National Cancer Institute 

CARCINOGENESIS 

Technical Report Series 

No. 39 

1978 

BIOASSAY OF 

LASIOCARPINE 

FOR POSSIBLE CARCINOGENICITY 

CAS No. 303-34-4 

NCI-CG-TR-39 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 

Public Health Service 

National Institutes of Health 

BIOASSAY OF 
 

LASIOCARPINE 
 

FOR POSSIBLE CARCINOGENICITY 
 

Carcinogenesis Testing Program 
 

Division of Cancer Cause and Prevention 
 

National Cancer Institute 
 

National Institutes of Health 
 

Bethesda, Maryland 20014 
 

U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 
 

Public Health Service 
 

National Institutes of Health 
 

DHEW Publication No. (NIH) 78-839 
 

BIOASSAY OF LASIOCARPINE 
 

FOR POSSIBLE 
 

CARCINOGENICITY 
 

Carcinogenesis Testing Program 
 

Division of Cancer Cause and 
 

Prevention 
 

National Cancer Institute 
 

National Institutes of 
 

Health 
 

CONTRIBUTORS: This report presents the results of the bioassay of 
 

lasiocarpine for possible carcinogenicity, conducted for the
 


Carcinogenesis Testing Program, Division of Cancer Cause and



Prevention, National Cancer Institute (NCI), Bethesda, Maryland.
 


The bioassay was conducted by Stanford Research Institute, Menlo
 


Park, California, initially under direct contract to NCI and 
 

currently under a subcontract to Tracer Jitco, Inc., prime



contractor for the NCI Carcinogenesis Testing Program. 
 

The experimental design and doses were determined by Drs. R. R.
 
 1,2 3 3
 Bates , D. C. L. Jones3

, D. P. Sasmore , G. W. Newell , and R. M. 
 

Elashoff4

, and Mr. W. E. Davis3. The principal investigator was 
 

Dr. D. C. L. Jones; the technical supervisor of animal treatment,
 


observation, and data handling was Mr. W. E. Davis; necropsy and
 


tissue fixation were supervised by Dr. D. P. Sasmore. 
 

Histologic examinations were performed by Dr. V. J. Rosen5

, and



the diagnoses included in this report represent his
 


interpretation. Neoplasms and compound-related hyperplastic 
 

lesions were reviewed by Dr. W. M. Busey6

, who also prepared the 
 

interpretive pathology summary included in this report. 
 

Animal pathology tables and survival tables were compiled at EG&G
 


Mason Research Institute7

. The statistical analyses were
 


performed by Dr. J. R. Joiner8

, using methods selected for the 
 

bioassay program by Dr. J. J. Gart9

. Chemicals used in this



bioassay were analyzed at Stanford Research Institute, and the



analytical results were reviewed by Dr. C. W. Jameson8

. 
 

iii 
 
This report was prepared at Tracor Jitco8

 under the direction of
 


NCI. Those responsible for the report at Tracor Jitco were Dr.
 


Marshall Steinberg, Director of the Bioassay Program; Dr. J. F. 
 

Robens, toxicologist; Dr. R. L. Schueler, pathologist; Ms. L. A.



Waitz and Mr. W. D. Reichardt, bioscience writers; and Dr. E. W.
 


Gunberg, technical editor, assisted by Ms. Y. E. Presley. 
 

The statistical analysis was reviewed by one or more members of
 


the Mathematical Statistics and Applied Mathematics Section of



NCI9

: Dr. John J. Gart, Mr. Jun-mo Nam, Dr. Hugh M. Pettigrew, 
 

and Dr. Robert E. Tarone. 
 

The following other scientists at the National Cancer Institute 
 

were responsible for evaluating the bioassay experiment, 
 

interpreting the results, and reporting the findings: 
 

Dr. Kenneth C. Chu 
 

Dr. Cipriano Cueto, Jr. 
 

Dr. J. Fielding Douglas 
 

Dr. Dawn G. Goodman 
 

Dr. Richard A. Griesemer 
 

Mr. Harry A. Milman 
 

Dr. Thomas W. Orme 
 

Dr. Robert A. Squire10



Dr. Jerrold M. Ward 
 

1

Carcinogenesis Testing Program, Division of Cancer Cause and 
 

Prevention, National Cancer Institute, National Institutes 
 

of Health, Bethesda, Maryland. 
 

2

Now with the Office of the Commissioner, Food and Drug 
 

Administration, Rockville, Maryland. 
 

3

Stanford Research Institute, Menlo Park, California. 
 

4

Department of Biomathematics, Center for the Health Sciences, 
 

University of California, Los Angeles, California. 
 

iv 
 
5

Department of Pathology, David M. Brotman Memorial Hospital, 
 

3828 Hughes Avenue, Culver City, California. 
 

6

Experimental Pathology Laboratories, Inc., P.O. Box 474, 
 

Herndon, Virginia. 
 

7

EG&G Mason Research Institute, 1530 East Jefferson Street, 
 

Rockville, Maryland. 
 

8

Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville, 
 

Maryland. 
 

9

Mathematical Statistics and Applied Mathematics Section, 
 

Biometry Branch, Field Studies and Statistics, Division of 
 

Cancer Cause and Prevention, National Cancer Institute, 
 

National Institutes of Health, Bethesda, Maryland. 
 

10Now with the Division of Comparative Medicine, Johns Hopkins 
 

University, School of Medicine, Traylor Building, Baltimore, 
 

Maryland. 
 

SUMMARY



A bioassay of lasiocarpine for possible carcinogenicity was



conducted by administering the test chemical in the diet to



Fischer 344 rats.



Groups of 24 rats of each sex were administered lasiocarpine at
 


one of three doses, either 7, 15, or 30 ppm, for 104 weeks.



Matched controls consisted of groups of 24 untreated rats of each
 


sex. All surviving rats were killed at 104 weeks.



Mean body weights of the high-dose male and female rats were
 


lower than those of the matched-control groups throughout most of
 


the study, while weights of the mid-dose rats were lower only
 


during the second year, and weights of the low-dose groups were
 


unaffected. There was a positive dose-related trend in mortality
 


for both sexes, with none of the high-dose animals, only five of
 


the mid-dose animals, 23 of the low-dose animals, and 43 of the
 


matched controls surviving to termination of the study. In spite
 


of these early deaths, all male rats except one low-dose animal
 


and one high-dose animal developed tumors, and among the females,
 


23 low-dose and 22 mid-dose animals developed tumors. Time­


adjusted analysis of the incidence of tumors was performed in the
 


female rats.



In male rats, there was a positive dose-related trend (P < 0.001)
 


in the incidence of angiosarcoma of the liver; furthermore, the
 


incidences in the mid- and high-dose groups, but not that in the
 


low-dose, were significantly higher (P < 0.001, both groups) than
 


that in the controls (controls 0/24, low-dose 5/24, mid-dose



11/24, high-dose 13/24). In females, the incidences in both the
 


low- and mid-dose groups, but not that in the high-dose, were
 


significantly higher (P = 0.002 and P = 0.005, respectively) than
 


that in the controls (controls 0/24, low-dose 8/24, mid-dose



7/24, high-dose 2/9). Metastatic angiosarcomas were present in



the lungs from a few of the rats in all three treated groups of
 


both sexes.



In both male and female rats, there was a positive dose-related



vii


 

trend in the combined incidence of hepatocellular carcinoma and
 


adenoma of the liver (males, P = 0.003; females, P < 0.001);
 


furthermore, the combined incidence of these tumors in the high­


dose females, but not those in the low- and mid-dose, was



significantly higher (P < 0.001) than that in the controls
 


(controls 0/24, low-dose 5/24, mid-dose 1/24, high-dose 7/9). The
 


P-value of the combined incidence in the high-dose males (P =



0.025) is above the 0.016 level required by the Bonferroni
 


inequality criterion, when multiple comparison is considered
 


(controls 0/24, low-dose 0/24, mid-dose 3/24, high-dose 5/24).
 


Nodular hyperplasia was observed in additional animals of each
 


treated group of each sex. Thus, lasiocarpine was associated with
 


proliferative lesions of hepatocytes as well as with



angiosarcomas arising from endothelial cells of the liver.



The combined incidence of lymphoma or leukemia was significant in
 


both the low- and mid-dose female groups (P < 0.018), but not in
 


the high-dose group, perhaps because of the early deaths in this
 


group (controls 2/24, low-dose 9/24, mid-dose 11/24, high-dose
 


1/23). The incidences of these tumors in the males were not



significant.



It is concluded that under the conditions of this bioassay,
 


lasiocarpine was carcinogenic in Fischer 344 rats producing
 


hepatocellular tumors and angiosarcomas of the liver in both



sexes and hematopoietic tumors in female animals.



viii


TABLE OF CONTENTS



Page



I. Introduction ............................................... 1



II. Materials and Methods ...................................... 3



A. Chemical ............................................. 3



B. Dietary Preparation .................................. 3



C. Animals .............................................. 4



D. Animal Maintenance ................................... 5



E. Subchronic Studies ................................... 6



F. Design of Chronic Studies ............................ 7



G. Clinical and Pathologic Examinations ................. 7



H. Data Recording and Statistical Analyses .............. 9



III. Results - Rats ............................................ 15



A. Body Weights and Clinical Signs ..................... 15



B. Survival ............................................ 15



C. Pathology ........................................... 18



D. Statistical Analyses of Results ..................... 21



IV. Discussion ................................................ 25



V. Bibliography .............................................. 29



APPENDIXES



Appendix A Summary of the Incidence of Neoplasms in



Rats Fed Lasiocarpine in the Diet ................ 31



Table Al Summary of the Incidence of Neoplasms in



Male Rats Fed Lasiocarpine in the Diet.... ....... 33



Table A2 Summary of the Incidence of Neoplasms in



Female Rats Fed Lasiocarpine in the Diet ......... 37



Appendix B Summary of the Incidence of Nonneoplastic



Lesions in Rats Fed Lasiocarpine in the Diet ..... 41



Table Bl Summary of the Incidence of Nonneoplastic



Lesions in Male Rats Fed Lasiocarpine



in the Diet ...................................... 43



ix


Page 

Table B2 Summary of the Incidence of Nonneoplastic 

Lesions in Female Rats Fed Lasiocarpine 

in the Diet ....................................... 46 

Appendix C Analyses of the Incidence of Primary Tumors 

in Rats Fed Lasiocarpine in the Diet .............. 49 

Table C1 Analyses of the Incidence of Primary Tumors 

in Male Rats Fed Lasiocarpine in the Diet ......... 51 

Table C2 Analyses of the Incidence of Primary Tumors 

in Female Rats Fed Lasiocarpine in the Diet ....... 55 

Table C3 Time-adjusted Analyses of the Incidence of 

Primary Tumors in Female Rats Fed Lasiocarpine 

in the Diet ....................................... 60 

TABLES 

Table 1 Design of Lasiocarpine Chronic Feeding 

Studies in Rats ................................... 8 

FIGURES 

Figure 1 Growth Curves for Rats Fed Lasiocarpine 

in the Diet ....................................... 16 

Figure 2 Survival Curves for Rats Fed Lasiocarpine 

in the Diet ....................................... 17 

x


I. INTRODUCTION



Lasiocarpine (CAS 303-34-4; NCI C01478) is a pyrrolizidine
 


alkaloid that is found in the seeds of Heliotropium lasiocarpum,
 


Heliotropium europaeum, and several other plant species, all



members of the family Boraginaceae (IARC, 1976). Its hepatotoxic
 


effects in sheep and cattle grazing on Heliotropium plants have
 


been studied in Australia. Acute liver dystrophy in man, caused
 


by H. lasiocarpum poisoning, was reported in the U.S.S.R. between
 


1931 and 1945 (Bull et al., 1968). Lasiocarpine has been reported
 


to be carcinogenic in rats following its intraperitoneal



injection (Svoboda and Reddy, 1972).



