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4A187 273 CHRONIC MAMMALIAN TOxICOLOGICAL EFFECTS OF LAP I./!NRSTEMATER(U) SRI INTERNATIONAL MENLO PARK CAJ MI BROWN ET AL JUN 81 DAMD17-9-C-912i
UNCLASSIFIED FG 24/4 NL
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MICROCOPY RESOLUTION TEST CHARr
IATONAL BUREAU OF STANOARDS1963-A
n FILE (XWJAD_
* ~ i CHRONIC MAMMALIAN TOXICOLOGICAL EFFECTS OF LAP WASTEWATER
Final Report
by
Janice M. Brown, Ted A. Jorgenson, and Ronald J. Spanggord
UJune 1983
Supported by
U.S. ARMY MEDICAL RESEARCHAND DEVELOPMENT COMMAND
Fort DetrickFrederick, Maryland 21701 -. .
Attention:- Jesse J. Barkley, Jr. FetT EProject Officer EL - A
Contract No. DAMD 17-79-C-9121 S NoV 61
SRI Project LSU-8846 A
SRI InternationalMenlo Park, California 94025
if Approved for public release; distribution unlimited
The findings in this report are not to be construed as an officialDepartment of the Army position unless so designated by other authorizeddocuments.
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IISIGNATURE PAGE
3 Submitted by:
I Janice M. Brown, BiologistMammalian Toxicology Department
Ted A. Jo ge on (Study Director)j Mammalian Toxicology Department
j Ronald J. Spanggord, Director (Study Director)Bio-Analytical Chemistry ProgramIApproved by:
David C. L. Joe ctorToxicology Laboratory
L k O -C,/ HW. A. Skinner, Vice resident
Life Sciences Division
I
IDist INSPECTED
Di i
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*cA
S2CU~kITY CLASSIFICATION OF THIS PAGE Whoa, Doa~tmdJeri
REPOT DCUMNTATON AGEBEFORE COMPLETING FORM1REPORT NUMBER 2.GOVT ACCESSION NO. 3. RECIPIENT'S CATALOG NUMBER
4. TITLE (and Subtiitle) S. TYPE OF REPORT & PERIOD COVERED
CHRONIC MAMMALIAN TOXICOLOGICAL EFFECTS OF Final Report
LAP WASTEWATER September 19 79-July 1982S. PERFORMING 010. REPORT NUMBER
__________________________________________ LSU-88467. AUTHOR(q) S. CONTRACT OR GRANT NUMBER(&)
Janice M. Brown, Ted A. Jorgenson, DAMD17-79-C-9121Ronald J. Spanggord
S. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT. PROJECT, TASK
SRI International AREA & WORK UN IT NUMBERS333 Ravenswood AvenueMenlo Park, California 94025 _____________
1IL CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE
U.S. Army Medical Research and Development Commanc June 1983Fort Detrick, Frederick, Maryland 21701 IS. NUMBER OF PAGES
___________________________________________ 253
14. MONITORING AGENCY NAME & ADDRESS(if different from Controlling Office) IS. SECURITY CLASS. (of this rmport)
Unclassified
I~.DECLASSI FICATION/ DOWNGRADINGSCHEDULE
16. DISTRIBUTION STATEMENT (of thiso Report)
Approved for public release; distribution unlimited.
17. DISTRIBUTION STATEMENT (of th. abstract entered i Block "0, it differmnt heom Report)
18. SUPPLEMENTARY NOTES
It. K EY WORDS (ConIhiuo an rovers. side If necesar and idenify by Mlock ambor)
2 ,4,6-trinitrotoluene (TNT); 1 ,3,5-trinitrohexahydro-1 ,3,5-triazine (RDX);LAP (load, assumble, and pack).
20. ABSTRACT (Coanthiu an reverse side if necesarny mid Ideantli by block number)
The objective of these studies was to provide a comprehensive definitionof the long-term toxicological effects of LAP wastewater (TNT/RDX in a ratioof 1.6 to 1) on Fischer-344 rats with respect to possible lesions at thebiochemical and cellular levels Acute oral LD50 values were determined
D ON 4 1473 EDITIOM OF I NOV 61 IS O990L T.CBFE
SEURTYCLSSIFICATO OTIA G (Won D-e. Etrod
1 UNCLASSIFIEDC U IrT Y C L A SI FIC A TIO N O F T IS P AG E (lhm , D Oa E-t- l r i
contained 75 rats of each sex. Because an excessive number o males receivingI200 mg/kg/day of LAP exhibited convulsions and death, the high-dose level wasreduced to 100 mg/kg/day for both sexes after 12 weeks on test. However,
* aggressive behavior, convulsions, and death continued to occu , particularly3in the males, and the entire dose level (consistinma f--W - e and 69 female
survivors) was terminated after 33 weeks on test. -Iales at the mid-dose levelshowed an increase in mortality compared with controls at 104 weeks (studytermination); these rats also exhibited convulsions and aggressive behavior.Low-dose males and females at both the low- and mid-dose levels showed littleresponse with regard to behavioral or clinical abnormalities, food consumption,and mortality; however, body weights in these groups were significantlydecreased throughout the study.
Effects on both the myelocytic and erythrocytic series were seen in bothsexes at the mid-dose level.) Clinical chemistry parameters were affected toSegree in females at the ow- and mid-dose levels, a dose response was
& in glucose, uric acid, U triglycerides, LDH, total iron, A/G ratio,Na C0 2 , and biluribin. Only LDH was significantly decreased in males atboth levels.
Treatment-related chan in relative organ weights consisted of an increasein liver weight in all treated s, possibly reflecting an induction ofhepatic enzymes to metabolize LAP, an ncrease in kidney weight in malesat all dose levels, reflecting kidney damage. The principal LAP-inducedlesions observed as histopathological examination were degenerative changes inthe eye and urinary system and decreased cellularity of the lymphoid tissues.These changes were generally restricted to the mid- and high-dose groups andwere usually more pronounced in the males.
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UNICLA.SSIFIEDI SCuRITY CLASSIFCATIO N O ru e P G Er Mlon D efa EenftmE )
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UII£ EXECUTIVE SUMMARY
Confirmatory oral LD5O and 14-day range-finding feeding studies on LAPwere conducted with Fischer-344 rats. The results of these studies wereused to establish dose levels for the chronic phase of this program, theobjective of which was to provide a comprehensive definition of the long-term toxicological effects of LAP with respect to possible lesions at thebiochemical and cellular levels.
In the LD50 study, six dose levels of LAP were tested with ten malesand ten females at each level. The results of this study showed thesingle-dose oral LD50 to be 294 and 325 mg/kg for male and female Fischer-344 rats, respectively.
1For the 14-day range-finding and chronic studies, LAP was incorporatedinto the diet by dissolving the compound in acetone, roto-evaporating theacetone, and adding 3% corn oil to the extract. In the 14-day range-finding study, five concentrations of LAP in the diet were tested with tenanimals of each sex at each level. Consistently reduced food consumptionand body weight gain were seen at the 0.5 and 0.7% dietary levels for bothsexes; mortality also occurred at both levels. Occasionally, food consump-tion, body weights, gross behavior, and hematological parameters wereadversely affected at the 0.3% level, but they were rarely affected at the1 0.1 and 0.05% levels.
