the cyclohexane and nitromethane extracts were diluted to 800 ml with isooctane and these solutions biologically tested. These represented a SO1 Arthur
S. La Pine
and
Co.,
Chicago,
Ill. 113
31
30 29 30 30
30 30
50 50
8
0 d 9 6
0 *
; 8
Chromatographic adsorbatef Nitromethane fraction*
Cyclohexane fractionf
Isooctanee
a b e d e f s tr
TUMOR
TABLE AMBER
I
40 27
19 18
27 25 45 45
21 18 15 16
16
27 27 27 27
25
29
32 16
12 11
11 2 3 6
12
14
33 27
14
23
37
16
50
70
PETROLATUM
27
Survivors at weeks”
WITH
30
INDUCTION
26 3 10 31 8 4 -
14 3 5 12
2
2
-
5 3
-
3”
2a
Tumors?
Total
FRACTIONS
-
ITS
-
2
1
Tumor bearing animals
AND
of
-
-
-
-
-
1
1 9 5 1
9
Carcinomas
number
Twice-weekly skin applications to Swiss mice. After the beginning of the treatment. All observed tumors including benign papillomas and keratoacanthomas and malignant squamous cell carcinomas, Tumors that were observed grossly for at least 4 weeks and then regressed. Three drops (approximately 60 microliters) per application. The material from 40 kg of petrolatum was diluted to 800 ml with isooctane. One drop (approximately 20 microliters) One papilloma on eyelid. One papilloma under chin.
in isooctane)C
30
0
(15%
40
d
30
(15% in isooclane)e Chromatograpbic filtrate
Sex
0
Treatmenta
Petrolatum
Initial number of mice
SKIN
per
7
application.
-
-
3 -1
-
-
1 2
Regressions”
62 55 17 49 81 48 63
-
-
100 69
Average latent period for all tumors (weeks)
.G
E:
c 2 z R 5
k
G
H
SKIN
TUMORIGENESIS
BY
PETROLATUM
115
fold concentration of the component compared with the original petrolatum. To detect any possible synergistic effect, equal volumes of the solutions of the nitromethane and cyclohexane extracts were mixed, evaporated to half the volume, and this solution was tested biologically. To complete the tests, the petrolatum itself and the aromaticfree fraction obtained as the filtrate from the silica gel columns were dissolved in isooctane to make a 15% solution and applied to mouse skin. Biological Each of the five solutions was tested by skin application to male and female Swiss mice of the colony of this Institute (Shubik et al., 1962). A group of 30 males and 30 females was used for each test. A control group of 50 males and 50 females received skin applications of isooctane only. The animals were bred randomly and housed not more than ten to a cage in plastic cages containing granular cellulose bedding. They were given a commercial mouse diet” and water ad Zibitum. The animals were 7-l 1 weeks old at the beginning of treatment. The solutions were applied to the shaved interscapular region of the mice, and the treated area was subsequently kept free of hair by clipping with scissors. The treatments consisted of twice-weekly applications of the solutions from droppers that delivered approximately 20 microliters per drop. Each application consisted of 1 drop of the solutions of the aromatic concentrate and its subfractions, or 3 drops of the petrolatum itself, the aromatic-free petrolatum (chromatographic filtrate), and the isooctane. Treatment was continued throughout the life of each animal. Skin tumors and other lesions in the treated area and all other visible lesions were recorded when present and the progress of each lesion was charted on graph paper. Histological confirmation of each lesion was made after autopsy was performed on all animals. RESULTS
AND
DISCUSSION
The treatments produced varying degrees of epidermal hyperplasia; none with the chromatographic filtrate, moderate with the petrolatum itself or with the cyclohexane fraction, and marked with the nitromethane fraction and with the chromatographic aromatic adsorbate. Skin crusts developed on the animals treated with the chromatographic adsorbate and with the cyclohexane fraction, and only occasionally in other groups, Some extensive skin ulcers were also seen in the chromatographic adsorbate group. The incidence of internal tumors appeared within the limits observed in untreated or solvent-treated controls (Shubik et al., 1962). A significant number of skin tumors was induced in three of the groups.” The survival rates and the results of the biological tests for skin tumorigenesis are given in Table 1. The petrolatum itself showed no significant tumorigenic effect ; the 2 Rockland Mouse Diet, Abrams a A report of all observed findings gross diagnosis of each skin tumor the tumor itself and its subsequent pathological diagnosis of the tumors of the experiment). This report is tiotti.