This bioassay of lasiocarpine was conducted as a part of a larger
 


study that was designed to assess the combined effects of a group
 


of known or suspected carcinogens. Only the results of the study
 


of the administration of lasiocarpine are reported herein.



1



II. MATERIALS AND METHODS



A. Chemical



Lasiocarpine is an alkaloid which is the 7((2,3-dihydroxy-2-(lmethoxyethyl)-3-methyl-l-oxobutoxy)methyl)-2,3,5,7a-tetrahydro-l 

H-pyrrolizin-1-yl ester (1S,(1α(Z),-7(2S,3R)7aα) of 2-methyl-2­

butenoic acid. The chemical was obtained in a single batch from 

Chemasea, Inc., Sidney, Australia. The purity of the batch was 

determined to be 97% by analyses at Stanford Research Institute. 

The melting point (capillary) was 92.5-93.5°C uncorrected 

(literature: 96.5-97°C), and the elemental analysis (C, H, N) was 

correct for C21H33NO7 the molecular formula of lasiocarpine. The 

identity of the chemical was confirmed by nuclear magnetic 

resonance, infrared, and ultraviolet spectra, which were in 

agreement with the structure and matched the spectra given in the 

literature. 

The lasiocarpine was stored at -4°C in capped bottles.



B. Dietary Preparation



All diets were formulated every 2 weeks using Low Fat Lab Chow®



(Ralston Purina Co., St. Louis, Mo.). A stock diet containing 500
 


ppm lasiocarpine was first prepared by grinding the alkaloid to a
 


fine powder and then mixing by hand a weighed amount of the



3


powdered material with a small amount of feed. Corn oil and more
 


feed were then added to give a final concentration of 500 ppm
 


lasiocarpine and 3% corn oil (Staley Manufacturing Company,



Orange, Calif.). Final mixing was accomplished with a Hobart



blender. Each stock diet was analyzed for content of lasiocarpine
 


by a method involving extraction and quantitation by gas-liquid
 


chromatography. Concentrations of 500 ppm ± 10% were considered
 


acceptable for use in preparing the test diets. Lasiocarpine at
 


500 ppm in the stock diet was found to be stable when held in rat
 


feeders at room temperature for a 2-week period.



To obtain test diets having appropriate concentrations of



lasiocarpine, the stock diet was diluted, as required, with



control diet containing 3% corn oil and mixed in a Hobart blender
 


for 7 minutes. The stock mixtures were stored in plastic bags at
 


-4°C. Test diets were stored in covered plastic containers at



room temperature.



C. Animals



Male and female Fischer 344 rats, obtained through contracts of
 


the Division of Cancer Treatment, National Cancer Institute, were
 


used in these bioassays. The rats were obtained from Simonsen
 


Laboratory, Gilroy, California. On arrival at the laboratory, all
 


animals were quarantined for 2 weeks as an acclimation period.



4


Following this period, all males gaining less than 25 grams, all
 


females gaining less than 15 grams, and all unhealthy animals were
 


culled. The remaining animals were assigned to cages, one per



cage, until each cage contained three animals. Cages were then



numbered and assigned to control and treated groups using a



computer-generated randomization table. Rats were ear-clipped for
 


individual identification.



D. Animal Maintenance



All animals were housed in temperature- and humidity-controlled
 


rooms. The temperature was maintained at 22°C with a range from
 


21-24°C, and the relative humidity was maintained at approximately
 


45%. The room air was changed 10 times per hour and was maintained
 


under positive pressure relative to the access halls. Fluorescent
 


lighting provided illumination 12 hours per day. Food and water
 


were available ad libitum. Drinking water was softened, filtered,
 


sterilized with ultraviolet light, and supplied by means of an



automatic watering system.



The rats were housed three per cage in polycarbonate cages



equipped with disposable polyester woven filter tops. Autoclaved



®



hardwood chips (Iso-Dri , Becton, Dickinson, and Carworth,



Warrensburg, N. Y.) were used as bedding. The cages were changed,



5


washed, and provided with fresh bedding twice per week. Filter
 


tops were replaced once per month.



Rats fed lasiocarpine were housed in the same room as rats treated
 


with N-methyl-N'-nitro-N-nitrosoguanidine (CAS 70-25-7); N-butyl­


N-(4-hydroxybutyl)nitrosamine (CAS 3817-11-6); nitrilotriacetic



acid, trisodium salt, monohydrate (CAS 18662-53-8); aflatoxin B1



(CAS 1162-65-8); cycasin (CAS 14901-08-7); or N-nitrosodi­


pentylamine (CAS 13256-06-9).



E. Subchronic Studies



Subchronic feeding studies were conducted with male and female



Fischer 344 rats to estimate the maximum tolerated dose of



lasiocarpine, on the basis of which low, mid, and high



concentrations (hereinafter referred to as "low doses", "mid



doses", and "high doses") were determined for administration in
 


the chronic studies. In the subchronic studies, lasiocarpine was
 


added to feed in concentrations of 5, 10, 20, 40, 80, or 160 ppm.
 


Treated and control groups each consisted of 15 male and of 15
 


female rats. The chemical was provided in feed to the treated



groups for 8 weeks.



At the end of 2 weeks, the animals receiving 160 and 5 ppm were
 


killed, because of appreciable body weight loss in the 160-ppm
 


group and lack of effect in the 5-ppm group. In the remaining



6


groups there were few deaths (1/15 males at 20 ppm and 1/15



females at 80 ppm). Weight gain of males at the end of 8 weeks was
 


unaffected at 10 and 20 ppm, 80% of that of controls at 40 ppm,
 


and 23% of that of controls at 80 ppm. In females, weight gain was
 


unaffected at 10 and 20 ppm, 72% of that of controls at 40 ppm,
 


and 14% of that of controls at 80 ppm. There were no gross lesions
 


at necropsy; however, slight hepatocellular pleomorphism was noted
 


at 40 ppm. The low, mid, and high doses for the chronic studies
 


were set at 7, 15, and 30 ppm.



F. Design of Chronic Studies



The design of the chronic studies is shown in table 1.



G. Clinical and Pathologic Examinations



All animals were observed daily for signs of toxicity, and animals
 


that were moribund were killed and necropsied. Animals were



weighed individually every other week for 12 weeks, and once every
 


fourth week for the remainder of the study. Palpation for masses
 


was carried out at each weighing.



The pathologic evaluation consisted of gross examination of major
 


organs and tissues from killed animals and from animals found



dead. The following tissues were routinely examined microscopical­


ly from both control and treated animals: lungs and bronchi,



7


in Diet

(ppm)

Male

Low-Dose 24 7 104

Mid-Dose 24 15 104

High-Dose 24 30 88d

Female

Low-Dose 24 7 104

Mid-Dose 24 15 104

Table 1. Design of Lasiocarpine Chronic Feeding Studies in



Rats



Sex and 

Treatment 

Group 

Matched-Control 

Initial 

No. of 

Animalsa 

24 

Lasiocarpine b 

0 

Time on Study 

Treatedc 

(weeks) 

Untreated 

(weeks) 

104 

Matched-Control 24 0 104 

High-Dose 24 30 69e 

a

All animals were approximately 54 ± 2 days of age when placed
 


on study.



b
All diets contained 3% corn oil.



c

A11 animals were started on study within 2 days of each other.



d

All high-dose males died by week 88.



e

All high-dose females died by week 69.



8


spleen, liver, kidney, pituitary, and testis. In addition, tissues
 


of the stomach, urinary bladder, thyroid, uterus, ovary, and brain
 


were examined from a majority of the controls; these tissues were
 


taken from treated animals only if a lesion was found at necropsy.
 


Occasionally, additional tissues were examined microscopically.
 


Gross lesions from all animals were also examined microscopically.
 


The different tissues were preserved in 10% buffered formalin,
 


embedded in paraffin, sectioned, and stained with hematoxylin and
 


eosin. Special staining techniques were utilized when indicated
 


for more definitive diagnosis.



A few of the tissues from some animals were not examined,
 


particularly from those animals that died early. Also, some



animals were judged to be in such an advanced state of autolysis
 


as to preclude histopathologic evaluation. Thus, the number of



animals from which particular organs or tissues were examined



microscopically varies, and does not necessarily represent the



number of animals that were placed on study in each group.



H. Data Recording and Statistical Analyses



Pertinent data on this experiment have been recorded in an auto­


matic data processing system, the Carcinogenesis Bioassay Data



System (Linhart et al., 1974). The data elements include descrip­


tive information on the chemicals, animals, experimental design,



9


clinical observations, survival, body weight, and individual
 


pathologic results, as recommended by the International Union



Against Cancer (Berenblum, 1969). Data tables were generated for
 


verification of data transcription and for statistical review.



These data were analyzed using the statistical techniques
 


described in this section. Those analyses of the experimental
 


results that bear on the possibility of carcinogenicity are



discussed in the statistical narrative sections.