Based on the data from the 14-day range-finding study, dietary levelsequivalent to dose levels of 12.5, 50, and 200 mg/kg/day were selected forthe chronic study.
In the chronic study, each treatment group contained 70 rats of eachsex. The vehicle control group consisted of 75 rats of each sex thatI. received a 3% corn oll/roto-evaporated acetone/feed mixture in which theoriginal acetone content was equal to that used to dissolve the LAP at thehighest treatment level. After just one week on test, food consumption andbody weights were reduced at the mid- (50 mg/kg/day) and high- (200mg/kg/day) dose levels. During the first 12 weeks of the study, the high-dose males had severe convulsions that resulted in multiple abrasions and10% of the rats had died by Week 12. Therefore, the high-dose level wasreduced by 50% at that time. Body weights in the females and food consump-tion in both sexes began to increase and eventually exceeded controlvalues. Convulsions and abnormal aggression continued to occur in bothsexes, as did mortality in the males. After 33 weeks on test, all sur-vivors in the high-dose groups were terminated. At the mid-dose level,
convulsions and abnormal aggression were also exhibited, with the malesbeing more severely affected. Mortality was significantly increased in
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these males. Rats in the low-dose groups (12.5 mg/kg/day) showed nophysiological or behavioral abnormalities and little response with regardto food consumption; body weights, however, were significantly decreased.
Results of the hematology analyses indicated that LAP had an effect onboth the myelocytic and erythrocytic series of both sexes at the mid-doselevel, although clinical chemistry parameters did not reflect the degree ofkidney damage seen histologically in the mid-dose males. In the low-dosemales, only LDH was significantly decreased. In the females, numerouschanges in clinical chemistry parameters were seen, beginning at the low-dose level. Parameters significantly decreased included glucose, uricacid, K+ , triglycerides, LDH, total iron, and the A/G ratio; Na+, C02, andtotal bilirubin were significantly increased at this level compared with
controls.
Among the treatment-related changes in relative organ weights were anincrease in liver weight in all treated groups, possibly reflecting aninduction of hepatic enzymes to metabolize LAP, and an increase in kidney
weight in the treated males, reflecting kidney damage.
The principal biologically significant LAP-induced toxic lesionsobserved at histopathological examination were degenerative changes in theeye and urinary system and decreased cellularity of the lymphoid tissue.I' These changes were generally restricted to the mid- and high-dose groupsand were usually more pronounced in the males.
From the results of these studies, the ambient water quality criterionfor protection of human health from ingestion of LAP through water or con-taminated aquatic organisms was determined to be 4.26 mg/L.
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IIf QUALITY ASSURANCE UNIT
Final Report Statement
I SRI International assures the quality and integrity of this
project, which includes an acute oral LDsO study, a 14-day range
finding feeding study, and a two-year chronic study for the U.S.
Army Medical Research and Development Command researching the
I effects of the test compound, LAP wastewater, in rats. Dose levels
for the two-year chronic toxicology study were established based on
3 the results of the LD5o and 14-day studies. The two-year study was
undertaken to determine long-term effects on rats.
SA table representing the experimental phases inspected and
inspection dates immediately follow. The raw data audit was completed
on January 24, 1983 and the draft final report audit was completed on
January 26, 1983. The results of these audits were communicated to
the Study Director and SRI management on January 27, 1983.
The final report accurately describes the methods and standard
operating procedures and reflects the raw data of the study. Any
deviations from the approved protocol and standard operating procedures
were made with proper authorization and documentation. The draft
final report and final report were audited and reviewed on January 26,
and July 22, 1983, respectively. The results of the draft and final
3 report reviews were communicated to the Study Director and SRI
management on January 27 and July 22, 1983,,respectively.
Quality #ubpce Director
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Quality Assurance Unit
Final Report Statement(continued)
Q A Inspection Schedule
Date communicatedto the Study Director
Experimental Phase Date Inspected and SRI Management
Blood chemistries & Feb. 25, 1982 Mar. 1, 1982terminal necropsy
Terminal necropsy Feb. 17, 1982 Feb. 17, 1982
Cage/feeder change, Dec. 18, 1981
animal weight, clinical observations Dec. 17, 1981
I Histology Dec. 7, 1981 Dec. 9, 1981
I Cage/feeder change,animal weight, clinical observations Aug. 27, 1981 Aug. 27, 1981
Cage/feeder change, May 28, 1981
animal weight, clinical observations May 21, 1981
3 12-Month necropsy Feb. 13, 1981 Feb. 18, 1981
Cage/feeder change, Nov. 20, 1980 Nov. 20, 1980animal weight, clinical observations
Chronic feeding June 5, 1980 June 6, 1980
Initiation inspection Feb. 19, 1980 Feb. 28, 1980
LDso Jan. 9, 1980 Jan. 9, 1980KRange-finding necropsy Jan. 9, 1980 Jan. 9, 1980
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I ACKNOWLEDGMENTS
This program was conducted in the Life Sciences Division under thedirection of Dr. David C. L. Jones, Director, Toxicology Laboratory. Theexperimental work In toxicology was directed by Ted A. Jorgenson, Director,Mammalian Toxicology Department. The chemical and analytical work wasdirected by Dr. Ronald J. Spanggord, Director, Bio-Analytical ChemistryProgram. Dr. Earl F. Meierhenry, Director, Pathology Services Program, andDr. Daniel P. Sasmore, former Director of Pathology, were responsible forthe histopathological evaluation of tissues. Dr. Harold S. Javitz,Director, Data Design and Analyses Department, provided statisticalanalyses. Ophthalmic examinations were conducted by Dr. Daniel P. Sasmore,Dr. Earl F. Neierhenry, and Dr. Lewis H. Campbell, VeterinaryOphthalmologist. Carol J. Rushbrook, Toxicologist, provided scheduling,coordination, data summary and evaluation, and supervision of the MammalianToxicology Department staff; Janice M. Brown, Biologist, subsequentlysummarized and evaluated the data, supervised the technical staff, andprepared the final report. Sherry J. Hanen, Director, Quality AssuranceUnit, reviewed the study. Technical assistance and support were providedby SRI staff chemists (Rodney Keck, Daniel Combs, Michael Regalia, KarynRaab, David Burris, and Irina Kusnezov), histologists (Barbara Kirkhart andEileen Paskert), computer programmers (Larry Walters and Lorraine Martin),and biological technicians (Peter Gribling, Sandra Phillips, Juan Dulude,Kathleen Dulude, John Wharton, Robert Harding, Ernestine Seay, LoreliBrown, Steven Halperin, Mark Gilbert, Janet Cortopassi, Kristi Fitzgerald,James Thompson, Mary Mittiga, Richard Romero, Janice Schindler, Jean Yee,Claudia Bouton, and Richard Sartor).