Small Stock Breeders, Chicago, Ill. was prepared, including the following data for each animal: observed during the life of the animal with the time at which and histochanges were first seen; time and mode of death; found at autopsy (lime is given in weeks from the beginning available to interested investigators on request from Dr. Saf-
WILLIAM
116
LIJINSKY,
ET
AL.
tumor incidence in this group was little different from that of controls treated with isooctane alone. The chromatographed petrolatum (after removal of aromatic material) also gave entirely negative results. On the other hand, the aromatic portion of the petrolatum was definitely carcinogenic to mouse skin, albeit at a concentration 50 times that at which it was present in the starting material. This is not to be regarded as too drastic a test for a material that might come into contact with humans, since a safety factor for human use is usually set at least as low as 1(/c of the maximum ineffective dose in animal tests (Joint FAO/WHO Expert Committee on Food Additives, 1958, 1961; Staff of the Division of Pharmacology, Food and Drug Administration, U. S. Department of Health, Education, and Welfare, 1959). The adsorbed aromatic material from the petrolatum (constituting 1.2%) produced 29 tumors on 29% of the animals, including 9 carcinomas. The concentrate of polynuclear compounds (nitromethane fraction), which constituted only 0.15% of the petrolatum, produced 41 tumors, including 10 carcinomas, on 28% of the animals. The cylohexane fraction, which might have contained some unextracted polynuclear compounds, produced 12 tumors (6 carcinomas) on 13% of the animals. This biological effect can in no way be accounted for by the identified components in the solution of the nitromethane fraction. No known carcinogenic polynuclear compounds were identified in the petrolatum (Lijinsky et al., 1963). Some methylpyrenes were present in the pyrene fraction, but could be a maximum of only 0.015% in the test solution. Such a concentration of even a potent carcinogen would not produce so large a number of tumors. There seems no doubt that we are faced here with an unknown carcinogen or carcinogens, possibly heterocyclic compounds. Attempts are being made to fractionate the material further and to identify the carcinogenic compounds. This report represents the biological testing of only one sample of an amber petrolatum (N.F. grade). This sample would not pass the U. S. Food and Drug Administration test4 for purity of petroleum waxes for food use. This might be an exceptional sample. On the other hand, further investigation of this type of material seems advisable. By analogy with the studies of highly refined paraffin waxes, with their wholly negative biological results (Shubik et al., 1962), it is probable that the more refined white petrolatums (U.S.P. grade) would present no such problem. SUMMARY A sample of amber petrolatum has been resolved into aliphatic and aromatic fractions. The aromatic portion was separated into two components by solvent partition. The aromatic portion and the two subfractions were tested in isooctane solution at 50 times their concentration in the petrolatum, and all showed significant carcinogenicity to mouse skin. The petrolatum itself, tested as a 15% solution in isooctane, showed no significant tumorigenic activity. The tumorigenic activity detected in the fractions cannot be ascribed to any of the compounds so far identified in the original sample. ACKNOWLEDGMENTS This investigation was supported by Public Health Service Research Grant No. CA-05170 from the National Cancer Institute. We wish to acknowledge the contribution of Drs. F. Cefis and A. Sellakumar in the pathological examination of the animals, and the technical assistance of Miss P. Johns and Messrs. R. Feldman, T. Safavi and J. Vergara in the performance of the experiments. 4 21
CFR
121.1156
SKIN
TUMORIGENESIS
BY
PETROLATUM
117
REFERENCES (1958). Procedures for the Testing JOINT FAO/WHO EXPERT CO~~MITTEE ON FOOD ADDITIVES. of Intentional Food Additives to Establish Their Safety fov Use. World Health Organization Technical Report Series No. 144. JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES. (1961). Evaluation of the Carcinogenic Hazards of Food Additives. World Health Organization Technical Report Series No. 220. LIJINSKY, W., and RARA, C. R. (1961). Polycyclic aromatic hydrocarbons in commercial solvents. Toxicol. Appl. Pharmacol. 3, 469-473. LIJIZVSKY, W., DOMSKY, I., MASON, G., RAMAHI, H. Y., and SAFAVI, T. (1963). The chromatographic determination of trace amounts of polynuclear hydrocarbons in petrolatum, mineral oil and coal tar. Anal. Chem. 35, 952-956 (Erratum p, 1397). SHUBIK, P., SAFFIOTTI, U., LIJINSKY, W., PIETTI~IZ! G., RAPPAPORT, H., TOTH, B., RAHA, C. R., TOMATIS, L., FELDZVAN, R., and RAMAHI, H. (1962). Studies on the toxicity of petroleum waxes. Toxicol. Appl. Pharmacol. 4, Suppl., l-62. STAPF OF THE DIVISION OF PHARMACOLOGY FED AND DRUG ADMINISTRATION, U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE. (1959). Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Assoc. Food Drug Officials U.S., Bureau of Food and Drugs, Texas State Dept. of Health, Austin, Texas.