Probabilities of survival were estimated by the product-limit
 


procedure of Kaplan and Meier (1958) and are presented in this
 


report in the form of graphs. Animals were statistically censored
 


as of the time that they died of other than natural causes or were



found to be missing; animals dying from natural causes were not
 


statistically censored. Statistical analyses for a possible dose­


related effect on survival used the method of Cox (1972) for



testing two groups for equality and Tarone's (1975) extensions of
 


Cox's methods for testing for a dose-related trend. One-tailed P
 


values have been reported for all tests except the departure from
 


linearity test, which is only reported when its two-tailed P value



is less than 0.05.



The incidence of neoplastic or nonneoplastic lesions has been



given as the ratio of the number of animals bearing such lesions



10


at a specific anatomic site (numerator) to the number of animals
 


in which that site is examined (denominator).



The purpose of the statistical analyses of tumor incidence is to
 


determine whether animals receiving the test chemical developed a
 


significantly higher proportion of tumors than did the control
 


animals. As a part of these analyses, the one-tailed Fisher exact
 


test (Cox, 1970) was used to compare the tumor incidence of a



control group with that of a group of treated animals at each dose
 


level. When results for a number of treated groups (k) are



compared simultaneously with those for a control group, a



correction to ensure an overall significance level of 0.05 may be
 


made. The Bonferroni inequality (Miller, 1966) requires that the P
 


value for any comparison be less than or equal to 0.05/k. In cases
 


where this correction was used, it is discussed in the narrative
 


section. It is not, however, presented in the tables, where the
 


Fisher exact P values are shown.



The Cochran-Armitage test for linear trend in proportions, with



continuity correction (Armitage, 1971), was also used. Under the
 


assumption of a linear trend, this test determines if the slope of
 


the dose-response curve is different from zero at the one-tailed
 


0.05 level of significance. Unless otherwise noted, the direction
 


of the significant trend is a positive dose relationship. This



11


method also provides a two-tailed test of departure from linear
 


trend.



A time-adjusted analysis was applied when numerous early deaths
 


resulted from causes that were not associated with the formation
 


of tumors. In this analysis, deaths that occurred before the first
 


tumor was observed were excluded by basing the statistical tests
 


on animals that survived at least 52 weeks, unless a tumor was



found at the anatomic site of interest before week 52. When such
 


an early tumor was found, comparisons were based exclusively on



animals that survived at least as long as the animal in which the
 


first tumor was found. Once this reduced set of data was obtained,
 


the standard procedures for analyses of the incidence of tumors
 


(Fisher exact tests, Cochran-Armitage tests, etc.) were followed.



When appropriate, life-table methods were used to analyze the



incidence of tumors. Curves of the proportions surviving without
 


an observed tumor were computed as in Saffiotti et al. (1972). The
 


week during which an animal died naturally or was sacrificed was
 


entered as the time point of tumor observation. Cox's methods of
 


comparing these curves were used for two groups; Tarone's
 


extension to testing for linear trend was used for three groups.
 


The statistical tests for the incidence of tumors which used life­


table methods were one-tailed and, unless otherwise noted, in



12


the direction of a positive dose relationship. Significant



departures from linearity (P < 0.05, two-tailed test) were also
 


noted.



The approximate 95 percent confidence interval for the relative
 


risk of each treated group compared to its control was calculated
 


from the exact interval on the odds ratio (Gart, 1971). The



relative risk is defined as Pt/Pc where pt is the true binomial
 


probability of the incidence of a specific type of tumor in a



treated group of animals and pc is the true probability of the
 


spontaneous incidence of the same type of tumor in a control
 


group. The hypothesis of equality between the true proportion of a
 


specific tumor in a treated group and the proportion in a control
 


group corresponds to a relative risk of unity. Values in excess of
 


unity represent the condition of a larger proportion in the



treated group than in the control.



The lower and upper limits of the confidence interval of the



relative risk have been included in the tables of statistical
 


analyses. The interpretation of the limits is that in



approximately 95% of a large number of identical experiments, the
 


true ratio of the risk in a treated group of animals to that in a
 


control group would be within the interval calculated from the
 


experiment. When the lower limit of the confidence interval is
 


greater than one, it can be inferred that a statistically signifi­


13


cant result (P < 0.025 one-tailed test when the control incidence
 


is not zero,P<0.050 when the control incidence is zero) has
 


occurred. When the lower limit is less than unity, but the upper
 


limit is greater than unity, the lower limit indicates the absence
 


of a significant result while the upper limit indicates that there
 


is a theoretical possibility of the induction of tumors by the
 


test chemical, which could not be detected under the conditions of
 


this test.



14


III. RESULTS - RATS
 


A. Body Weights and Clinical Signs



Mean body weights of high-dose male and female rats were lower
 


than those of all other groups throughout most of the study



(figure 1). Weights of mid-dose animals were lower than those of
 


controls only during the second year, and weights of low-dose
 


groups were unaffected by lasiocarpine. Between weeks 80 and 88,
 


surviving treated and control animals were anorexic and lost



weight from a suspected viral infection. No treatment was given,
 


and the animals apparently recovered by the next weighing period
 


(week 92).



Mortality was not usually preceded by any clinical signs;



occasionally, however, pale mucous membranes, general weakness,
 


abdominal distention, or emaciation were observed before deaths
 


occurred.



B. Survival



The Kaplan and Meier curves estimating the probabilities of



survival for male and female rats fed lasiocarpine in the diet at
 


the doses used in this study, together with those of the



controls, are shown in figure 2.



In both sexes, the Tarone test result for positive dose-related



15


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1-

l: 

5:! 

w 

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0 

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~ 

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1-

l: 

5:! 

w 200 

~ 

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Ill 

z 

~ 

w 

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20 

0 10 20 

6 

6 

MALE RATS 

0 MATCHED CONTROL 

0 LOW DOSE 

0 MID DOSE 

6 HIGH DOSE 

50 60 

--------~T~IM~ ~S~ ~~~~7~o---- OY IWEEKSI :s~o ----~----~--- 90 

100 

30 40 

6 

50 60 

TIME ON STU OY !WEEKS) 70 

30 40 80 

FEMALE RATS 

0 MATCHEDC ONTROL 

0 LOW DOSE 

0 MID DOSE 

6 HIGH DOSE 

90 100 

110 

110 

Figure 1. Growth Curves For Rats Fed Lasiocarpine In The Diet 

16


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~ vf 

I 

0 LOW DOSE 1 - 0Mt000SE . A HIGH DOSE v~ h. 

i --h 6 .. 1 

0 15 30 45 60 75 90 105 120 135 

TIME ON STUDY (WEEKS) 

Figure 2. Survival Curves For Rats Fed Lasiocarpine In The Diet 

17


trend in mortality is significant (P < 0.001), and departure from
 


linear trend is observed (P < 0.001), due to the steep increase
 


in mortality occurring in the treated groups, especially the



high-dose groups. In male rats, 88% of the controls, 54% of the
 


low-dose group, 17% of the mid-dose group, and none of the high­


dose group survived to termination of the study. In females, 92%
 


of the controls, 42% of the low-dose group, 4% of the mid-dose
 


group, and none of the high-dose group survived to termination of
 


the study. Since over 50% of the female high-dose group died
 


before week 52 on study, the statistical analysis of female rats
 


was done using only those animals surviving more than 52 weeks.
 


The numbers of such female rats were 24 in the control, 24 in the
 


low-dose, 23 in the mid-dose, and 9 in the high-dose groups.
 


Since a tumor occurred in the hematopoietic system at week 39 in
 


female rats, the time-adjusted analysis at this site is based on
 


animals surviving 39 weeks or more. All male rats, except one



low-dose and one high-dose animal, developed tumors. In female
 


rats, 23/24 low-dose and 22/24 mid-dose rats developed tumors,
 


but the early deaths of the high-dose females may have reduced
 


the incidences of late—appearing tumors in that group.



C. Pathology



Histopathologic findings on neoplasms in rats are summarized in



18


Appendix A, tables Al and A2; nonneoplastic lesions are



summarized in Appendix B, tables B1 and B2.



A variety of neoplastic processes not related to treatment were
 


observed in both the control and treated rats. The incidences of
 


these neoplasms were comparable among the control and treated
 


groups. The following neoplasms were randomly distributed
 


throughout the control and treated groups: squamous-cell



carcinoma of the skin, alveolar/bronchiolar adenomas of the lung,
 


and endometrial stromal polyps of the uterus. Lymphomas and



leukemias were found more frequently in treated than in control
 


rats.



Angiosarcomas were observed in the livers from several of the
 


male and female lasiocarpine-treated rats. In the males, the



incidence of this neoplasm of the liver occurred in a dose­


related fashion, whereas in the females, the incidence was



greater in the low- and mid-dose groups than in the high-dose
 


group. This inverse dose relationship can probably be explained
 


by the increased deaths in the high-dose females compared with
 


either the controls or low-dose females. Metastatic angiosarcomas
 


were occasionally seen in the lungs from the rats in all three
 


treated groups. The angiosarcomas were either single or multiple
 


in the liver and were characterized by a proliferation of



neoplastic endothelial cells, which in some



19


cases exhibited a high degree of anaplasia. Large blood-filled
 


cysts lined by neoplastic endothelial cells frequently were



present. The neoplastic endothelial cells sometimes assumed a



cuboidal shape and contained large basophilic nuclei. In some



instances, sheets of proliferating endothelial cells were present
 


in the parenchyma of the liver, destroying the hepatic



architecture. Adenomas and/or nodular hyperplasia of the



hepatocytes were also frequently seen in the liver. The areas of
 


nodular hyperplasia contained swollen hepatocytes with large,



bizarre nuclei and possessed tinctorial properties distinctly
 


different from the surrounding liver tissue. In some instances,
 


the foci were compressing the surrounding liver parenchyma. The
 


hepatocellular adenomas morphologically resembled neoplastic
 


nodules (Squire and Levitt, 1975) and were usually composed of
 


large, eosinophilic hepatocytes containing bizarre nuclei. These
 


adenomas were large, occupied several liver lobules, and



frequently compressed the surrounding liver parenchyma. In both
 


sexes, hepatocellular carcinomas were found only in the high-dose
 


animals: two in the males and one in the females.