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TABLE OF CONTENTS
ISIGNATURE PAGE ......................... ...... .............. . I
3 REPORT DOCUMENTATION PAGE ....................... ..... .... ........ 2
EXECUTIVE SUMMARY ................................................. 4
QUALITY ANC STATEMENT ........................................ 6
ACKNOWLEDGMENTS ................................................. 8
LIST OF FIGURES .............. ...... *............... 0LIST OF TABLES ... o...... o , ooo.. o - oo ol o 11
LIST OF APPENDICES ...... ..... ... .... ......... ................ 14IINTRODUCTIONoo. o....o oo oo. oooo..o.... oooo 15
MATERIALS AND METHODS ............................................. 16
Compound Identification and Purity Analyses .................... 16Animalsood ... s .. .. ......... .......... . ............... 18
So eg............ ......... oi.oo.o..o...... ... 46Housing nsan ...... o.r... ......... ... . 0... . ... 18Feed .O ...c .......a ...ti on ... o. o ............... 18Water ........................ ..... ......... 20
1Dose Preparation and Analyses ..... 21Acute Oral LD50 Study ..o........ o.oooooooo~ 2114-Day Range-Finding Studyo.... .............. 22
IChronic Study ............... Oo........... ..0 .0.....0. 26
RESULTS AND DISCUSSION ........... ........ ... ............. 28
Act rlL5 Study ..o.......... o.oo..,,,,,O, 28
14-Day Range-Finding Studyoo.. o....... ... ....... .. ... 28Chronic Study ..o... .. 0.. ............ .. . ....... 32
SDose Level Determinations ... o..... ........ ....... 32
Food Consumptiono.0...... .. . .... .. ........ 42Body Weight~o ....................... ...... o........ 46
ClnclSgsand Mortalityo oo.. .. o- ooo o 49
Ophthalmic Examnain and Histopathology ..o.... ....... 50Hematologyoo..... o... ........... ....... .. ........... 58Organ Weightoo. . ... . . .... ... .... ............ 67Histopathologyo.t.. ... .... .... .. . ... ... 0.......... 76
Water Quality Criteria ..o...o... .. ...... 0......... 160
iREFERENCES ... o... .. .... .... ... o. .. .... .... ..... 162
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LIST OF FIGURES
5 LD50 Study
1. Probit Curve for Male Rats Treated Orally with LAP........... 31
I 14-Day Range-Finding Study
2. Average Body Weights of Male Rats Treated with LAP ........... 343. Average Body Weights of Female Rats Treated with LAP ......... 34
Chronic Study
4. Variation in Food Consumption Relativeto Control Males (%) ....... 0..... ................. . ........ 44
3 5. Variation in Food Consumption Relativeto Control Females (Z) ....................................... 45
6. Variation in Body Weight Relativeto Control Males (%) .... ........... ............ .. . . .... 47
7. Variation in Body Weight Relativeto Control Females (Z) ............. .............. .... ....... 48
5 8. Mortality of Male Rats Treated with LAP ...................... 52
9. Mortality of Female Rats Treated with LAP.................... 54
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LIST OF TABLES
I LD50 Study
1. Acute Oral Toxicity of LAP Administered to Male and FemaleFischer-344 Rats .................... 0................... .... 29
2. Average Body Weights (g) of Rats Treated Orally withSLAP ................ s.............. ...e ... ........... *... 30
14-Day Range-Finding Study
3. Body Weights (g) of Male and Female Rats Treated with LAP .... 33
4. Weekly Body Weight Gain (g) of Male
and Female Rats Treated wit LAP............................. 35
5. Food Consumption (g/day) of Maleand Female Rats Treated with LAP............................. 36
6. Food Consumption (g/kg body weight/day) of Maleand Female Rats Treated with LAP ............................. 37
7. Hematology Analyses of Rats Treated with LAP ................. 38
3 8. Clinical Chemistry Analyses of Rats Treated with LAP ......... 39
9. Gross Necropsy Findings in Male Rats Treated with LAP........ 40
10. Gross Necropsy Findings in Female Rats Treated with LAP ...... 41
Chronic Study
11. Average Dose of LAP, TNT, and RDX Received by Male and
Female Rats Treated with LAP for up to 2 years ............... 43
12. Mortality of Male Rats Treated with LAP ...................... 51
1 3. Mortality of Female Rats Treated with LAP .................... 53
14. Results of Ocular Examination of Rats Treated withLAP for One Year....... ........ 6 ......................... ..... 55
15. Results of Ocular Examination of Male Rats Treated withLAP for Two Years ............................................ 56
16. Results of Ocular Examinaton of Female Rats Treated withLAP for Two Years ............................................ 57
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I3 17. Summary of Microscopic Ocular Lesions in Male Rats [Dead or
Moribund (13-24 Months) and Terminal NecropsyJ ............... 59
18. Summary of Microscopic Ocular Lesions in Female Rats [Dead
or Moribund (13-24 Months) and Terminal Necropsy] ............ 61
19. Hematology Analyses of Male and Female Rats Treatedwith LAP (1-Year Necropsy) ................................... 63
20. Clinical Chemistry Analyses of Male and Female RatsTreated with LAP (1-Year Necropsy) ........................... 64
21. Hematology Analyses of Male and Female Rats Treated5 with LAP (Terminal Necropsy)................................ 65
22. Clinical Chemistry Analyses of Male and Female RatsTreated with LAP (Terminal Necropsy) ......................... 66
23. Average Body Weights, Organ Weights, and Weight Ratiosof Male Rats Treated with LAP (1-Year Necropsy) .............. 68
24. Body Weights, Organ Weights, and Weight Ratios of MaleRats Treated with LAP (Terminal Necropsy) .................... 69
25. Average Body Weights, Organ Weights, and Weight Ratiosof Male Rats Treated with LAP (Found Dead or Moribund) ....... 70
3 26. Average Body Weights, Organ Weights, and Weight Ratios ofMale and Female Rats Treated with 200/100 mg/kg/day of LAP(Terminal Necropsy after 33 Weeks on Test) ................... 71
27. Average Body Weights, Organ Weights, and Weight Ratiosof Female Rats Treated with LAP (1-Year Necropsy) ............ 73
U 28. Average Body Weights, Organ Weights, and Weight Ratiosof Female Rats Treated with LAP (Terminal Necropsy) .......... 74
29. Average Body Weights, Organ Weights, and Weight Ratios
of Female Rats Treated with LAP (Found Dead or Moribund) ..... 75
30. Incidence of Tumors in Male Rats Treated with LAP ............ 77
31. Incidence of Tumors in Female Rats Treated with LAP .......... 79
32. Summary of Microscopic Lesions in High-Dose Male Rats(Dead or Moribund, 1-33 Weeks) .............................. 81
33. Summary of Microscopic Lesions in High-Dose Male Rats
(Terminal Necropsy, Week 34) ................................. 86
34. Summary of Microscopic Lesions in High-Dose Female Rats(Terminal Necropsy, Week 34).............. .................. 93
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35. Summary of Microscopic Lesions in Male and Female Rats(Dead or Moribund, 1-12 Months)............................. 98
36. Summary of Microscopic Lesions in Male Rats (1-YearSNecropsy) ............................................ 0........ 104
37. Summary of Microscopic Lesions in Female Rats(1-Year Necropsy) ............................................ 108
38. Summary of Microscopic Lesions in Male Rats [Dead orMoribund (13-24 Months) and Terminal Necropsy] ............... 112
39. Summary of Microscopic Lesions in Female Rats [Dead orMoribund (13-24 Months) and Terminal Necropsy] ............... 137
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I LIST OF APPENDICES
A. Average Weekly Dose Received (mg/kg/day), Percentage ofIntended Dose, and Cumulative Weekly Dose Received forMale Rats Treated vith I ..... .......... *... s.*..... ...... 166
ifB. Average Weekly Dose Received (mg/kg/day), Percentage ofIntended Dose, and Cumulative Weekly Dose Received forFemale Rats Treated with LAP ... .. .. . . . .. . . . ... . .. *. .0o . . .. 172
IC. Analyses of Diet Preparations of LAP......... ............. 178
D. Average Weekly Food Consumption (g/day) of Male RatsTraeIihLP.... ...... *00. ........ 8E. Average Weekly Food Consumption (g/day) of Female
Rats Treated with LAP. .. .. . .. .. . .. .. .. 0 .. . .. . .. .. . . . . .... 190
F. Average Weekly Food Consumption (g/kg body weight/day)of Male Rats Treated with LAP............ *o............ o..... 199
G. Average Weekly Food Consumption (g/kg body weight/day)
of Female Rats Treated with LAP............... 0.............. 208
H. Average Weekly Body Weights (g) of Male Rats Treated
it LAverage Weekly .... Body Weiht (g) of Female...... Rats Trete
Is Aveg Weekly Body Weight (g) of ale Rats Treatedwith LAP .............. ................... ... s**... o....... 235
K. Weekly Body Weight Gain (g) of FMale Rats Treatedwith LAP... ..................... 0000 ...... ........... *.... 244
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I INTRODUCTION
The overall objective of this research was to provide a comprehensivedefinition of the long-term toxicological reactions of rodents to LAP(load, assemble, and pack) wastewater, a mixture of the munitions waste-water components TNT and RDX in a ratio of 1.6:1. Specific objectives wereto identify, verify, and determine the specificity of possible lesions atthe biochemical and cellular levels and to further elucidate dose-responserelationships. These data will constitute a significant part of the over-all data base necessary to evaluate the potential hazards of these waste-waters to human health and to define the limits of relative safety.
