Drug and Chemical Toxicology
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Tumorigenicity of Nickel Subsulfide in Strain A/J Mice Daniel A. McNeill, Clarence E. Chrisp & Gerald L. Fisher To cite this article: Daniel A. McNeill, Clarence E. Chrisp & Gerald L. Fisher (1990) Tumorigenicity of Nickel Subsulfide in Strain A/J Mice, Drug and Chemical Toxicology, 13:1, 71-86, DOI: 10.3109/01480549009011070 To link to this article: http://dx.doi.org/10.3109/01480549009011070
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DRUG A N D CHEMICAL TOXICOLOGY, 13(1), 71-86 (1990)
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TUMORlGENlClTY OF NICKEL SUBSULFIDE IN STRAIN A/J MICE Daniel A. McNeill', Clarence E. Chrisp2, and Gerald L. Fisher'*3 'Battelle, 505 King Avenue, Columbus, Ohio 43201 2Laboratory Unit for Animal Medicine, University of Michigan, Ann Arbor, Michigan 481 09
ABSTRACT The pulmonary tumor response of Strain A mice has been reported to be a rapid and efficient predictor of carcinogenic potential for a variety of chemicals. The route of exposure has usually been by intraperitoneal injection (i.p.) of solubilized materials. We compared intratracheal (i.1.) instillation as a more representative route typical of human exposures, with i.p. injection of nickel subsulfide, a potent animal carcinogen. Animals were sacrificed either 20 weeks after the first dosing, or were held until 45 weeks after the first dosing. Urethane, a positive control, produced a significant increase in pulmonary tumor response after i.t. instillation as well as i.p. injection. For nickel subsulfide treated animals, there was no evidence of a dose-related increase in pulmonary tumor response in any i.p. or i.1. treatment group when compared with age-matched controls. INTRODUCTION Nickel and various nickel compounds have long been implicated in human lung diseases. Epidemiological investigations of nickel workers have demonstrated an increased risk of nasal and lung
S~nderrnan'~) docu-
30whom correspondence should be sent Present address: Sandoz Research Inst., 59 Route 10, E. Wanover, NJ 07936 71 Copyright 0 1990 by Marcel Dekker, Inc
72
McNEILL, CHRISP, AND FISHER
mented 327 cases of lung cancer and 115 cases of nasal cancer among nickel workers world-wide. This high incidence of occupational cancer associated with nickel and nickel compounds has led to many investigations of the carcinogenicity of nickel compounds in animal studies.
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Most attention has been directed towards nickel dust and insoluble nickel compounds such as nickel oxide and nickel subsulfide. Nickel subsulfide has been shown to be strongly carcinogenic by a number of routes of administration and in a variety of animal model^(^‘^). For most of the studies, however, routes of exposure (intraperitoneal, intramuscular, intraocular, and intrarenal injections) have been used that differ significantly from environmental exposures. In the present report, we describe the effects of intratrachealexposure to nickel subsulfide in Strain A mice. lntratracheal instillation approximates inhalation exposure.
Several
investigators have shown that, while initial deposition patterns of intratracheally delivered material differ from inhaled material, homogeneity of delivered dose clearance kinetics of deposited materials from lungs are similar for both In addition, intratracheal instillation offers significant advantages in terms of a) quantification of delivered dose, b) simplicity of technique with resultant reduced stress on the animal, c) rapid delivery of large doses of material to the lung, and d) greatly reduced cost relative to inhalation exposures. The pulmonary tumor response of Strain A mice has been reported to be a rapid and efficient predictor or carcinogenic potential for a variety of chemical^'^) The route of exposure has usually been by intraperitoneal injection of solubilized materials. While this route has produced dose-response relationships for a variety
73
TUMORICENICITY OF NICKEL SUBSULFIDE
of compounds, it is not a likely route in terms of environmental or industrial exposure. For this reason we compared intratracheal instillation as a more representative route, typical of human exposures, with intraperitoneal injection. Our goal was the development of a rapid lung tumor model using a well defined
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metal carcinogen. Animals were sacrificed either 20 weeks after the first dosing, which is the standard protocol for the tumorigenicity assay, or were held until 45 weeks after the first dosing in order to observe possible late effects due to the
relative insolubility of the nickel subsulfide. MATERIALS AND METHODS Nickel subsulfide (Ni3S2)was supplied by the International Nickel Company, Inc. (INCO), Sheridan Park, Mississauga, Ontario, Canada. Information supplied by INCO stated that Ni,S2 was the only major phase detected by X-ray diffraction with no minor phases detected. Chemical analysis of the Ni,S,
showed nickel
to be 73.7% by weight and sulfur to be 26.3% by weight.