In conclusion, the results of this microscopic examination



indicate that the administration of lasiocarpine at the three



doses used in this study had a carcinogenic effect in the liver
 


of Fischer 344 rats. This effect was manifested by the induction



20


of angiosarcomas, several of which metastasized to the lung. No
 


angiosarcomas were present in any of the tissues examined from
 


the control rats, and this neoplasm is rare in the Fischer 344
 


rat. Lesions diagnosed as adenomas or nodular hyperplasia were



seen only in the treated rats, indicating a proliferative
 


hepatocellular effect. This finding lends support to the



conclusion that lasiocarpine had a carcinogenic effect in the



liver.



D. Statistical Analyses of Results



Tables C1-C3 in Appendix C contain the statistical analyses of



the incidences of those primary tumors that occurred in at least
 


two animals in one group and with an incidence of at least 5% in
 


one or more of the treated groups. The analysis of female rats is
 


based on those animals surviving 52 weeks or more; however, the
 


analysis of the incidence of lymphoma and the combined incidence
 


of lymphoma and leukemia is based on animals that lived 39 weeks
 


or more, because such a tumor appeared in this system at week 39.



In male rats, the Cochran-Armitage test results for positive
 


dose-related trend in the proportions of angiosarcoma of the



liver is significant (P < 0.001). The Fisher exact test shows
 


that the incidences in the mid-, and high-dose groups are



21


significantly higher than the incidence in the matched-control
 


group (P < 0.001, and P < 0.001, respectively), and the lower
 


limits of the 95% confidence intervals for these relative risks
 


are greater than one. The Fisher exact comparison of incidences
 


in the low-dose groups with incidences in the matched-control
 


groups show a probability level of 0.025, which is above the
 


0.016 level required by the Bonferroni inequality criterion when
 


multiple comparison is considered.



In female rats, the Fisher exact test shows that the incidences
 


of angiosarcoma of the liver in both the low- and mid-dose groups
 


are significantly higher than that in the controls (P = 0.002 and
 


P = 0.005, respectively). The incidence of this tumor in the



high-dose group is not significant, due to the low number of
 


animals in the denominator of that group; nevertheless, the



incidence is 2/9 (22%). The statistical evidence suggests that
 


the incidence of this tumor in rats is associated with



lasiocarpine at the doses used in this experiment.



When the analyses of the incidence of leukemia and of the



combined incidence of leukemia and lymphoma in female rats are



performed, the Fisher exact test shows that the incidences in the
 


low- and mid-dose groups are significantly higher (P < 0.025)
 


than those in the corresponding matched controls. The time­


adjusted incidence of these tumors in the high-dose group is 1/9



22


(11%) for leukemia alone and 1/23 (4%) for the combination of



lymphoma and leukemia. Statistical tests on the incidence of



these tumors in the male rats are not significant. Although the
 


combined incidence in the mid-dose group indicates a P value of
 


0.030 when compared to the matched-control incidence, this



probability level is higher than the 0.016 level required by the
 


application of the Bonferroni inequality criterion to the results
 


of multiple comparison under this situation.



In both sexes, hepatocellular carcinomas are found only in the
 


high-dose groups. None of the Fisher exact test results in either
 


sex are significant. However, the combination of adenoma, NOS



(not otherwise specified), and hepatocellular carcinoma indicates
 


a significant difference in incidence (P < 0.001) between the
 


high-dose group and the controls in females. The Fisher exact



comparison of incidences between the high-dose and matched­


control groups in male rats indicate a probability level of



0.025, which is above the 0.016 level required by the Bonferroni
 


inequality criterion, when multiple comparison is considered. The
 


result of the Cochran-Armitage test for positive dose-related
 


trend is also significant for both sexes (P = 0.003, males; P <
 


0.001, females). The principle contribution to these significant
 


results is made by the adenomas, NOS. The overall statistical
 


conclusion is that there is a dose relationship
 


23


between this chemical and the occurrence of these liver tumors in
 


female rats.



Significant results in the negative direction are observed in the
 


incidence of interstitial-cell tumor of the testis in male rats
 


and in the incidence of chromophobe adenoma of the pituitary in
 


female rats, where the incidences in the controls exceed those in
 


the treated groups. These significant negative results may be



explained by the severe mortality of the treated animals.



24


IV. DISCUSSION



The high and mid doses of lasiocarpine used in this bioassay were
 


toxic, as shown by the decrease in body weights and decrease in
 


survival. Mean body weights of the high-dose groups were lower



than those of matched-control groups throughout most of the



study, while mean body weights of the mid-dose groups were lower
 


only during the second year, and weights of the low-dose groups
 


were unaffected. There was a positive dose-related trend in



mortality for both sexes, and none of the high-dose animals
 


survived to termination of the study. Time-adjusted analysis of
 


the incidence of tumors was performed in the female rats.



Angiosarcomas occurred at several sites in treated rats of both
 


sexes (males: 5 low-dose, 11 mid-dose, 14 high-dose; females: 8
 


low-dose, 9 mid-dose, 2 high-dose). However, most of these



occurred in the liver. In male rats, there was a positive dose­


related trend (P < 0.001) in the incidence of angiosarcoma of the
 


liver; furthermore, the incidences in the mid- and high-dose
 


groups, but not that in the low-dose, were significantly higher
 


(P < 0.001, both groups) than that in the controls (controls
 


0/24, low-dose 5/24, mid-dose 11/24, high-dose 13/24). In



females, the incidences in both the low- and mid-dose groups, but
 


not that in the high-dose, were significantly higher (P = 0.002



and P = 0.005, respectively) than that in the controls



25


(controls 0/24, low-dose 8/24, mid-dose 7/24, high-dose 2/9).
 


Metastatic angiosarcomas were observed in the lungs from a few of
 


the rats of both sexes in all three treated groups (males: three
 


low-dose, five mid-dose, and seven high-dose; females: three low­


dose, four mid-dose, and one high-dose). No angiosarcomas were



present in control rats and this neoplasm is rare among Fischer
 


344 rats.



In both male and female rats, there was a positive dose-related
 


trend in the combined incidence of hepatocellular carcinoma and
 


adenoma of the liver (males, P = 0.003; females, P < 0.001);
 


furthermore, the combined incidence of these tumors in the high­


dose females, but not those in the low- and mid-dose, was



significantly higher (P < 0.001) than that in the controls
 


(controls 0/24, low-dose 5/24, mid-dose 1/24, high-dose 7/9). The
 


P-value of the combined incidence in the high-dose males (P =



0.025) is above the 0.016 level required by the Bonferroni
 


inequality criterion, when multiple comparison is considered
 


(controls 0/24, low-dose 0/24, mid-dose 3/24, high-dose 5/24).
 


Nodular hyperplasia was observed in additional animals of each
 


treated group of each sex. Thus, lasiocarpine was associated with
 


proliferative hepatocellular lesions as well as with the



angiosarcomas arising from endothelial cells of the liver.



The combined incidence of lymphoma or leukemia (controls 2/24,



26


low-dose 9/24, mid-dose 11/24, high-dose 1/23) was significant in 

both the low- and mid-dose female groups (P ≤ 0.018), but not in 

the high-dose group, perhaps because of the early deaths in this 

group. The incidences of these tumors in the males were not 

significant. 

Previous studies have also shown that the liver is the target
 


organ for lasiocarpine. Rats injected with lasiocarpine developed
 


centrilobular necrosis that sometimes appeared several weeks or
 


months after the treatment (Schoental and Magee, 1957). Midzonal
 


hemorrhagic necrosis was reported to occur in injected rats in
 


acute toxicity studies (Bull et al., 1958). In chronic toxicity
 


studies, injected rats developed enlarged livers which consisted
 


almost entirely of regenerated parenchyma (Bull et al., 1968;
 


Svoboda et al., 1971); this alteration was believed to be related
 


to the prolonged antimitotic effect of the chemical. Svoboda and
 


Reddy (1972) found carcinomas of the liver and squamous-cell
 


carcinomas of the skin in Fischer 344 rats that were injected
 


with lasiocarpine twice per week for 4 weeks, and then injected
 


once per week for 52 weeks.



It is concluded that under the conditions of this bioassay,



lasiocarpine was carcinogenic in Fischer 344 rats producing



hepatocellular tumors and angiosarcomas of the liver in both



sexes and hematopoietic tumors in female animals.



27



V. BIBLIOGRAPHY



Armitage, P., Statistical Methods in Medical Research. John Wiley
 


& Sons, Inc., New York, 1971, pp. 362-365.



Berenblum, I., ed., Carcinogenicity Testing; A Report of the



Panel on Carcinogenicity of the Cancer Research Commision of
 


the UICC, Vol. 2. International Union Against Cancer,



Geneva, 1969.



Bull, L. B., Culvenor, C. C. J., and Dick, A. T., The



pyrrolizidine alkaloids. Frontiers of Biology. Vol. 9,



North-Holland Publishing Co., Amsterdam, 1968, pp 13-17,



221-227.



Bull, L. B., Dick, A. T., and McKenzie, J. S., The acute toxic
 


effects of heliotrine and lasiocarpine, and their N-oxides,
 


on the rat. J. Path. Bact. 75:17-25, 1958.



Cox, D. R., Regression models and life tables. J. R. Statist.
 


Soc. B 34(2);187-220. 1972.



Cox, D. R., Analysis of Binary Data, Methuen & Co. , Ltd.,



London, 1970, pp. 48-52.



Gart, J. J. , The comparison of proportions: a review of



significance tests, confidence limits and adjustments for
 


stratification. Rev. Int. Statist. Inst. 39(2);148-169,



1971.



International Agency for Research on Cancer, Some naturally
 


occurring substances. IARC Monographs on the Evaluation of
 


the Carcinogenic Risk of Chemicals to Man, Vol. 10, World
 


Health Organization, Geneva, 1976, pp 281-290.



Kaplan, E. L. and Meier, P., Nonparametric estimation from



incomplete observations. J. Am. Statist. Assoc. 53:457-481,



1958.



Linhart, M. S., Cooper, J. A., Martin, R. L. , Page, N. P., and
 


Peters, J. A., Carcinogenesis bioassay data system. Comp.



and Biomed. Res. 7: 230-248, 1974.



Miller, R. G., Jr., Simultaneous Statistical Inference, McGraw-
 

Hill Book Co., New York, 1966, pp. 6-10.



29


Saffiotti, U. , Montesano, R., Sellakumar, A. R. , Cefis, F. and
 


Kaufman, D. G., Respiratory tract carcinogenesis in hamsters
 


induced by different numbers of administrations of benzo (a)
 


pyrene and ferric oxide. Cancer Res. 32;1073-1081, 1972.