The studies undertaken were (1) confirmatory acute oral LD5O and 14-day range-finding studies of LAP in rats; (2) evaluation of the effect ofchronic dietary administration of LAP in rats, and (3) related analyticalchemistry studies. These studies were performed in accordance with the FDAGood Laboratory Practice Regulations and the "Guide for Laboratory AnimalFacilities and Care," as promulgated by the Committee on the Guide forLaboratory Animal Resources, National Academy of Sciences-National ResearchCouncil.
Preliminary animal studies (LD50 and 14-day range-finding) were con-ducted between 12/13/79 and 1/10/80. The chronic toxicity study wasinitiated on 2/14/80; all test animals were terminated by 3/5/82. Uponcompletion of the final report, all records from this project will be per-manently transferred to SRI's Records Retention Center.
A copy of the statistical evaluation of the LD50 study, hematology,and clinical chemistry data for the 14-day study, and hematology, clinicalchemistry, and organ weight data for the chronic study are on file with theUSAMDC.
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MATERIALS AND METHODS
I Compound Identification and Purity Analyses
Sufficient quantities of 2,4,6-trinitrotoluene (TNT), Lot #VOL76K014-341, and 1,3,5-trinitrohexahydro-l,3,5-triazine (RDX), Lot #HOL-435-0039,were received from the U.S. Army at SRI's explosives magazine in Tracy, CA,in November 1979. Bulk chemicals were stored at room temperature and weretransferred to SRI in limited quantities as needed. Both TNT and RDX werecharacterized by chemical analysis as follows:
3 TNT
TNT was identified by its mass spectrum, infrared spectrum, andnuclear magnetic resonance spectrum. All spectra were in agreement withthe 2,4,6-trinitrotoluene structure.
The purity of TNT was evaluated through its elemental composition andits homogeneity was established by gas chromatography. The results of
elemental analysis are as follows:
5C H N
Calculated: 37.00 2.22 18.50
3 Found: 37.15 2.25 18.18
It should be noted that nitro compounds tend to give low nitrogenresults. The carbon and hydrogen results suggest that the TNT is 98.9 to100.4% pure.
The purity of TNT was evaluated by gas chromatography under thefollowing conditions:
Instrument: Hewlett-Packard 5711A gas chromatograph
Column: 1.8 M x 2 am Glass column packed with 10% DC-200 on100/120 mesh Gas Chrom Q
I Temperature: 100 to 200*C at 4*/min
Flow rate: 25 ml/min
Detection: Flame ionization
Retention time: 22.4 min.
One major peak eluted at 22.04 min and represented 99.92 of total peakareas. A minor peak representing 0.06% of total peak areas eluted at 18.28
min and was identified by mass spectrometry as 2,4-dinitrotoluene. Another
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I minor peak eluting at 24.53 min and representing 0.05% of total peak areaswas identified as 4-amino-2,6-dinitrotoluene.
I Based on the above data, the TNT used in this study is the 2,4,6-isomer with a purity of > 98.7%.
RDXprpRDX was identified by its mass spectrum, infrared spectrum, and
nuclear magnetic resonance spectrum. All spectra were consistent with theproposed structure for RDX.
3 The purity of RDX was estimated by elemental analysis and high-pressure liquid chromatography. The results of elemental analysis are as
follows:
IC H N
3 Calculated: 16.21 2.72 37.84
Found: 16.36 2.64 37.14
It should be noted that nitro compounds tend to give low nitrogenresults. The carbon and hydrogen results suggest that the RDX is 97.1 to100.9% pure.
The purity of RDX was evaluated by high-pressure liquid chromatographyunder the following conditions:
IInstrument: Spectra-Physics Model 3500B liquid chromatograph
5Column: C1 8 Radial Pak A (Waters Associates)
Solvent: Acetonitrile/methanol/water (10/30/60)
3 Flow rate: 2.0 ml/min
Detection: UV at 254 nm
3 Retention time: 8.32 min.
A major component eluted at 8.32 min (RDX) and a minor component eluted at5.78 min. The minor component was identified as HiNX by cochromatography
with an authentic standard. Based on the response to the UV detector, HMXwas present at 5.1% on a weight basis. Three additional evaluations ondifferent samples from the same batch of RDX showed that HMX was present at9.1, 10.5, and 7.3%. The average HMX concentration was calculated to be8.0 ± 2.3%.
We conclude from the above data that munition-grade RDX contains HNXas an impurity that is inhomogeneously distributed in the sample matrix and
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Icontributes to 5.1 to l0.5Z of the total sample weight. The elemental datado not account for such a large impurity since the elemental ratios for HMXare identical to those of RDX.
Animals
5 Source
Initially, young-adult, Fischer-344 rats of both sexes (40 to 70 g)were purchased from Simonsen Laboratories, Inc., Gilroy, CA, for both thepreliminary and chronic studies. Because the automatic watering system innew racks acquired for the chronic study was not properly flushed by theinstaller, animals for the chronic study were exposed during quarantine totoxic residues of the glues used in the assembly of the racks. Theseanimals were terminated, the problem was corrected, and a replacement ship-ment of young-adult Fischer-344 rats of both sexes in the same weight rangewere purchased for the chronic study from Charles River BreedingLaboratories, Inc., Portage, MI, because Simonsen Laboratories could notprovide a replacement shipment in time for initiation of the chronic study.
* Housing
All rats were held in an air-conditioned animal room for 5 to 7 daysof acclimation for the preliminary studies and for 2 weeks for the chronicstudy. They were housed five per cage in hanging polycarbonate shoeboxcages containing hardwood chip bedding (AbsorbDri) supplied by Gardena Seedand Feeding Corp., Vernon, CA. Animals in each cage were individuallyidentified by ear notches; an identification card on each cage was color-coded to indicate dose level.