The material as supplied by INCO had a particle size distribution with a mass median diameter (MMD) of 13.3 pm and a corresponding geometric standard deviation (ug) of 2.2. Particle size was reduced by grinding the material in a hardened steel grinding chamber of a Spex Model 8000 mixer mill (Spex Industries, Metuchen, NJ). After grinding, the fine particles were separated from the remaining coarse material by settling the material in an Andreasson pipet. Light microscopy of the recovered particles showed a calculated MMD of 1.8 p m with a corresponding ug of 1.6. X-ray diffraction analysis of the Ni,S,
before and after grinding and
settling in alcohol demonstrated no change in the diffraction pattern.
74
McNEILL, CHRISP, AND FISHER
Dose PreDaration Suspensions of nickel subsulfide were prepared in sterile, 0.9% phosphate buffered saline (PBS)in an injection vial. The suspension was then sealed and sonicated for 10 minutes to disaggreate and assure uniform distribution of
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particles in the suspension. Fresh material was suspended for each dosing period. Solutions of urethane (Mallinckrodt; St. Louis, MO) were prepared in sterile 0.9%
PBS. Delivery Svstem For intratracheal instillation (i.t.), a Gilson Pipetrnan 20 pI micropipet was standardized using Fe,O,
particles (0.4 to 3.7 pm) in 0.9% PBS. A 1 ” x 23 gauge
sterile blunt needle was attached to the pipet and a 20 pI sample of the Fe,O, suspension withdrawn. For intraperitoneal injection, a sterile tuberculin syringe (0.5 ml) was used to deliver 0.1 ml to each animal.
Instillation Techniaue Strain A/J mice (8 to 10 weeks of age) were anesthetized with methoxyflurane for the tumorigenicity studies. As soon as the mice reached a stage of deep, even respiration, they were removed and placed on a specially made holder as described by Ho and Furst(*). The effects of the anesthesia lasted for approximately 2 minutes before the animal was fully awake and ambulatory. A fiber optic system was used to illuminate the buccal cavity of the anestl-,e-
-
tized animal and a dissecting scope gave sufficient magnification : allow insertion of a sterile blunt needle (1” x 23 gauge) into the trachea. The tip of the needle was inserted just anterior to the first bifurcation and the test article
~ 3 slowly s
delivered. A 20 pI phosphate buffered saline solution was found to give excellent survival and was large enough to minimize sample-to-sample variation.
TUMORIGENICITY OF NICKEL SUBSULFIDE
75
Tumor Assay At necropsy, lungs were removed and the airways perfused with 10%
buffered formalin and placed in a vial of 10% buffered formalin for about 1 week. The lungs were then trimmed of excess tissue and all 5 lung lobes removed from
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the bronchial tree. Each lobe was then examined by the same observer under a dissecting microscope for adenomas. Animals Male A/J mice, 8-10 weeks old, were ordered from Jackson Laboratories, Bar Harbor, Maine. Upon arrival animals were housed 5 per cage on Absorb-dri@ bedding. Water, containing 0.2 mg/ml tetracycline hydrochloride (American Cyanamide Co.), and feed (Purina Laboratory Chow) were available ad libitum. Animals were quarantined for three weeks and viral screening was negative on randomly selected animals. EXPERIMENTAL DESIGN Based on initial 4-week range finding studies, mice were administered two dosages of nickel subsulfide using three dosage regimens (once per week, once every 2 weeks, or once every 3 weeks) by two routes of administration, intraperitoneal (i.p.) injection or intratracheal (i.t.) instillation. The protocol is summarized in Table 1. Thirty mice were assigned to each treatment group. Ten mice from each treatment group were sacrificed 20 weeks after the first treatment with the exception of mice receiving Ni,S,
once every 2 weeks. All surviving mice
were sacrificed 45 weeks after the first treatment. Lung adenomas were observed and counted with a microscope and histology was performed on randomly selected adenomas to verify the type of tumor. Historically, urethane has been used to
McNEILL, CHRISP, AND FISHER
76
TABLE 1 . Treatment Schedule for N,iS,
Vehicle, or Urethane Exposures
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Dose (mQ/KQ) Urethane N,S i, Frequency
Untreated Controls
Saline i.p. i.t.