Schoental, R. and Magee, P. N., Chronic liver changes in rats
 


after a single dose of lasiocarpine, a pyrrolizidine



(Senecio) alkaloid. J. Path. Bact. 74:305-319, 1957.



Squire, R. A. and Levitt, M., Report of a workshop on



classification of specific hepatocellular lesions in rats.
 


Cancer Res. 35:3214-3223, 1975.



Svoboda, D. J. and Reddy, J. K., Malignant tumors in rats given
 


lasiocarpine. Cancer Res. 32:908-911. 1972.



Svoboda, D. J., Reddy, J. K., and Bunyaratvej, S., Hepatic
 


megalocytosis in chronic lasiocarpine poisoning. Amer. J.
 


Path. 65(2);399-410. 1971.



Tarone, R. E., Tests for trend in life table analysis.



Biometrika 62(3):679-682, 1975.



30


APPENDIX A 
 

SUMMARY OF THE INCIDENCE OF NEOPLASMS 
 

IN RATS FED LASIOCARPINE IN THE DIET 
 

31 
 

------------------------------------------------------------------------------------------------

TABLE A1. 


SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS 


FED LASIOCARPINE IN THE DIET 


CONTROL LOW DOSE MID DOSE HIGH DOSE 

AHIMALS 

AHIMALS 

ANIMALS 

lNITIALLY IN STUDY ~ECFOPSIED 

EXA~INED HISTOPATHOLOGICALLY 

24 

24 

24 

24 

211 

211 

24 

211 

24 

INTEGU~ENTARY SYSTEM 

*SKIN 

SQlAMOUS CtLL CARCINOMA 

(24) (24) 1 (4%) 

(24) {24) 

*SUECUT TISSUE 

SARCCMA, NOS 

( 24) (24) (24) 

1 (4~) 

(24) 

~ESPIRA!CRY SYSTEM 

HUNG 

ALVECLAR/BRONCHIOLAR ADENOMA 

PHECCHROMOCYTOMA, METASTATIC 

SARCCMA, NOS 

ANGICSARCOIIA 

ANGIOSARCOMA, METASTATIC 

(23) 

2 (9%) 

1 ( 4%) 

(24) 

1 

3 

(4%) 

( 13%) 

(24) 

5 (21%) 

(24) 

1 

1 

7 

(4") 

(4%) 

(29%) 

HEMATOFOIETIC SYSTEM 

*MULTIPlE ORGANS 

MAliGNANT LYMPHOMA, NOS 

UNCIFFERENTIATED lEUKEMIA 

GRANULOCYTIC LEUKEMIA 

(24) 

1 (4%) 

1 (4%) 

1 ( 4 %) 

(21.1) 

2 (8%) 

(4%) 

(24) 

3 

2 

6 

( 13%) 

(8%) 

(25%) 

(24) 

1 (4 'li:) 

(8'li:) 

4 ( 17'li:) 

tLYIIPH NOI:E 

LEICHCSARCOtlA, ME'IASTAHC 

ANGIOSARCOMA, METASTATIC 

(2) (4) 

(25'll:) 

( 10) 

1 

1 

( 1 0") 

(10%) 

(8) 

UIVE!i 

GRA!iULOCYTIC LE UK EIIIA 

(2 3) 

1 (4%) 

(24) (23) (23) 

f!HYIIUS 

ANGICSARCOIIA, METASTATIC 

( 1) ( 1) 

1 ( 100%) 

(2) 

CIRCULATCRY SYSTEII 

__ !!ONE 

-----------------· I NUIIBER OF 

* NU~BER CF 

ANIMALS 

ANIMALS 

WITH TISSUE NEC~OP~IEt EXA~INEt MICROSCOPICALLY 

33 

TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

=====~~=~===============================-------------------------------------------------------

CONTROL LOW DOSE MID DOSE HIGH DOSE 

DIGESTJVE SYSTEM 

HIVEF (23) (24) (23) (2 3) 

ADHCNA, NOS 3 (13") 3 (13ll) 

HEFA'IOCFLLULAF. CARCINOMA 2 (9%) 

LEI C MYOSARCOMA, MEHSTA'IIC (4%) 1 ( 4%) 

A~GICMA 1 (4ll) 

ANGICSHCOMA 5 (21%) 11 (48ll) 13 (5711) 

4DUCDHUM (3) (2) (2) 

ACENCCARCINOMA, NOS 1 (50%) 

SARCOMA, NOS (33%) (50") 

LEIOMYOSARCOMA (33%) (50%) (50%) 

Fl ERCADENO~JA (33%) 

ICOLO~ (2) 


ADE~CMATUUS POLYP, NOS 1 (50%) 


URINARY SYSTEI! 

IKlDNEY (23) (24) (24) (2 4) 


ANGICSARCOI!A, METASTATIC 1 (4%) 


#KICNH;CCRTEX (23) (24) (24) ( 24) 


ADHCMA, NOS 1 (4%) 


ENDOCRirE SYSTEM 

HliU l'IAPY ( 22) (2 3) (23) (24) 


CH&CrOFHOBE ADENOMA 1 (4%) 


#ACRE NAL ( 1) ( 1) 


FHfCCHFOMOCYTOMA, MALIGIANI 1 ( 1 OO'ii) 


HHYRCH ( 16) ( 1) ( 1) 


C-CEll CARCINOMA 1 (100%) 


REFbOLUC1IVF SYSTEM 

UES':'IS (2<1) (24) (24) (24) 

INTEFSTITIAL-CLLL TCMOR 24 ( 100%) 21 (88%) 22 (92%) E (33%) 

!IEEVCU~ SYSTEM 

___ NONE--------------------------------------------------------­

f NU~BER OF ANIMALS WITH TISSUE IXAMINEC MICROSCOPICALLY * NOBBE~ CF ANIMALS NFCRGPSI![ 

34 

------- ------------------------------------------------------------------------------------------------- --------------- ------- ---

TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOWOOSE MID DOSE HIGH DOSE 

SPECIAL SENSE ORGANS 

*EYE/LACRIMAL GLAND 

UNtifFERENTIATED CARCINCMA 

(24) (24) (2!1) 

1 '""J (24) 

MUSCULOSKELETAL SYSTEM 

NCNE 

BODY CAVITIES 

II ONE 

ALL CTH~~ SYSTEMS 

*MULTiflE ORGANS 

MESCTHELIOMA. MALIGNANT 

(2!1) 

1 (4") 

(24) (24) (24) 

ANIMAL IISFOSITION SUMMARY 

A~IMALS INITIALLY IN STUDY 

NATU6AL DEATHi 

MORIEUND SACRIFICE 

SCHErUU'D SACRIfiCE 

ACCltENTALLY KillED 

TERMINAL SACRIFICE A~HAL IIISSING 

24 

3 

21 

24 

7 " 

13 

24 

6 

14 

4 

2!1 

10 

14 

I NUMBEB OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NOBBE& OF ANIMALS NECROPSIED ) 

35 

------------------------------------------------------------------------------------------------ -----------------------------

TABLE A1. MALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE MID DOSE HIGH DOSE 

IU!IvR ~lMMAR~ 

TCTAl A~IMALS wiTH PRIMARY lUMORS* 24 23 24 23 

TO!Al fRIMARY TUMORS 32 36 54 37 

TOTAL ANIMALS WITH BENIGN TUMCRS 24 21 23 10 

ICTAl BENIGN TUMOR! 26 23 28 12 

TCTAl ANIMALS WITH MAliGNANT TUMORS 6 12 20 23 

T011l MALIGNANT TUMORS 6 13 26 25 

IOIAl ANIMALS WITH SfCON~ARY 1UMORSl 5 6 i 

TOTAl SECO~DARY TUMORS 7 9 7 

TOTAl ANIMALS WITH TUMORS UNCERTAIN­


BENIG~ CR MALIGNANT 


TOTAl UNCERTAIN TUMORS 


TOTAl ANIMALS WITH TU!IORS UNCERTAI~­

PRIMARY CR METASTATIC 


TOTAl UNCERTAIN TUMORS 


* 	 PRIMARY TO!IORS: ALL TUMORS EXCEPT SECCNDARY TOKORS 

SECONtARY TOMORS: !IE1ASTATIC 1UMORS OR TUMORS INVASIVE INTO AN ADJACENT ORGAN 

36 

TABLE A2. 


SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS 


FED LASIOCARPINE IN THE DIET 


----------------------.....-------------------------------------------------------------------------· ---.---------- --- ­ CONTROL LOW DOSE MID DOSE HIGH DOSE 

ANI~AL~ INITIALLY IN STUDY 24 24 24 :<4 

ANiftALS NECROPSIED 24 24 211 :<4 ANl~ALS EXA~INED HISTOfATHOLOGICALLY 24 24 211 23 

INTEGU~ENTARY SYSTEM 

*SKIN (211) {24) (24) (24) 


~QCA~OUS CELL CARCINOMA 1 { 4%) 


*SUBCUT TISSUE ( 24) {24) {24) (24) 


AN GICSARCOIIA 1 {4%) 


RESPIR~TORY SYSTEM 

HUNG (24) (23) (24) (23) 

ALVECLAR/BRONCHIOLAF ADENOMA 1 (4%) 

ANGICSARCOIIA 2 (8%) 

ANGIOSARCOMA, METASTATIC 3 { 13%) 4 (17%) (4%) 

HEMATOFOlETIC SYSTEM 

*MULTlFLE ORGANS ( 24) (24) (24) (24) 

~AIIGNANT LYMPHOMA, NO~ 1 (4 :c) 2 (8%) 3 (1H) 

UN~IFFERhNTIATED LEUKEMIA 1 (II :II) 

LYIIFHOCYTIC LEUKEMIA 1 (II%) 

GRANULOCYTIC LEUKEI!IA (Ill) 6 (251) 7 (29%) 1 (II%) 

ITHYIIUS ( 1) (5) 


ANGlCSARCOMA, METASTATIC 1 (1001) 


ClRCULATCEY SYSTEM 

NONE 

DIGESTIVE SYSTEM 

#LIVE!' (24) (22) (24) 123) 

__A]l.Jj!;Jlk-li~-----------· ---------2-~----~1~14:ulu.>------~~ 

I NUIIBEE OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUIIBFF OF ANIMALS NECROPSIED 

37 

---------------- ----------------------------------------------------------------------------------------------- ---------

TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE MID DOSE HIGH DOSE 

HEFA~CCELLULAR CARCINOM~ 

ANGIOSARCOMA 8 (36%) 7 (29ll) 

(4ll) 

(9ll) 

JSTCMAC t 

S(iiJAMCTJS CELL PAPILLOMA 

ADENCCAFCINOMA, NOS 

ADENCM!ITOUS POLYP, NOS 

(2 3) 

1 ( 4%) 

(4) 

1 

2 

(25%) 

(50%) 

( 1) (8) 

URINARY ~YSTJlM 

#UEINAFY BLADDER ( 16) ( 1) ( 1) 

FAfiiLARY CARCINOMA 1 ( 6") 

ENDOCRI~F SYSTEM 

#FITU I'IARY (24) (24) (23) (23) 

CHECMCFHOBE ADENOMA 11) ( 4 2ll) 5 (21%) 

EASCFHII ADENOMA 1 (4%) 

!iEPRCDlC'IIVF SYSTEM 

*MAMI!AFY GLAND (24) (24) (24) (24) 

-~DEH~A, NOS 4 (17%) 

ADE1CCARCINOMA, NOS (4%) 

#UTERUS (22) ( 15) ( 12) (2) 

CAECJNCMA-IN-SITU, NOS 1 (7%) 

ADENCCAFCINOMA, NOS 1 (5%) 1 (7%) 

ENLOMETRIAL STROMAL fOLYP 6 (27") 9 (60%) 4 (33%) 

ENLGr.ETFIAL STiiOM AI. SARCOMA 1 (5%) 

CARCINOSARCOMA 1 (7i) 

NERVOU~ SYSTEM 

~CNE 

SPECIAl SENS£ ORGANS 

NONE 

MUSCULC~KEitTAL SYS'IEM 

_].Q_lig___________________________________________________________________ 

# NUMBER OF ANIMALS WITH TISSUE EXAMINED MIC&OSCOPICALLY * NUMBER OF ANIMALS NECROPSIED 

38 

------------------------------------------------------------------------------------------------

TABLE A2. FEMALE RATS: NEOPLASMS (CONTINUED) 

CONTROL LOW DOSE MID DOSE HIGH DOSE 

BODY CAVITIES 

NONE 

ALL CTHE~ SYSTEMS 

AI:HCSE 'IISSUE 


ANGICSARCOMA, METASTATIC 


ANI~AL [lSFOSITION SU~MARY 

ANIMAlS INITIALLY IN STUDY 24 24 24 24 


NATUFAI DEATHiil 5 7 14 


MORIEUND SACRIFICE 2 9 16 10 


SCHE[ULED SACRIFICE 


ACCIDENTALLY KILLED 
 TER~INAL SACRIFICE 22 10 


ANHAL MISSING 


• INCLUDES AUTOLYZED ANIMALS 

TUIICR SUlMAH 

TOTAl ANIMALS WITH PRIMARY TU~CRS* 18 22 22 8 

TOTAl PRIMARY TUMORS 27 45 26 10 

TOTAl ANI~ALS WITH BENIGN TUMCRS 17 16 4 6 

TOTAl BENIGN TU"ORS 21 23 5 6 

TOTAL JNIMALS WITH ~ALIGNANT TU"ORS 6 19 20 4 

TOTAl MALIGNANT TUMORS 6 22 21 4 

TOTAl ANIMALS WITH SECONDARY TUMORSI 3 4 1 

TOTAl SECONDARY TUMORS 4 4 2 

TOTAL ANIMALS WITH TU~ORS UNCERTAIN­


BENIG~ CR MALIGNANT 


TOTAl UNCERTAIN TUMORS 


TOTAl ANIMALS WITH TUMORS UNCERTAIN­


FfiiMARY CR METASTATIC 


TOTAL UNCERTAIN TUMORS 


* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TU"ORS 

I SECO.[ARY TUMORS: METASTATIC TUMORS OR TUMORS IBVASIVE INTO AN ADJACENT ORGAN 

----~-----------------------------------------------------------------------------------------

39 


 

APPENDIX B 
 

SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC 
 

LESIONS IN RATS FED LASIOCARPINE IN THE DIET 
 

41 
 

TABLE 81. 


SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS 


FED LASIOCARPINE IN THE DIET 


-------------------------------------------------------------------------------------------------· CONTROL LOW DOSE MID DOSE HIGH DOSE 

ANiftALS INITIALLY IN STUDY 

ANI~ALS ~ECFOPSIED 

ANIMALS EX~MINED HlSTOfA!HOLOGICALLY 

<4 

24 

24 

24 

24 

24 

24 

24 

24 

INTEGUMENTARY SYSTEM 

HONE 

RESFIRAiCRY SYSTEM 

HUNG CO~GESTION, NOS 

INFlAMMATION, INiERSTITIAl AB~CISS, NOS 

(23) 

2 (91) 

(24) 4 

1 

(171) 

(4%) 

(24) 5 (211) (~4) 

3 (13:11) 

1 (41) 

HEHATOFOIETIC SYSTEM 

ISPLEEN 

CONGESTION, NOS 

INFARCT, NOS 

HEMATOPOIESIS 

(23) 

1 (41) 

(24) 

2 (8%) 

(23) 

1 

1 

(41) 

(4%) 

(23) 

ILYHPE NODE 

CONGESTION, NOS 

HYFHPLASIA, NOS 

(2) (4) 

(25%) 

(1 0) 

(10%) 

{8) 2 (25ll) 

ITHYI!US 

CONGESTION, NOS 

(1) (1) (2) 

2 (100%) 

CIRCULATCRY SYSTEM 

IIONE 

DIGESTIVE SYSTEI! 

I LIVE~ 

_..l.Bl.lA.III!ATIO!I. GRANULOI!AiOUS 

(23) (24) 

2 !811 

(23) C~3) 

I IIUI!BEn * NUI!BEE OF 

OF 

ANIMALS 

ANIMALS 

WiiH TISSUE 

NECROPSIED 

EXAMINED MICROSCOPICALLY 

43 

TABLE 81. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

----------------------------------------------------------------------------·-------------------­ CONTROL LOW DOSE MID DOSE 

GRAlULOI'IA, NOS 

INFLAI'II'IATION, FOCAL GRANULOI'I

PELICSIS HEPATIS 

INFARCT, NOS HYfE~PLASIA, NODULAR 

ATOU 2 (9%) 2 

1 

4 

(8%) 

(4%) 

(17%) 

(4%) 

(4%) 

*I!ILE tUCT 

INFLAMMATION, CHRONIC 

(24) (24) 

1 (4%) 

(24) 

tSTOI'IACE 

DIVERTICULUM 

ERGS ION 

(23) 

1 ( 4'll) 

(3) 

2 {6 7%) 

(2) 

(50'll) 

UJiiNARY SYSTEM 

tKIDN! Y (23) (24) (24) 

INFAFCT, HEALED 1 (4%) 

ENCOCR!NE SYSTEM 

tThYRC!D ( 16) ( 1) (1) 

CYS'I, NOS 1 (6%) 

REfRODOC!IVF SYSTEM 

HROS'IAH ( 1) 

HE~CFRHAGE 1 ( 100%) 

UES'IIS (24) (24) (24) 

ATECfHY, NOS 1 (4%) 1 (4%) 

NERVOUS SYSTEM 

tBRAIN (23) 

CONGESTION, NOS 

HEMCBRHAGE 

SPECIAL SENSE ORGANS 

NCNE 

MUSCULCSKElFTAL SYSTEM 

_.I!Qll____________________________________________ 

t NUMBER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY 

* NUMB!~ OF ANIMALS NECROPSI!L 

HIGH DOSE 

1 (4%) 

fi (26'll) 

(24) 

{4) 

(25%) 

(24) 

(24) 

1 (4%) 

(2) 

44 

---------------

TABLE 81. MALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

-----------------------------------------------------------------------------------------------· CONTROL LOW DOSE MID DOSE HIGH DOSE 

BOI:Y OVlTlES 

*INGUINAl REGION (24) (24) (24) (24) 

NECFCSIS, FAT 1 (4~) 4 (17~) 4 (17~) 1 (41) 

ALL OTEEB SYSTEMS 

iHORAX 


HEMCHHAGE 


SPECIAl tCRFHOLOGY SUMMARY 

NCNE 

f NOMBEF·CF ANIMALS WiiH !!~SUE EXAMINED MICROSCOPICALLY * NUMBEE Of ANIMALS NECECFSIED 

45 

TABLE 82. 


SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS 


FED LASIOCARPINE IN THE DIET 


------------------------------------------------------------------------------------------------~ 

CONTROL LOW DOSE MID DOSE HIGH DOSE 

ANitAU HITIALLY IN S~UDY 

ANI~ALS ~ECROPSIED 

ANI~ALS EXAMINED HISTOPATHOlOGICAllY 

24 

24 

24 

24 

24 

24 

24 

24 

24 

24 

24 

23 

INTEGU~E~TARY SYSTEM 

NCNE 

RESPIRA!CRY SYSTEM 

ILUNG/ ERONC HUS 

BRCNCHIECTASIS 

(24)

, (4") 

(23)

, (4") 

(24) (2 3) 

HUNG CO~GISTION, NOS INFLA~MATION, INTERSTITIAL 

GRANULOMA, NOS 

HEMCSIDEROSIS 

(24) 

4 

1 

(17") 

(4") 

(23) 

2 

1 

(9") 

(4") 

( 24) 

3 

1 

1 

(13") 

(4") 

(4") 

'2:!) 

E (26") 

HEMATOPOIETIC SYSTEM 

IISPLEB 

CONGESTION, NOS 

FIERCSIS 

HEIIA!OPOIESIS 

ILYIIPH NOI:F 

lY!fHANGIECTASIS 

GRANULOMA, NOS 

(.24) 

(5) 

1 (20") 

(24) 

3 

(2) 

1 

( 13") 

(50") 

(24) 

1 

2 

(6) 

(4") 

(8") 

(2 3) 

1 (4") 

t~ESENTERIC L. NODE 

:EDEIIA, NOS 

HYPERPLASIA, NOS 

(5) ( 2) (6) 

1 

1 

(17") 

(17") 

tTHYIIOS 

HHHPLASIA, NOS 

( 1) (5) 

1 (20~) 

CIRCULAiCRY SYSTEII 

__l!Qn____________ 

t NUIIBE~ OP ANIMALS WITH TISSUE EXAIIINED ~ICROSCOPICALLY * NUIIBE~ OE ANIMALS NECROPSIEt 

46 

TABLE 82. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 

-------------------------------------------------------------------------------------------------· CONTROL LOW DOSE MID DOSE HIGH DOSE 

DIGESTIVE SYSTEft 

ILIVH GRA~UICMA, NOS 

FEIICSIS HEPATIS 

NEC!iCSIS, fOCAL 

Ill F ARCT, NOS 

HYIEFFLASIA, NODULAR 

(24) 

8 

2 

( 331) 

(8:11) 

(22) 

6 

(5:11) 

(2H) 

(24) 

10 (421) 

(23) 

13 

(41) 

(571) 

fSTCI!ACf. 