Skin
AND
APPLIED
Tumorigenesis WILLIAM
8,
PHARMACOLOGY
by
LIJINSKY,
(1966)
113-ll’i
an
USIBERTO
Extract
of Amber
Petrolatum
SAFFIOTTI,
AND
SHUBIK
Division of Oncology, The Chicago Medical
Institute School,
Received
February
PHILIPPE
for Medical Research, Chicago, Illinois 60612
10, 1965
An extensive study of refined petroleum waxes (Shubik et aE., 1962) showed complete absenceof carcinogenicity when they were tested biologically. Simultaneous chemical studies revealed no carcinogenic polynuclear hydrocarbons in them, although somenoncarcinogenic polynuclear hydrocarbons were present in concentrations ranging up to 0.6 ppm. Since someamber or yellow petrolatums were found to contain total polynuclear hydrocarbons at concentrations of the order of ten times as much as in refined paraffin waxes, a study of the biological effects of petrolatum was also undertaken. The results of chemical analysis of ten samplesof petrolatum have already been reported (Lijinsky et al., 1963) and no known carcinogenic compoundswere detected. One samplewas selected for detailed biological study, fractionated into aromatic and nonaromatic portions and the fractions, as well as the petrolatum itself, were tested by topical application to mouseskin. These tests were analogousto those carried out with petroleum waxes, the results of which were entirely negative (Shubik et al., 1962). METHODS
Chemical
A sampleof petrolatum (N.F. grade; amber) was obtained,l and the concentration of polynuclear hydrocarbons determined spectrophotometrically (Sample 10, Table 1, Lijinsky et aZ., 1963). Forty kg of this petrolatum were chromatographed on silica gel. Five-kg portions were mixed with 5 liters of hot isooctaneand filtered through a 28 X S cm column of silica gel (100-200 mesh), the waxy material was washed into the filtrate with l-2 liters of hot isooctane, and the adsorbedaromatic material eluted with 1.5 liters of benzene. All solvents were freed of polynuclear compounds by chromatography before use by the method of Lijinsky and Raha (1961). All the benzene eluates were combined, and the solvent was removed by distillation under reduced pressure. The oily residue weighed 483 g ( 1.2% of the starting material). The polynuclear compoundswere further concentrated by partition between cyclohexane (500 ml) and nitromethane (4 X 500 ml) in which the polynuclear compounds passedinto the nitromethane (Lijinsky et al., 1963). The nitromethane was removed by distillation on a steambath under nitrogen in WZCUO, leaving a pale yellow oily residue (58 g or 0.15% of the starting material). The cyclohexane was likewise distilled, leaving a dark brown oil. Both the cyclohexane and nitromethane extracts were diluted to 800 ml with isooctane and these solutions biologically tested. These represented a SO1 Arthur
S. La Pine
and
Co.,
Chicago,
Ill. 113
31
30 29 30 30
30 30
50 50
8
0 d 9 6
0 *
; 8
Chromatographic adsorbatef Nitromethane fraction*
Cyclohexane fractionf
Isooctanee
a b e d e f s tr
TUMOR
TABLE AMBER
I
40 27
19 18
27 25 45 45
21 18 15 16
16
27 27 27 27
25
29
32 16
12 11
11 2 3 6
12
14
33 27
14
23
37
16
50
70
PETROLATUM
27
Survivors at weeks”
WITH
30
INDUCTION
26 3 10 31 8 4 -
14 3 5 12
2
2
-
5 3
-
3”
2a
Tumors?