50.0 i.p. i.t.
0.053 i.p. i.t.
i.p.
__
29b 29b
__ __
2gb 2Sb
2gb 2gb
__
2gb 29a
__ __
2gb 2ga
2gb 2ga
-_
29b 29c
__
2gb 2gb
2gb 2gb
5x (1 /3 wk for 15 wk) 8X (1/2 wk for 15 wk) 15X
__
0.160
i.t.
(1 wk for 15 wk)
a
Sacrificed 45 weeks after the initial treatment. Ten mice from each group were sacrificed 20 wks after the initial treatment; the remaining mice were sacrificed at 45 wks. Nine mice sacrificed 20 wks after the initial treatment
induce lung adenomas in Strain A mice by the i.p. route of exposure(g). It was used in this study as a positive control with both routes of administration. Statistical Analvsis
A 2x2 contingency table was used to evaluate adenoma incidence with the method described by Fisher to compute the exact test of significance. Treated
TUMORIGENICITY OF NICKEL SUBSULFIDE
77
and control comparisons of the number of adenomas per mouse were performed using a two-tailed Student t-test with an a-value of 0.025. R ESU LTS
On the basis of range-finding studies, mice were exposed to vehicle control,
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0.053 or 0.160 mg/kg of Ni,S,
by either the intraperitoneal or intratracheal route
using three dosage regimens; once per 3 weeks for 15 weeks, once per 2 weeks for 15 weeks, or once per week for 15 weeks (Table 1). The positive control, urethane, was administered once. There were no significant differences between the mean final body weights for any group by each route of exposure at either the 20 or the 40 week sacrifice. All animals appeared normal throughout the course of the study. The early deaths that occurred were probably due to anesthesia rather than toxicity since they occurred during or shortly after dosing. After 20 weeks there was no significant difference between incidence of adenomas in controls and in animals given nickel subsulfide intraperitoneally at any dose regimen (Table 2). However, there was an apparent decrease in spontaneous adenoma incidence in mice intratracheally administered nickel subsulfide (Table 3). Since no significant difference in tumor incidence between the treatment regimen of nickel subsulfide (i.t.) was found, these data were pooled. Also because no significant difference was observed in tumor incidence in PBS controls or untreated controls, these data were pooled according to route of exposure. Subsequent comparison of the pooled control data to the pooled treatment data indicated that the (i.t.) nickel-treated animals had significantly fewer tumors (p < 0.05). Comparison of controls and low dose animals (0.053 mg/kg nickel subsulfide), was not statistically significant. The
McNEILL, CHRISP, AND FISHER
TABLE 2.
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Lung Adenomas in Strain A/J Mice 20 Weeks After lntraperitoneal Injections of Nickel Subsulfide or Urethane
Treatment Untreated Urethane PBS
Ni,S,
a
Mice with Lung Adenomas Survival
(%)
Av. No. of AdenomasIMouse c+ S.E.)