5 Feed
Prior to arrival of the animals, Purina Certified Rodent Chow #5002and noncertified Purina Rodent Chow #5001 were analyzed for pesticide resi-dues, PCBs, aflatoxins, heavy metals, and nitrate and nitrite anions and acomparison was made.
I Feed samples were extracted according to official AOAC methods.1 Theextracts were analyzed for chlorinated organic pesticides by glass-gcapillary gas chromatography under the following conditions:
Instrument: Varian Model 3740 gas chromatograph
3 Column: SP-2100 30 M Glass capillary column
Temperature: 220*C
IFlow rate: 0.8 al/mi N2
Detection: Electron capture at 300*C
Injector port temperature: 250C
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Attenuation: 4 Range 10-12.
Analyses for organophosphate pesticides were performed by packed-
column gas chromatography under the following conditions:
5 Instrument: Hewlett-Packard Model 5730A gas chromatograph
Column: 1.8 M x 2 mm Glass column packed with 10Z DC-200 on5 Gas Chrom Q
Temperature: 220"C
3 Flow rate: 25 ml/mi N2
Detection: Alkali-flame ionization
I I"Jector port temperature: 250"C.
The results of these analyses are as follows:
RodentLaboratory CertifiedChow 5001 Rodent Chow Max. for 5002 Feed
Chemical (ppm) 5002 (ppm) as Certified (ppm)
3 Heptachlor 0.005 0.008 0.05
Aldrin 0.001 not detected 0.05
5 Heptachlor epoxide not detected 0.006 0.05
Chlordane not detected 0.021 0.05
3 DDE not detected 0.015 0.15
Dieldrin 0.001 0.001 0.05
Endrin not detected 0.001 0.05
Phorate not detected 0.004 0.5
Diazinon 0.001 0.001 0.5
Disulfoton not detected 0.003 0.5
Methyl parathion 0.002 0.004 0.5
3 Malathion 0.072 0.110 0.5
Parathion 0.003 0.013 0.5
3 Ethion not detected not detected 0.5
Aflatoxins B, B22 G1 , and G in feed were analyzed by high-pressureliquid chromatography. The results showed that no aflatoxins were present3 at concentrations greater than 1 ppb.
519
II Feed samples were analyzed for nitrate and nitrite by high-pressure
liquid chromatography using an anion exchange column and ultraviolet detec-tion at 210 nm.3 Neither anion was found at concentrations greater than100 ppb.
The noncertified diet did not contain contaminant levels greater thanthose stated or determined for the certified diet; however, the USAMRDCspecified the use of Purina Certified Rodent Chow #5002. Diet was suppliedad libitum in all studies. Subsequent information on feed content wasprovided by the analytical data sheet provided by Purina with each ship-
3 ment.
Water
U Water supplied by the San Francisco Water District was available tothe animals through an automatic watering system. Annual San FranciscoWater Department analyses of partial chemical, coliform, and inorganiccontent performed during the course of these studies were within acceptablelimits and are on file at SRI. At SRI, the nonfluorinated district wateris passed first through an activated charcoal bed and then through cationand anion exchanger columns. Each of these columns can process up to80,000 gallons of water, and they are changed approximately every 5weeks. A conductivity light that goes out if the resistance of the water
Cis less than 50,000 ohms is used to determine when the columns needreplacement. The deionized water than passes through a 7.5-4 filter andpast an ultraviolet lamp. The water in the automatic watering system(except in the "drops" and racks) is recirculated constantly past the lampand through a mixed bed column and a 5-i filter to remove any accumulatedmetals and particulates. A conductivity light that goes out when the waterresistance is less than 200,000 ohms is used to determine when this columnneeds replacement.
Prior to the start of the studies, water from the automatic wateringsystem was analyzed at SRI. On 30 November 1979, one water sample wasevaluated for pesticide residues, and another sample was cultured for bac-teria. No bacterial colonies grew from the sample. For the pesticideanalyses, one liter of water was extracted with diethyl ether, concentratedin a Kurderna-Danish apparatus, and analyzed by glass capillary gas chroma-tography using electron capture detection. None of the pesticides listedbelow were observed above the level of 0.1 ppb.
Chlorinated Pesticides Organophosphate Pesticides
lleptachlor PhorateAldrin Diazinonfleptachlor epoxide DisulfotonChlordane Methyl parathionDDE MalathionDieldrin ParathionEndrin Ethion
We were unable to get a radioactivity analysis from the supplier, butwe believe that the ion exchange treatment would remove any radionuclidesas well as stable ions.
I
U During the course of these studies, water from the automatic systemwas further analyzed annually by Stoner/McIntosh Laboratories, Santa Clara,CA, for heavy metals (detected as HiS precipitable material), oxidizableorganics, and coliforms. Results of these analyses were within acceptablelimits and are on file at SRI.
We conclude that the water supplied to the animals was free of knowncontaminants that would influence the results of these samples.
3 Dose Preparation and Analyses
For preparation of oral suspensions or diets, the RDX was firstfiltered from its water slurry and air-dried. For the oral intubationstudy, the LAP (1.6:1 TNT/RDX) was suspended in acetone and then in cornoil to achieve the necessary concentration; the acetone was removed byrotary evaporation. For the feeding studies, LAP was dissolved in acetoneand added to 3% corn oil; the acetone was again removed by rotary evapora-tion. This compound/residual acetone/corn oil mixture was thenincorporated into the finely ground rodent chow and mechanically mixed.Diets were stored at 4*C until use. Chemical stability studies showed thatmixed diets were stable for two weeks at room temperature. Accordingly,diets were mixed every two weeks. In the 14-day range-finding studies, LAPwas administered as a constant percent in the diet; analyses of LAP concen-tration in the diet were performed on diet samples from the lowest andhighest dose levels. In the chronic studies, diet concentrations wereadjusted once every two weeks throughout the study, based on examination ofbody weight and food consumption trends, to achieve a close approximationof the mg/kg/day target dose levels. Diets prepared during the first 13weeks and one mixing in each quarter thereafter were analyzed for TNT andRDX by high-pressure liquid chromatography.4 Samples were saved from eachdiet mix regardless of whether it was analyzed. The corn oil used in thesestudies was Gregg's Refined Corn Oil supplied by Fast Food Supply Co.,Inc., San Jose, CA, or Domestic Cheese Co., Inc., San Francisco, CA. Eachbatch of corn oil was assayed for peroxide levels by iodometric titration.5
3 Acute Oral LD5O Study
Ten males and ten females, weighing between 52 and 95 g, were assignedto a vehicle control and six compound treatment levels using a weight-sorting randomization procedure. All animals were fasted overnight beforereceiving a single oral dose of the test compound by gavage, prepared aspreviously described. Controls received a corn oil-acetone mixture fromwhich the acetone had been evaporated. The six treatment (LAP) groupsreceived one of the following dose levels: 150, 300, 450, 600, 750, or 900mg/kg. The animals were closely observed immediately after treatment andseveral times later that day. Daily observations for physiological andbehavioral responses and mortality were continued for 14 days after treat-ment. Body weights were recorded initially and then weekly during the 2-week observation period. Animals that died during the study werenecropsied for evidence of gross pathological changes; all survivors werenecropsied at the end of the observation period. No tissues were retained.