ERCS ION 

(23) (4) 

1 (251) 

(1) (€) 

fGAST HC MUCOSA 

ERCSlON 

(23) (4) ( 1) (E) 

1 (13:11) 

fSlALI INTESTINE HE~CHHAGE 

(1) 

1 ( 100'11) 

tCECUII 

CONGESTION, NOS 

EDEI!A, NOS 

INFlAMIIATION, CHRONIC 

( 1) ( 1) 

(1001) 

(5) 

1 

3 

1 

(20'11) 

(601) 

(201) 

URINARY SYSTEM 

tKIDII.EY 

BEIICSICEROSI S 

(24) (24) (23) 

fURINAFY BLADDER 

HUCFRHAGE 

(16) ( 1) ( 1) 

1 (1001) 

ENDOCRINE SYSTEM 

HITUI!ARY CO~GlSTION, 

HEMCiiRHAGE 

NOS 

(24) 

1 ( 41) 

(24) (23) 

1 (41) 

REPRODlCliVE SYSTEM 

fU'IER U~ (2:2) 

____lil!~~,.ET~R~A~--------------------~~,jj!L___ 

( 15) (12) (2) 

f NUMBEii 

* IIUIIBEii 

OF 

CF 

ANIMALS 

ANIMALS 

WITH TISSUE 

NECROPSIED 

EXAMINED MICROSCOPICALLY 

47 

------------------------------------------------------------------------------------------------

TABLE 82. FEMALE RATS: NONNEOPLASTIC LESIONS (CONTINUED) 


----------------- ----------------------- -----· 

CONTROL LOW DOSE MID DOSE HIGH DOSE 

FYCH'IRA 4 (18%) 1 (7%) 

A'IROPHY, NOS (50,;) 

IUTERUS/ENDOMET~IUM ( 2 2) ( 15) (12) (2) 


IN HAIII!ATION, NOS 1 (7%) 


HYfEBPlASIA, CYSTIC 1 (5%) 3 (25%) 


4IOVARY (20) (2) ( 1) 


CYS'I, NOS 4 (20%) 


AB~CESS, NOS (100%) 


--------------------------------------------~---------------------------------------------------· 

NERVOU~ SYSTEM 

liCNE 

SPECIAl SENSE O~GANS 

NONE 

MUSCULCS~ELETAL SYS'IEI! 

NONE 

BODY CA VI'IIES 

NCNE 

ALL CTEEB SYSTEMS 

NONE 

SPECIAl ~CRPHOLOGY SUMMARY 

NC USION REPOR'IED 1 

AU'IC/NECROPSY/NO HISTO 1 

I NUMEEF OF ANIMALS WI'IH 'IISSUE EXAI!INED I!ICEOSCOPICALLY * NUMBEE OF ANIMALS NECROPSIED 

48 

APPENDIX C 
 

ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS
 


IN RATS FED LASIOCARPINE IN THE DIET 
 

49 
 

Table C1. Analyses of the Incidence of Primary Tumors in Male Rats 
 

Fed Lasiocarpine in the Dieta



51 
 

Topography: Morphology Hematopoietic System: Lymphomab P Valuesc,d Relative Risk (Matched Control)f 

Matched Control 

1/24 (4) 

N.S. 

Low Dose 

2/24 (8) 

N.S. 

2.000 

Mid Dose 

3/24 (13) 

N.S. 

3.000 

Lower Limit 0.111 0.265 

Upper Limit 112.815 150.246 

Weeks to First Observed Tumor 103 88 80 

Hematopoietic System: 

Leukemiab 

P Valuesc,d 

Relative Risk (Matched Control)f 

3/24 (13) 

N.S. 

1/24 (4) 

N.S. 

0.333 

8/24 (33) 

N.S. 

2.667 

Lower Limit 0.007 0.739 

Upper Limit 3.801 13.700 

Weeks to First Observed Tumor 86 103 82 
Hematopoietic System: 

Departure from Linear Trende P = 0.045 

52 
 

Table C1. Analyses of the Incidence of Primary Tumors in Male Rats 
 

Fed Lasiocarpine in the Dieta 


(continued) 


Matched Low Mid 

Topography: Morphology Control Dose Dose 

Lymphoma or Leukemiab 4/24 (17) 3/24 (13) 11/24 (46) 

P Valuesc,d N.S. N.S. P = 0.030 Relative Risk (Matched Control)f 0.750 2.750 

Lower Limit 0.121 0.968 

Upper Limit 3.951 9.911 

Weeks to First Observed Tumor 86 88 80 

Liver: Hepatocellular 

Carcinomab 

P Valuesc,d 

0/24 (0) 

N.S. 

0/24 (0) 

N.S. 

0/24 (0) 

N.S. 

Relative Risk (Matched Control)f -­ -­ 

Lower Limit -­ -­ 

Upper Limit -­ -­ 

Weeks to First Observed Tumor -­ -­ -­ 
Table C1. Analyses of the Incidence of Primary Tumors in Male Rats 
 

53 
 

Fed Lasiocarpine in the Dieta 


(continued) 


Matched Low Mid 

Topography: Morphology Control Dose Dose Liver: Hepatocellular Carcinoma 


b 0/24 (0) 3/24 (13) 

or Adenoma, NOS 0/24 (0) 


c,d
 P Values P = 0.003 N.S. N.S. 

Relative Risk (Matched Control)f -- Infinite 

Lower Limit -- 0.622 

Upper Limit -- Infinite 

Weeks to First Observed Tumor -­ -­ 104 

Liver: Angiosarcomab 0/24 (0) 5/24 (21) 11/24 (46) 

P Valuesc,d P < 0.001 P = 0.025 P < 0.001 

Relative Risk (Matched Control)f Infinite Infinite 

Lower Limit 1.309 3.478 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor -­ 87 74 
Testis: Interstitial-Cell Tumorb

Departure from Linear Trende P = 0.040 

54



Table C1. Analyses of the Incidence of Primary Tumors in Male Rats 

Fed Lasiocarpine in the Diet a 

(continued) 

Matched Low Mid 

Topography: Morphology Control Dose Dose 

24/24 (100) 21/24 (88) 22/24 (92) 8

P Valuesc,d P < 0.001 (N) N.S. N.S. P 

Relative Risk (Matched Control)f 

Lower Limit 

0.875 

0.000 

0.917 

0.000 

0

0

Upper Limit 1.143 1.091 3

Weeks to First Observed Tumor 86 87 74 

a

Treated groups received doses of 7, 15, or 30 ppm. 
 

b

Number of tumor-bearing animals/number of animals necropsied (percent). 
 

c

Beneath the incidence of tumors in the control group is the probability level for the Coc

test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the incidenca treated group is the probability level for the Fisher exact test for the comparison of group with the matched-control group when P < 0.05; otherwise, not significant (N.S.) is 

d

A negative trend (N) indicates a lower incidence in a treated group than in the control ge

The probability level for departure from linear trend is given when P < 0.05 for any comp

f

The 95% confidence interval of the relative risk between each treated group and the match

control group. 
 
Table C2. Analyses of the Incidence of Primary Tumors in Female Rats 
 

Fed Lasiocarpine in the Dieta



55 
 

Topography: Morphology Lung: Angiosarcomab 

Matched Control 

0/24 (0) 

Low Dose 

0/24 (0) 

Mid Dose 

2/24 (8) 

P Valuesc,d N.S. N.S. N.S. 

Relative Risk (Matched Control)f -­ Infinite 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor -­

-­-­-­

0.305 

Infinite 

68 

Hematopoietic System: 

Lymphoma, NOSb 

P Valuesc,d 

Malignant 

1/24 (4) 

N.S. 

2/24 (8) 

N.S. 

3/24 (13) 

N.S. 

Relative Risk (Matched Control)f 2.000 3.000 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 104 

0.111 

112.815 

70 

0.265 

150.246 

39 
Departure from Linear Trende P = 0.003 

Departure from Linear Trende P < 0.001 

56 
 

Table C2. Analyses of the Incidence of Primary Tumors in Female Rats 
 

Fed Lasiocarpine in the Dieta 


(continued) 


Matched Low Mid 

Topography: Morphology Control Dose Dose Hematopoietic System: 

Leukemiab 1/24 (4) 7/24 (29) 8/24 (33) 

P Valuesc,d N.S. P = 0.024 P = 0.013 

Relative Risk (Matched Control)f 7.000 8.000 

Lower Limit 1.010 1.212 Upper Limit 297.414 333.388 7

Weeks to First Observed Tumor 95 72 63 

Hematopoietic System: Lymphoma 

or Leukemiab 

P Valuesc,d 

2/24 (8) 

N.S. 

9/24 (38) 

P = 0.018 

11/24 (46) 

P = 0.004 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor 95 

4.500 

1.072 

38.367 

70 

5.500 

1.395 

45.074 

39 
Table C2. Analyses of the Incidence of Primary Tumors in Female Rats 
 

Fed Lasiocarpine in the Dieta 


(continued) 


Matched Low Mid 

Topography: Morphology Control Dose Dose Liver: Hepatocellular 

Carcinomab 0/24 (0) 0/24 (0) 0/24 (0) 

P Valuesc,d N.S. N.S. N.S. 

Relative Risk (Matched Control)f -­ -­ 

Lower Limit -­ -­ 

Upper Limit -­ -­ 

Weeks to First Observed Tumor -­ -­ -­ 

57 
 Liver: Hepatocellular Carcinoma or Adenoma, NOSb 0/24 (0) 5/24 (21) 1/24 (4) 

P Valuesc,d P = 0.013 P = 0.025 N.S. 