_-
10110
10
0.10 (.lo)
50
1
10110
90"
1.6 (.29)'
0
15
10110
30
0.30(.15)
0
8
10110
40
0.50 (.22)
0
5
10110
10
0.10 (.lo)
0.053
15
10110
40
0.60 (.31)
0.053
8
10110
20
0.20 (.13)
0.053
5
10110
20
0.20 (.13)
0.160
15
10110
20
0.30 (.21)
0.160
8
10110
10
0.10 (.lo)
0.160
5
1011 0
40
0.50(.22)
Dose No. of (mglkg) Treatments
__
Significantly different from pooled controls (p c 0.05).
79
TUMORIGENICITY OF NICKEL SUBSULFIDE
TABLE 3.
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Lung Adenomas in Strain NJ Mice 20 Weeks After lntratracheal Instillations of Nickel Subsulfide or Urethane
Mice with Lung Adenomas (%) Survival
Av. No. of Adenomas/Mouse (+ S.E.)
Treatment
Dose (mg/kg)
No. of Treatments
Untreated
__
__
10/10
20
0.30 (.21)
50
1
lob0
80a
1.2 (.29)a
0
15
10/10
30
0.30 (.21)
0
5
10/10
40
0.50 (.22)
0.053
15
10/10
0
0.053
5
10/10
20
0.20 (.13)
0.160
15
lob0
1oa
0.10 (.lo)
0.160
5
10/10
0
0
Urethane
PBS
Ni,S,
a
0
Significantly different from pooled controls (p c 0.05).
urethane treated animals from either intratracheal or intraperitoneal routes, had significantly more tumors than the respective pooled controls (p c 0.05). At 20 weeks, the tumor yield was 1.6 tumors/l.4 mg of urethane injected intraperitoneally and 1.2 tumors/l.4 mg administered intratracheally. The remaining animals were held an additional 25 weeks and were sacrificed 45 weeks after the first dose to determine iflung adenoma development required
McNEILL, CHRISP, AND FISHER
80
TABLE 4.
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Lung Adenomas in Strain AIJ Mice 45 Weeks After lntraperitoneal Injections of Nickel Subsulfide or Urethane
Mice with Lung Adenomas Survival (“A)
Av. No. of AdenomasIMouse & S.E.)
Treatment
Dose (mglkg)
No. of Treatments
Untreated
__
__
19119
80
1.27 (.21)
50
1
10110
90
2.3 (.45)’
0
15
19/19
63
1.0 (.24)
0
8
19119
58
0.95 (.22)
0
5
19119
68
1.0 (.23)
0.053
15
19/19
47
0.89 (30)
0.053
8
19/19
53
0.82 (.19)
0.053
5
19119
63
0.84 (.18)
0.160
15
18/19
50
0.67 (.18)
0.160
8
19/19
63
1.21 (.24)
0.160
5
19119
53
0.95 (.26)
Urethane PBS
N,iS,
a
Significantly different from pooled controls (p c 0.05).
a longer latency period. There was no significant difference between incidence of adenomas in controls compared to animals given nickel subsulfide intraperitoneally (Table 4) or intratracheally (Table 5) at any dose level, although there was an increase in tumor incidence with time across all groups. Urethane
81
T U M O R I G E N I C I T Y OF N I C K E L S U B S U L F I D E
TABLE 5.
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Lung Adenomas in Strain A/J Mice 45 Weeks After lntratracheal Instillations of Nickel Subsulfide or Urethane
Treatment Untreated Urethane PBS
Ni,S,
a
Dose No. of (mglkg) Treatments
Mice with Lung Adenomas
Av. No. of AdenomasIMouse S.E.)
Survival
(%)
__
19119
63
1.0 (.21)
50
1
10110
goa
2.0 (.42)'
0
15
17/19
47
0.71 (.21)
0
8
25/29
64
0.96 (.18)
0
5
19119
68
1.11 (.23)
0.053
15
15/19
47
0.60 (.19)
0.053
8
28/29
54
0.86 (.21)
0.053
5
19/19
63
1.16 (.27)
0.160
15
18119
56
0.89 (.25)
0.160
8
18/29
61
1.0 (.23)
0.160
5
19119
58
0.84 (.19)
__
Significantly different from pooled controls (p < 0.05).
treated animals demonstrated an increase (p < 0.01) in adenomas per mouse (2.0 adenoma/mouse) compared to control and nickel-treated animals.