3 21
ILD50 values were calculated by computer-generated statistical
programs. The probit method 6 was used for the males, and the binomialmethod using linear interpolation of log doses provided a point estimate ofthe LD50 for the females. The results of these studies were used to deter-3 mine dose levels for the 14-day range-finding study.
3 14-Day Range-Finding Study
Ten male and ten female rats, weighing between 46 and 94 g, wereassigned to each of six experimental groups (vehicle control, 0.05, 0.1,0.3, 0.5, and 0.7% in the diet). For assignment to treatment groups, ratswere weight-sorted into cages, and the cages were assigned to groups usingcomputer-generated sets of random numbers. The compound was administeredthrough the diet, which was prepared as described earlier. The vehiclecontrol group received a feed/corn oil mixture prepared as described aboveexcept that the acetone contained no LAP. Body weights and food consump-tion were recorded weekly. The animals were observed daily forphysiological and behavioral responses and mortality for 14 days. Grossnecropsies were performed on rats that died during the study and on
g survivors at the end of the 2-week observation period.
Hematology and clinical chemistry determinations were conducted on allsurvivors at the termination of the study. The following analyses wereIperformed by Peninsula Medical Laboratory, Menlo Park, CA.
Erythrocyte and Leukocyte Counts (RBC, WB). A Coulter ElectronicParticle Counter with 100 g aperture was used. The instrument wasstandardized daily in a three-step process, as follows. The electronicswere first checked in a standard procedure for proper functioning. Then
the instrument was standardized for erythrocyte and leukocyte counts (aswell as hemoglobin and hematocrit) against a 4C control standard (CoulterElectronics, Inc.). Finally, two blood samples that had been kept from theprevious day and refrigerated were rerun for erythrocyte and leukocyte3 counts. Each test blood sample was counted in duplicate.
Hemoglobin (Hb). Hemoglobin was determined in the Coulter counter ascyanmethemoglobin. Cyanmethemoglobin standards were supplied by CoulterElectronics, Inc., as part of the 4C control standard. Each blood samplewas measured in duplicate.
Hematocrit (Rct). Hematocrit was calculated by the equation Hct - RBC3 (106/m) x MCV (g,)
Mean Corpuscular Volume (HCV). MCV was determined in the CoulterCounter after (daily) standardization by the Wintrobe microhematocritmethod. MCV on each test sample was determined in duplicate, and Hct wascalculated from the average according to the above equation.
Mean Corpuscular Hemoglobin (HCH). MCH was calculated as follows:
MCH Hgb (gn %) x 10
RBC (106/mm3)
322
I
Mean Corpuscular Hemoglobin Concentration (MCHC). MCHC was calculatedas follows:
MCHC (gin Z) - Hgb (gi %) x 100
Differential Leukocyte Counts. Leukocytes were stained with Wright'sstain for examinatioi and counting under the microscope. Cell typesidentified and counted were polymorphonuclear cells, band cells,lymphocytes, monocytes, eosinophils, and/or basophils.
3 Clinical chemistry tests, representing a SMAC-20 profile as describedin the Technicon manual (Technical Publication No. UA3-0306B3, March 1976),were performed using the Technicon SMAC high-speed, computer-controlledbiochemical analyzer (Technicon Instruments Corp., Tarrytown, New York).Standardization for each test was made on every forty-eighth tube (sixthrack), using the Technicon SMAC References I and II (FDA-approved) and theprocedures outlined in the bulletins accompanying these references (Nos.4060-A-R8-6/Rll-7-2 and 4060 B-R4-7/Rll-7-2, Technicon Instruments Corp.).
Glucose (mg 2). Blood glucose levels were determined in sera fromfasted animals by a modification of the procedures of Gochman andSchmitz.9 The method basically involves oxidation of glucose with glucose
oxidase to produce H202, which then reacts with 3-methyl-2-benzothiazolinone hydrazone and dimethylaniline indicators to produce anintensely colored indamine dye for determination in the colorimeter at370C.
BUN (mg %). The Technicon method used was a modification of theprocedure described by Marsh et al. 1 0 This method entails the hydrolysisof diacetyl monoxime to diacetyl in relatively weak acid solutions and itssubsequent reaction with urea in the presence of ferric ions and thiosemi-carbazide to intensify the color (analysis at a wavelength of 520 nm).
Creatinne (mg %). Creatinine was analned by an automatedadaptation" of the original method of Jaffe in which the creatinine isallowed to react with saturated picric acid in alkaline solution at 37"C.3 Analysis in the colorimeter was performed at 505 rn.
Uric Acid (mg 2). Uric acid was determined by the method of Sobrinho-Simoes, as modified by Musser and Ortigoza. The method is based on thereduction of a phosphotungstate complex to a phosphotungstite complex withaddition of hydroxylamine to intensify the color (observations were made at660 nm).
Sodium (meg/L). The sodium ion content of sera was determinedpotentiometrically, using a sodium ion-selective glass electrode.
15
Potassium (meq/L4. Potassium was determined with a potassium ion-selective electrode.
Carbon Dioxide (meg/L). The method for determining carbon dioxide wasbased on the automated procedure of Skeggs and Hochstrassser.1 ? Carbon
3 23
I
I dioxide, released first by acid, was determined from the decrease in thered color of an alkaline phenolphthalein solution (at 550 nM).
U Chloride (mg/L). Chloride was determined colorimetrically using theautomated method of Morgenstern et al.1 8 In this method, Hg (SCN)2 reactswith chloride ions in the presence of ferric ions to produce red Fe (SCN)3(observed at 480 nm).
Calcium (mg %). Calcium was determined compleximetrically using analkallne solution of 8-hydroxyquinoline.1 9 The complex produces a pinkcolor with a maximum absorption at 570 nm.
Phosphorus (mg %). Inorganic phosphorus wag determined by thephosphosolybdate method of Daly and Ertinghausen"0 as modified for theautomatic analyzer by Amador and Urban.21 The unreduced phosphomolybdate
complex absorbs at 340 na, and the amount of phosphorus present can bedetermined by difference.
Balance (Na-[Cl + C02fl. Electrolyte balance is the numericalI difference of Na' concentration and the sum of the concentrations of C1-
and of dissolved C02.
3 m Cholesterol (m Z).2 Cholesterol was determined by the automatedmetho~d of Levine et a. 2 In this method (based originally on that of
Huang et al. 2 3 ), cholesterol and sulfuric acid react to form bi-cholestadienyl disulfonic acid, a green compound measured at 630 nm in the
* colorimeter.
Triglycerides (mg %). Analysis of serum triglycerides involves theenzymatic hydrolysis of the compound to glycerol anC free fatty acids.24 Asolution of glycerol kinase and pyruvate kinase in a second channelconverts glycerol to pyruvate, which in turn is reduced by NADH and lacticacid dehydrogenase to lactate (followed at 340 nm).
Total Bilirubin (mg 2). Determination of total bilirubin s sera,like triglycerides, involves a two-channel system for analysis." The
* bilirubin was reacted with a caffeine-containing diluent that formsazobilirubin. This solution was then mixed with a strongly alkaline sodiumpotassium tartate buffer and sulfanilic acid to yield a green complex,which can be quantified at 600 nm against a blank channel containing allreagents except for the diazo compound.