Relative Risk (Matched Control)f 

Lower Limit 

Upper Limit 

Weeks to First Observed Tumor -­

Infinite 

1.309 

Infinite 

104 

Infinite 

0.055 

Infinite 

82 
Departure from Linear Trende P = 0.002 

Table C2. Analyses of the Incidence of Primary Tumors in Female Rats 
 

58 
 

Fed Lasiocarpine in the Dieta 

(continued) 

Matched Low Mid 

Topography: Morphology Control Dose Dose 

Liver: Angiosarcomab 0/24 (0) 8/24 (33) 7/24 (29) 

P Valuesc,d N.S. P = 0.002 P = 0.005 

Relative Risk (Matched Control)f Infinite Infinite 

Lower Limit 2.382 2.021 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor -­ 84 68 Pituitary: Chromophobe 

Adenomab 10/24 (42) 5/24 (21) 0/24 (0) 

P Valuesc,d P < 0.001 (N) N.S. P < 0.001 (N) 

Relative Risk (Matched Control)f 0.500 0.000 

Lower Limit 0.160 0.000 Upper Limit 1.348 0.322 

Weeks to First Observed Tumor 84 94 -­ 
59 
 

Table C2. Analyses of the Incidence of Primary Tumors in Female Rats 
 

Fed Lasiocarpine in the Dieta



(continued) 
 

Matched Low Mid 

Topography: Morphology Control Dose Dose Uterus: Endometrial Stromal 
 6/24 (25) 9/24 (38) 4/24 (17) 

Polypb 


P Valuesc,d P = 0.004 (N) N.S. N.S. 

Relative Risk (Matched Control)f 1.500 0.667 

Lower Limit 0.568 0.158 

Upper Limit 4.264 2.440 

Weeks to First Observed Tumor 104 70 71 

aTreated groups received doses of 7, 15, or 30 ppm. 
 bNumber of tumor-bearing animals/number of animals necropsied (percent). 
 

cBeneath the incidence of tumors in the control group is the probability level for the Coc

Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the of tumors in a treated group is the probability level for the Fisher exact test for the cthat treated group with the matched-control group when P < 0.05; otherwise, not significaindicated. 
 

dA negative trend (N) indicates a lower incidence in a treated group than in the control geThe probability level for departure from linear trend is given when P < 0.05 for any comp

fThe 95% confidence interval of the relative risk between each treated group and the match

control group. 
 
Table C3. Time-adjusted Analyses of the Incidence of Primary Tumors in Female R

Fed Lasiocarpine in the Dieta



Matched Low Mid 

Topography: Morphology Control Dose Dose 

Lung: Angiosarcomab (52) 0/24 (0) 0/24 (0) 2/24 (8) 

P Valuesc,d N.S. N.S. N.S. 

Relative Risk (Matched Control)f -­ Infinite 

Lower Limit -­ 0.305 

Upper Limit -­ Infinite 

Weeks to First Observed Tumor -­ -­ 68 

60 
 Hematopoietic System: Malignant Lymphoma, NOSb (39) 1/24 (4) 2/24 (8) 3/24 (13) 

P Valuesc,d N.S. N.S. N.S. 

Relative Risk (Matched Control)f 2.000 3.000 

Lower Limit 0.111 0.265 

Upper Limit 112.815 150.246 

Weeks to First Observed Tumor 104 70 39 
Departure from Linear Trende P = 0.027 

Departure from Linear Trende P < 0.001 

Table C3.Time-adjusted Analyses of the Incidence of Primary Tumors in Female Rat

61 
 

Lasiocarpine in the Dieta 

(continued) 

Matched Low Mid 

Topography: Morphology Control Dose Dose 

Hematopoietic System: Leukemia b (52) 1/24 (4) 7/24 (29) 8/23 (35) 

P Valuesc,d N.S. P = 0.024 P = 0.009 

Relative Risk (Matched Control) f 7.000 8.348 

Lower Limit 1.010 1.267 

Upper Limit 297.414 346.345 18

Weeks to First Observed Tumor 95 72 63 Hematopoietic System: Lymphoma or 

Leukemiab (39) 2/24 (8) 9/24 (38) 11/24 (46) 

P Valuesc,d N.S. P = 0.018 P = 0.004 

Relative Risk (Matched Control) 

f 

4.500 5.500 

Lower Limit 1.072 1.395 

Upper Limit 38.367 45.074 

Weeks to First Observed Tumor 95 70 39 
Departure from Linear Trende P = 0.001 

Table C3. Time-adjusted Analyses of the Incidence of Primary Tumors in Female 

62 
 

Fed Lasiocarpine in the Dieta 


(continued) 


Matched Low Mid 

Topography: Morphology Control Dose Dose Liver: Hepatocellular Carcinomab 
 0/24 (0) 0/24 (0) 0/24 (0) 1/

(52) 


c,d
 P Values N.S. N.S. N.S. 

Relative Risk (Matched Control)f -- -- 

Lower Limit -- -- 

Upper Limit -- -- 

Weeks to First Observed Tumor -­ -­ -­ 

Liver: Hepatocellular Carcinoma or 

Adenoma, NOS b(52) 0/24 (0) 5/24 (21) 1/24 (4) 

P Valuesc,d P < 0.001 P = 0.025 N.S. 

Relative Risk (Matched Control) f Infinite Infinite 

Lower Limit 1.309 0.055 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor -­ 104 82 
(continued) 

Departure from Linear Trende P = 0.029 

Table C3. Time-adjusted Analyses of the Incidence of Primary Tumors in Female R

Fed Lasiocarpine in the Dieta



63 
 

Matched Low Mid 

Topography: Morphology Control Dose Dose Liver: Angiosarcomab (52) 0/24 (0) 8/24 (33) 7/24 (30) 

P Valuesc,d N.S. P = 0.002 P = 0.005 

Relative Risk (Matched Control)f Infinite Infinite 

Lower Limit 2.382 2.021 

Upper Limit Infinite Infinite 

Weeks to First Observed Tumor -­ 84 68 Pituitary: Chromophobe 

Adenomab (52) 10/24 (42) 5/24 (21) 0/24 (0) 

P Valuesc,d P = 0.001 (N) N.S. P < 0.001 (N) 

Relative Risk (Matched Control)f 0.500 0.000 

Lower Limit 0.160 0.000 

Upper Limit 1.348 0.322 

Weeks to First Observed Tumor 84 94 — 
64 
 

Table C3. Time-adjusted Analyses of the Incidence of Primary Tumors in Female R

Fed Lasiocarpine in the Diet3


(continued) 
 

Matched Low Mid 

Topography: Morphology Control Dose Dose 

Uterus: Endometrial Stromal Polyp b 

P Valuesc,d (52) 6/24 (25) N.S. 9/24 (38) N.S. 4/24 (17) N.S. 

Relative Risk (Matched Control)f 1.500 0.667 

Lower Limit 0.568 0.158 

Upper Limit 4.264 2.440 

Weeks to First Observed Tumor 104 70 71 

a

Treated groups received doses of 7, 15, or 30 ppm. 
 

b

Number of tumor-bearing animals/number of animals necropsied (percent). (Based on animals 

that lived at least as long as shown in parentheses after the description of the morpholo

c

Beneath the incidence of tumors in the control group is the probability level for the Coch

Armitage test when P < 0.05; otherwise, not significant (N.S.) is indicated. Beneath the of tumors in a treated group is the probability level for the Fisher exact test for the cthat treated group with the matched-control group when P < 0.05; otherwise, not significaindicated. 
 

d

A negative trend (N) indicates a lower incidence in a treated group than in the control gre

The probability level for departure from linear trend is given when P < 0.05 for any compa

f

The 95% confidence interval of the relative risk between each treated group and the matche

control group.


Review of the Bioassay of Lasiocarpine*for Carcinogenicity 
 

by the Data Evaluation/Risk Assessment Subgroup of the 
 

Clearinghouse on Environmental Carcinogens 
 

November 28, 1977 
 

The Clearinghouse on Environmental Carcinogens was established in 
 

May, 1976 under the authority of the National Cancer Act of 1971 (P.L. 
 

92-218). The purpose of the Clearinghouse is to advise on the National 
 

Cancer Institute's bioassay program to identify and evaluate chemical 
 

carcinogens in the environment to which humans may be exposed. The 
 

members of the Clearinghouse have been drawn from academia, industry, 
 

organized labor, public interest groups, State health officials, and 
 

quasi-public health and research organizations. Members have been 
 

selected on the basis of their experience in carcinogenesis or related 
 

fields and, collectively, provide expertise in organic chemistry, bio­


chemistry, biostatistics, toxicology, pathology, and epidemiology. 
 

Representatives of various Governmental agencies participate as ad hoc 
 

members. The Data Evaluation/ Risk Assessment Subgroup of the 
 

Clearinghouse is charged with the responsibility of providing a peer 
 

review of NCI bioassay reports on chemicals studied for carcinogenicity. 
 

In this context, below is the edited excerpt from the minutes of the 
 

Subgroup's meeting at which Lasiocarpine was reviewed. 
 

Lasiocarpine, a known experimental carcinogen, was tested in rats 
 

as part of another study designed to investigate the combined effects of 
 

chemicals. Under the conditions of test, Lasiocarpine induced 
 

angiosarcomas in both sexes of treated rats. Although the report 
 

indicated that it also caused granulocytic leukemia in the treated 
 

females, it was pointed out that the incidence was approximately the same 
 

as observed in female control animals from the Hexa-chlorophene study. 
 

One member emphasized the limited number of animals per group and the 
 

high early mortality. 
 

A motion was made that Lasiocarpine was carcinogenic under the 
 

conditions of test. It was added that the study was less than ideal in 
 

that the animal groups were smaller than the current standard and there 
 

was high mortality in the mid- and high-dose groups. The motion was 
 

seconded and approved unanimously. 
 

65 
 
Members present were:



Gerald N. Wogan (Chairman), Massachusetts Institute of



Technology



Lawrence Garfinkel, American Cancer Society



Henry C. Pitot, University of Wisconsin Medical Center



George Roush, Jr., Monsanto Company



Verald K. Rowe, Dow Chemical U.S.A.



Michael B. Shimkin, University of California at San Diego



Louise Strong, University of Texas Health Sciences Center



John H. Weisburger, American Health Foundation



*		 Subsequent to this review, changes may have been made in the



bioassay report either as a result of the review or other reasons.



Thus, certain comments and criticisms reflected in the review may



no longer be appropriate.



*U.S. GOVERNMENT PRINTING OFFICE: 1978 260-899/3018 1-3 66 



DHEW Publication No. (NIH) 78-839