There were no significant differences between the average number of adenomas per mouse for untreated and vehicle treated groups using either the intratracheal or intraperitoneal administration route. The controls (untreated and
a2
McNEILL, CHRISP, AND FISHER
PBS) were pooled according to dose route and compared to the respective treatment groups for that route. Urethane treatment (i.p.) resulted in a significant increase in adenomas per mouse (p < 0.05) compared to pooled controls. Other treated groups were not significantly different from control values. Animals treated
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intratracheally with urethane had significantly more adenomas per mouse (p < 0.05) compared to pooled controls.
No other intratracheally treated animals
had significantly different numbers of adenomas per mouse than pooled controls. DISCUSSION Urethane produced an increase in pulmonary tumor response, after intratracheal instillation as well as the usual intraperitoneal injection. Other investigators(79m10'12) have consistently demonstrated a dose response relationship for urethane in this tumor model system in which 1 mg per mouse produced 1 adenoma 20 weeks after intraperitoneal injection. In our study, a dose of 1.4 mg
of urethane by intraperitoneal injection yielded 1.6 adenomas per mouse after 20 weeks while intratracheal instillation of the same dose produced 1.2 adenomas
per mouse. Both results were significantly different (p < 0.05) from control tumor incidence. Urethane-treatedanimals sacrificed 45 weeks after dosing demonstrated 2.3 tumors per mouse for intraperitoneal injection and 2.0 tumors per mouse for
intratracheally instilled animals. These incidences were also significantly different than the untreated age-matched controls (p < 0.05). The tumor incidence in both control groups was also similar to previously published result^(^"^). At 20 weeks, 0.3 tumors per mouse were observed while at 45 weeks one tumor per mouse was observed.
TUMORIGENICITY OF N I C K E L SUBSULFIDE
83
While no work has been done on intratracheallydelivered materials in Strain A mice, inhalation has been used as a route of exposure. Strain A/J mice exposed to cigarette smoke by inhalation demonstrated a positive
Lynch('')
exposed the F, generation of Strain A mice crossed with Strain C mice to asbestos
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dust and a positive adenoma response was achieved. A positive response has also been obtained using aerosolized urethane both in terms of incidence of lung tumors (94% for treated vs. 41% for control) and average number of tumors per tumor bearing animal (57.5 for treated vs. 2.2 for controls)(").
Thus, the
pulmonary tumor response of Strain A mice can be produced by intraperitoneal injection or inhalation exposure, and on the basis of the present work, intratracheal instillation. For nickel subsulfide-treatedanimals there was no evidence of a dose-related increase in tumor incidence in any treatment group regardless of the route of exposure when compared with age-matched controls. Stoner('*) evaluated the pulmonary tumor response of various soluble metallic compounds injected intraperitoneally in Strain A/J mice. Four of the 13 compounds tested produced a significant increase in pulmonary adenomas, including nickel acetate. Our findings of a lack of tumorigenicity of nickel subsulfide, a most potent carcinogen, cast doubt on the utility of the Strain A mouse model for evaluation of potential metal carcinogens, at least by the route here tested. Smith('9)20)investigated the correlation between the Strain A tumor model and whole animal carcinogenesis studies as well as the reproducibility of the Strain A tumor model. Correlation with two-year bioassay programs was very poor with the Strain A tumor model correctly predicting carcinogenicity or lack thereof
04
McNEILL, CHRISP, AND FISHER
for 20 of 54 chemicals (37%) with 7/16 (44%) false positive tests and 27/38 (71%) false negative results. Our data further support the lack of correlation. ACKNOWLEDGEMENTS This work was supported by the Electric Power Research Institute. The au-
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thors are indebted to Ms. K. L. McNeill for support in manuscript preparation. REFERENCES Leonard, A,, G.B. Gerber, and P. Jacquet, “Carcinogenicity, Mutagenicity and Teratogenicity of Nickel,” Mutat. Res., 87, 1-15 (1981). Sunderman, F.W., Jr., “Carcinogenic Effects of Metals,” Fed. Proc., 37,40-46 (1978). Sunderman, F.W., Jr., “Nickel Carcinogenesis,” Dis. Chest, 54, 527-534 (1968). Sunderman, F.W., Jr., “Mechanisms of Nickel Carcinogenesis,” Scand. J. Work Environ. Health, l5, 1-15 (1989). Brain, J.D., D.E. Knudson, S.P. Sorokin, and M.A. Davis, “Pulmonary Distribution of Particles Given by lntratracheal Instillation or by Aerosol Inhalation,” Environ. Res., 11,13-33 (1976). Watson, J.A., A.A. Spritzer, J.A. Auld, and M.A. Guetthoff, “Deposition and Clearance Following Inhalation and lntratracheal Injection of Particles,” Arch. Environ. Health, l9, 51-58 (1969). Shimkin, M.B., and G.D. Stoner, “LungTumors in Mice: Application to Carcinogenesis Assay,“ Advan. Cancer Res., 21, 1-58 (1975). Ho, W., and A. Furst, “lntratracheal Instillation Method for Mouse Lungs,” Oncol., 27, 385-393 (1973).