SCOT (=U/ml). Serum glutamic-oxaloacetic transaminase (SOT) activitywas measured by ollowing the rate of change of NADH absorption at 340 nm
and 37"C produced by maleate dehydrogenase. The latter enzyme system wascoupled with GOT-catalyzed transamination of aspartic acid and a-keto-3 glutarate in the medium.2 6
SGPT (mU/ml). Serum glutamic-pyruvic transaminase (SOFT) activity wasmonitored in the same manner as SOT, except that alanine was substitutedfor aspartic acid and the coupling enzyme was lactate dehydrogenase.
2 6
*24
i
U LDH (mU/ml). Lactate dehydrogenase (LDH) activity was determineddirectly by monitoring the rate of change in absorption at 340 nm in the3 presence of added L-lactic acid and NAD.
18
Alkaline Phosphatase (mU/ml). The Technicon method for determinationof alkaline phosphatase involves the hydrolysis of stock -nitrophenylphosphate solutions by the enzyme in the presence of Mg2 to produce abright-yellow £-nitrophenol product (monitored at 410 nm and 37*C) at pH10.25.2
I Total Iron (mcg %). Serum iron was determined by reacting 3-(2-pyridyl)-5,6-bis-(4-phenylsulfonic acid)-l,2,4-triazine (trademarked asFerroZine) in the presence of ascorbic acid to liberate transferrin-boundiron.28 The FerroZine complex in a sodium acetate medium was measuredcolorimetrically at 560 nm.
Total Protein (g Z). The method for determination of total proteinwas based on the biuret method, automated for use with the Techniconanalyzer.
1 6
I Albumin (g %). The Technicon method utilizes the reactivity ofalbumin with bromcresol green (BCG) to form an albumin-BCG complex that canbe quantified colorimetrically at 630 nm.
2 9
Globulin (g %). Globulin is the difference between total protein andalbumin determinations.
A/G Ratio. The albumin-to-globulin (A/G) ratio was calculated indivi-dually for each animal sample, and the ratios were averaged for each groupby a computer program.
The following tissues were taken from all rats and preserved forpossible histological examination:
Brain (3 sections) Thyroids/parathyroids Spinal cord (if neuro-Heart Mesenteric lymph nodes logical signs were present)Liver Thymus (if present) SkinKidneys Lung and mainstream Colon (sigmoid)Spleen bronchi PancreasGonads Diaphragm Cervical lymph nodeAdrenals Esophagus Urinary bladderPituitary Stomach (cardiac and UterusEyes (if grossly pyloric) Prostate
abnormal) Small intestine SternumTrachea (duodenum, ileum, Abnormal tissue
jejunum) Mandibular lymph nodeCaecum Salivary glandMammary gland
I
I
Histopathological examinations were conducted on the testes of rats inboth the control and 0.5% treatment level.
U Body weight and hematology data were statistically analyzed by (1)Williams' test, 30,31 (2) Finney's ratio test,6 (3) a linear trend test,3 2
and (4) Bartlett's chi-square test 33 . Food consumption data wereI statistically analyzed by analysis of variance (ANOVA) and the t-test.34
The results of these studies were used to determine dose levels forthe chronic study.
* Chronic Study
Three dose levels were selected for the chronic study: 12.5, 50.0,and 200 mg/kg/day. Each treatment group consisted of 70 male and 70 femalerats. A vehicle control group contained 75 rats of each sex. For assign-ment to treatment groups, rats were weight-sorted into cages that wereassigned to groups using computer-generated sets of random numbers. Whenplaced on test, the male rats weighed between 95 and 142 g, and the femalesweighed 73 to 110 g. All animals were either 6 or 7 weeks of age. Indivi-dual body weights and food consumption by cage were recorded weekly. Atthis time, cages and racks were rotated within the animal room. Roomtemperature was recorded daily. The eyes of each animal were dilated andexamined initially, at I year, and again at the end of the 2-year exposureperiod. Only rats with normal eyes at the initial examination were3 randomized into test groups.
The rats were observed daily for physiological and behavioralresponses and mortality throughout the 24-month duration of the study.Starting in the ninth month of the study, the rats were palpated duringroutine weekly handling for evidence of internal masses. Moribund animalsand any animals discovered dead were necropsied and submitted for histo-pathological examination as described later in this report.
Because of signs of excessive toxicity in the male rats receiving 200mg/kg/day, the high-dose level was lowered to 100 mg/kg/day after 12 weekson test. Mortality continued to occur, and after 33 weeks on test, allremaining male and female rats at this level were necropsied. Organweights were taken and histopathology was performed on these animals; hema-tology and clinical chemistry evaluations were not conducted.
After 12 months on test, 10 males and 10 females from each treatmentgroup (including the vehicle control) were necropsied and examined forpathological changes. Animals for this interim evaluation were taken fromthe highest numbered cages per group, after a statistical assessment(analysis of variance) of body weights showed no rack- or cage-relatedeffects. After 24 months on test, all remaining animals were necropsiedover a 2-week period. Hematology and clinical chemistry evaluations, asdescribed for the 14-day range-finding study, were performed on all rats atthe interim necropsy (12 months) and on 20 rats per sex at the terminalnecropsy. A reticulocyte count was also included.
26
U
I At necropsy, performed on all rats, the tissues identified in the 14-day range-finding study were preserved. A section of the thoracic/lumbarspinal cord and vertebrae and the rectum were also taken from animals atthe terminal necropsy. Absolute organ weights of the brain, heart, liver,kidneys, spleen, and testes were recorded. Histopathological examinationwas performed on all preserved tissues.
The body and organ weight, hematology, and food consumption data wereanalyzed as described for the 14-day range-finding study.
* Lesions observed in the low- and mid-dose animals surviving more thanone year on study, and tumors with an incidence of 5% or greater in any
group, regardless of time of death, were statistically analyzed by Fisher'sexact probability test.
I:2
U
UII
II
5 7
UI3 RESULTS AND DISCUSSION
Acute Oral LD5O Study
Table 1 summarizes the acute oral toxicity of LAP in rats. The
majority of deaths were preceded by decreased activity, salivation, andlacrimation; convulsions and dyspnea were also frequently noted. Other
clinical signs or conditions noted in some animals prior to death includedprostration, humped back, cyanosis, hyperactivity, ataxia, nasal exudate,chromodacryorrhea, and opisthotonos. All rats receiving LAP had red urine
approximately 1 hour after treatment. In addition to the red urine, thesurvivors also had transient signs of decreased activity, salivation, rough
fur, and a humped appearance. Average body weights are presented in Table2; all survivors from the treated groups gained weight throughout the 2-
week observation perod. Necropsy of the animals that died revealed nogross abnormalities. Emphysema, atelectasis, petechial hemorrhage, and/or
congestion of the lungs were noted in a few of the rats surviving treat-ment.
One male in the vehicle control group died 12 days after treatment.Beginning 7 days after treatment, this animal had a humped appearance andwas emaciated; a bloody nasal exudate was also noted. Necropsy revealedmarked emphysema and moderate atelectasis and congestion of the lungs. All
other control rats appeared normal and gained weight throughout the 2-weekobservation period. An incidence of respiratory disease similar to that inthe treated animals was noted at necropsy of the surviving control rats.