Total
FRACTIONS
-
ITS
-
2
1
Tumor bearing animals
AND
of
-
-
-
-
-
1
1 9 5 1
9
Carcinomas
number
Twice-weekly skin applications to Swiss mice. After the beginning of the treatment. All observed tumors including benign papillomas and keratoacanthomas and malignant squamous cell carcinomas, Tumors that were observed grossly for at least 4 weeks and then regressed. Three drops (approximately 60 microliters) per application. The material from 40 kg of petrolatum was diluted to 800 ml with isooctane. One drop (approximately 20 microliters) One papilloma on eyelid. One papilloma under chin.
in isooctane)C
30
0
(15%
40
d
30
(15% in isooclane)e Chromatograpbic filtrate
Sex
0
Treatmenta
Petrolatum
Initial number of mice
SKIN
per
7
application.
-
-
3 -1
-
-
1 2
Regressions”
62 55 17 49 81 48 63
-
-
100 69
Average latent period for all tumors (weeks)
.G
E:
c 2 z R 5
k
G
H
SKIN
TUMORIGENESIS
BY
PETROLATUM
115
fold concentration of the component compared with the original petrolatum. To detect any possible synergistic effect, equal volumes of the solutions of the nitromethane and cyclohexane extracts were mixed, evaporated to half the volume, and this solution was tested biologically. To complete the tests, the petrolatum itself and the aromaticfree fraction obtained as the filtrate from the silica gel columns were dissolved in isooctane to make a 15% solution and applied to mouse skin. Biological Each of the five solutions was tested by skin application to male and female Swiss mice of the colony of this Institute (Shubik et al., 1962). A group of 30 males and 30 females was used for each test. A control group of 50 males and 50 females received skin applications of isooctane only. The animals were bred randomly and housed not more than ten to a cage in plastic cages containing granular cellulose bedding. They were given a commercial mouse diet” and water ad Zibitum. The animals were 7-l 1 weeks old at the beginning of treatment. The solutions were applied to the shaved interscapular region of the mice, and the treated area was subsequently kept free of hair by clipping with scissors. The treatments consisted of twice-weekly applications of the solutions from droppers that delivered approximately 20 microliters per drop. Each application consisted of 1 drop of the solutions of the aromatic concentrate and its subfractions, or 3 drops of the petrolatum itself, the aromatic-free petrolatum (chromatographic filtrate), and the isooctane. Treatment was continued throughout the life of each animal. Skin tumors and other lesions in the treated area and all other visible lesions were recorded when present and the progress of each lesion was charted on graph paper. Histological confirmation of each lesion was made after autopsy was performed on all animals. RESULTS
AND
DISCUSSION
The treatments produced varying degrees of epidermal hyperplasia; none with the chromatographic filtrate, moderate with the petrolatum itself or with the cyclohexane fraction, and marked with the nitromethane fraction and with the chromatographic aromatic adsorbate. Skin crusts developed on the animals treated with the chromatographic adsorbate and with the cyclohexane fraction, and only occasionally in other groups, Some extensive skin ulcers were also seen in the chromatographic adsorbate group. The incidence of internal tumors appeared within the limits observed in untreated or solvent-treated controls (Shubik et al., 1962). A significant number of skin tumors was induced in three of the groups.” The survival rates and the results of the biological tests for skin tumorigenesis are given in Table 1. The petrolatum itself showed no significant tumorigenic effect ; the 2 Rockland Mouse Diet, Abrams a A report of all observed findings gross diagnosis of each skin tumor the tumor itself and its subsequent pathological diagnosis of the tumors of the experiment). This report is tiotti.
Small Stock Breeders, Chicago, Ill. was prepared, including the following data for each animal: observed during the life of the animal with the time at which and histochanges were first seen; time and mode of death; found at autopsy (lime is given in weeks from the beginning available to interested investigators on request from Dr. Saf-
WILLIAM
116
LIJINSKY,
ET
AL.