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a5
Shimkin, M.B., R. Wieder, M. McDonough, L. Fishbein, and D. Swern, “Lung Tumor Response in Strain A Mice as a Quantitative Bioassay of Carcinogenic Activity of Some Carbamates and Aziridines,” Cancer Res., 29,2184-2190 (1969).
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Poirier, L.A., G.D. Stoner, and M.B. Shimkin, “Bioassay of Alkyl Halides and Nucleotide Base Analogs by Pulmonary Tumor Response in Strain A Mice,” Cancer Res., 35 1411-1415 (1975). Schut, H.A.J.,T.R. Loeb, and G.D. Stoner, “Distribution Elimination, andTest for Carcinogenicity of 2,4-Dinitrotoluene in Strain A Mice,” Toxicol. Appl. Pharmacol., 64, 213-220 (1982). Witschi, J.P., P.J. Hakkinen, and J.P. Kehrer, “Modification of Lung Tumor Development in A/J Mice,” Toxicol., 21, 37-45 (1981). Essenburg, J.M., “Further Study of Tumor Formation in the Lungs of the Albino Mice,” West. J. Surg. Obstet. Gynecol., fXj 161-163 (1957). Essenburg, J.M., M. Horowitz, and E. Gaffney, “The Incidence of Lung Tumors in Albino Mice Exposed to the Smoke from Cigarettes Low in Nicotine Content,” West. J. Surg. Obstet. Gynecol., 63, 265-267 (1955). Essenburg, J.M., A.M. Leavitt, and E. Gaffney, “The Effect of Arsenic in Tobacco on Primary Neoplasms of the Lungs of Albino Mice,” West. J. Surg. Obstet. Gynecol., 64, 35-36 (1956). Lynch, K.M., F.A. Mclver, and J.R. Cain, “Pulmonary Tumors in Mice Exposed to Asbestos Dust,” AMA Arch. Ind. Health, l5, 207-214 (1957). Leong, B.K.J., H.N. Macfarland, and W.H. Reese, “Induction of Lung Adenomas by Chronic Inhalation of Bis (Chloromethyl) Ether,” Arch. Environ. Health, 22, 663-666 (1971).
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(18) Stoner, G.D., M.B. Shimkin, M.C.Troxell, T.L Thompson, and L.S. Terry, “Test
for Carcinogenicity of Metallic Compounds by the Tumor Response in Strain A Mice,” Cancer Res., 36,1744-1747 (1976). (19) Smith, L.H., and H.P. Witschi, “The Mouse Lung Tumor Assay: A Final Re-
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port,” ORNL-5961, pp 1-62. Springfield, Vir:NTIS, U.S. Dept. Commerce (1983). (20) Smith, L.H., H.P. Witschi, and R.N. Maronpot, “Studies on the Mouse Lung
Tumor Assay as a Screen for Carcinogens,“ Toxicol. 4, 37 (1984).