tumor incidence in this group was little different from that of controls treated with isooctane alone. The chromatographed petrolatum (after removal of aromatic material) also gave entirely negative results. On the other hand, the aromatic portion of the petrolatum was definitely carcinogenic to mouse skin, albeit at a concentration 50 times that at which it was present in the starting material. This is not to be regarded as too drastic a test for a material that might come into contact with humans, since a safety factor for human use is usually set at least as low as 1(/c of the maximum ineffective dose in animal tests (Joint FAO/WHO Expert Committee on Food Additives, 1958, 1961; Staff of the Division of Pharmacology, Food and Drug Administration, U. S. Department of Health, Education, and Welfare, 1959). The adsorbed aromatic material from the petrolatum (constituting 1.2%) produced 29 tumors on 29% of the animals, including 9 carcinomas. The concentrate of polynuclear compounds (nitromethane fraction), which constituted only 0.15% of the petrolatum, produced 41 tumors, including 10 carcinomas, on 28% of the animals. The cylohexane fraction, which might have contained some unextracted polynuclear compounds, produced 12 tumors (6 carcinomas) on 13% of the animals. This biological effect can in no way be accounted for by the identified components in the solution of the nitromethane fraction. No known carcinogenic polynuclear compounds were identified in the petrolatum (Lijinsky et al., 1963). Some methylpyrenes were present in the pyrene fraction, but could be a maximum of only 0.015% in the test solution. Such a concentration of even a potent carcinogen would not produce so large a number of tumors. There seems no doubt that we are faced here with an unknown carcinogen or carcinogens, possibly heterocyclic compounds. Attempts are being made to fractionate the material further and to identify the carcinogenic compounds. This report represents the biological testing of only one sample of an amber petrolatum (N.F. grade). This sample would not pass the U. S. Food and Drug Administration test4 for purity of petroleum waxes for food use. This might be an exceptional sample. On the other hand, further investigation of this type of material seems advisable. By analogy with the studies of highly refined paraffin waxes, with their wholly negative biological results (Shubik et al., 1962), it is probable that the more refined white petrolatums (U.S.P. grade) would present no such problem. SUMMARY A sample of amber petrolatum has been resolved into aliphatic and aromatic fractions. The aromatic portion was separated into two components by solvent partition. The aromatic portion and the two subfractions were tested in isooctane solution at 50 times their concentration in the petrolatum, and all showed significant carcinogenicity to mouse skin. The petrolatum itself, tested as a 15% solution in isooctane, showed no significant tumorigenic activity. The tumorigenic activity detected in the fractions cannot be ascribed to any of the compounds so far identified in the original sample. ACKNOWLEDGMENTS This investigation was supported by Public Health Service Research Grant No. CA-05170 from the National Cancer Institute. We wish to acknowledge the contribution of Drs. F. Cefis and A. Sellakumar in the pathological examination of the animals, and the technical assistance of Miss P. Johns and Messrs. R. Feldman, T. Safavi and J. Vergara in the performance of the experiments. 4 21
CFR
121.1156
SKIN
TUMORIGENESIS
BY
PETROLATUM
117
REFERENCES (1958). Procedures for the Testing JOINT FAO/WHO EXPERT CO~~MITTEE ON FOOD ADDITIVES. of Intentional Food Additives to Establish Their Safety fov Use. World Health Organization Technical Report Series No. 144. JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES. (1961). Evaluation of the Carcinogenic Hazards of Food Additives. World Health Organization Technical Report Series No. 220. LIJINSKY, W., and RARA, C. R. (1961). Polycyclic aromatic hydrocarbons in commercial solvents. Toxicol. Appl. Pharmacol. 3, 469-473. LIJIZVSKY, W., DOMSKY, I., MASON, G., RAMAHI, H. Y., and SAFAVI, T. (1963). The chromatographic determination of trace amounts of polynuclear hydrocarbons in petrolatum, mineral oil and coal tar. Anal. Chem. 35, 952-956 (Erratum p, 1397). SHUBIK, P., SAFFIOTTI, U., LIJINSKY, W., PIETTI~IZ! G., RAPPAPORT, H., TOTH, B., RAHA, C. R., TOMATIS, L., FELDZVAN, R., and RAMAHI, H. (1962). Studies on the toxicity of petroleum waxes. Toxicol. Appl. Pharmacol. 4, Suppl., l-62. STAPF OF THE DIVISION OF PHARMACOLOGY FED AND DRUG ADMINISTRATION, U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE. (1959). Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Assoc. Food Drug Officials U.S., Bureau of Food and Drugs, Texas State Dept. of Health, Austin, Texas.