IThe acute oral LD50 of LAP in male Fischer-344 rats was calculated bythe probit method to be 294 mg/kg, with a 95% confidence interval of 200 to367 mg/kg (Figure 1). Since none of the doses given to the female groupsresulted in a mortality rate of 35% or less, the binomial method of linearinterpolation was the most appropriate method of statistical analysis.Based on this method, a point estimate of the acute oral LD50 for female
rats was calculated to be 325 mg/kg, with confidence limits of negativeinfinity to 450 mg/kg.
3 14-:.y Range-Finding Study
Red urine was observed in all rats receiving LAP in the diet. Ratsreceiving the two higher dietary levels (0.5 and 0.7%) showed clinicalUi signs of toxicity including rough fur, a humped appearance, and a decreasein activity. Three males receiving 0.5% of LAP in the diet died during the14-day study. Of the ten male and ten female rats receiving 0.7%, only one
male survived for the duration of the study; one male and two females werenecropsied early when found moribund, and the remainder were found dead.Rats receiving either the vehicle control or one of the three lower dose
levels of LAP (0.05, 0.1, and 0.3%) exhibited no clinical abnormalitiesduring the study.
28
3 Table 1
ACUTE ORAL TOXICITY OF LAP ADMINISTEREDTO MALE AND FEMALE FISCHER-344 RATS
3 LD5O Study
3 Dose Level No. Dead/ Time LD50Sex (mg/kg) No. Treated to Death (mg/kg)
3 Male 0 1/10 12 days
150 2/10 1-4 hrs
300 5/10 5-29 hrs 294
450 8/10 2-21 hrs (200-367)
600 10/10 1-21 hrs
750 10/10 2-50 hrs
900 10/10 1-4 hrs
Female 0 0/10 -
3 150 4/10 2-6 hrs
300 4/10 3-21 brs 325
450 9/10 1-28 hrs (neg. infinity-450)
600 10/10 3-27 hra
750 10/10 3-21 brs
900 10/10 1-4 hrs
I*95% Confidence limits in parentheses.I
3 29
| kf
AMA Q
Ui
I3 Table 2
AVERAGE BODY WEIGHTS (g) OF RATS TREATED ORALLY WITH LAP
3 LD5O Study
DoseSex (mg/kg) Day 0 Day 7 Day 14 Gain
Male 0 74.1 107.0 142.2 (9)* 68.1
150 71.4 103.4 (8) 137.3 (8) 65.9
300 74.1 100.0 (5) 137.6 (5) 63.5
450 73.7 106.5 (2) 139.5 (2) 65.8
600 71.1 ....
750 74.3
900 75.8 - w
Female 0 69.6 94.4 114.2 44.6
150 66.6 92.0 (6) 113.8 (6) 47.2
300 66.5 93.8 (6) 113.2 (6) 46.7
450 68.4 90.0 (1) 111.0 (1) 42.6
600 68.7 --..
750 69.1
900 69.5 --
*Number of survivors in parentheses.
wI
I
U1 3
UI
*3S*3* PROBIT METHOD as=aasLINEAR DOSE PMDEL
..............................................-
: LOWER LDSO UPPER: 95 PCT LIMIT ESTIMATE 95 PCT LIMIT
-----------------------------------------------200.1 294.0 367.3
. -----------------------------------------------
-~-----------4----------------+---------------100 + - -*+
90 + +
U80 + +
70 + +
60 + +
50 + * +
40 + +
30 + +
20 + +
U 104 . :0 +.+
0 - -
--4-------------.+----------. ---------------------LDOI LniO LD5 L050 LD75 LD90 Lnq9
-63.60 96.89 293.97 491.04 ,51!133 190.20 397.73
!FIGURE I PROBIT CURVE FOR MALE RATS TREATED ORALLY
WITH LAP--LD50 STUDY
3 31
I
I Table 3 sumarizes the body weight data during the 2 weeks of treat-ment. These data are illustrated in Figures 2 and 3 for males and females,respectively. After 1 week on test, the males weighed significantly lessthan the controls, beginning at the 0.1% level; females weighed signifi-cantly less compared with controls, beginning at the 0.3% level. Weeklybody weight gains after 1 and 2 weeks are presented in Table 4 in whichsignificant recoveries toward control values from Week 1 to Week 2 in bothsexes are shown.
Closely correlated with the body weight losses were decreases in foodconsumption in rats of both sexes. Table 5 presents the food consumptiondata based on grams of food consumed per day; Table 6 presents foodconsumption data based on grams of food consumed per day per kilogram ofbody weight. The results show a definite dose-related response during Week1, with indications of recovery toward control values during Week 2.
Results of the hematology and clinical chemistry analyses are pre-sented in Tables 7 and 8, respectively. In the males, hematologyparameters reflected high toxicity at the 0.5% level. Some parameters weresignificantly affected at all dose levels. At comparable levels, mostparameters appeared slightly less affected in the females. Again, someparameters were affected at all dose levels. Numerous clinical chemistry
values were significantly affected beginning at the 0.3% level for bothsexes. In the males, only triglycerides, LDH, and alkaline phosphatasewere signficantly affected (decreased) at the lowest dose level (0.05%).In the females, the lowest dose animals were not affected, and only thetriglycerides were significantly decreased at the 0.1% level.
Summaries of findings from the terminal necropsies of the males andfemales are presented in Tables 9 and 10, respectively. The testes of allseven male survivors receiving 0.05% of LAP in the diet and of the onesurviving male at the 0.7% level were smaller compared with controls. Theprostate and seminal vesicles of many of these rats also appearedsmaller. For a more specific evaluation of this finding, the testes fromcontrol rats and from rats at the 0.5% level were examined histo-logically. Evidence of immaturity and delayed development was seenhistologically, correlating with the 46% reduction in body weights of malerats after 2 weeks of treatment at this dose level, relative to controlmales (Figure 2). Degenerative or inflammatory changes were not detected.
3 Chronic Study
Dose Level Determinations
I Based on the data collected from the 14-day range-finding study,dietary levels equivalent to 12.5, 50, and 200 mg/kg/day were selected forthe chronic study. These dose levels meet the basic criteria of the EPAdose selection guidelines for both oncogenic and nononcogenic chroniceffects test standards. They allow for a dose-response analysis, with thehigh-dose level (HDL) resulting in toxicity, the intermediate dose beingone-half to one-quarter of the high dose, and the low dose being a "noobserved effect level" (no more than one-half of the intermediate dose).35
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Table 11 presents the average doses of LAP, TNT, and RDX receivedin the chronic study.
Weekly data on dose utilization and cumulative average doses of LAPreceived weekly for male and female rats are presented in Appendices A andB. Because of excessive toxicity in the males receiving 200 mg/kg/day, thehigh-dose level was reduced from 200 to 100 mg/kg/day for both sexes,beginning at Week 13. Excessive toxicity and mortality continued to occur,and all surviving high-dose rats were terminated after 33 weeks on test.Assuming that 100% of the intended compound was available in the diet, theaverage doses received over the 33-week study were 135.90 and 135.71mg/kg/day for the high-dose males and females, respectively. The weeklydoses received ranged from 86.77 to 232.58 mg/kg/day for the males, and
97.27 to 233.33 mg/kg/day for the females.
After 104 weeks on test, the average weekly doses received were within2% of the target doses of 12.5 and 50 mg/kg/day for the low- and mid-doselevels. The values for these groups were 12.40 and 50.11 mg/kg/day formales and 12.32 and 49.83 for females, respectively. Weekly doses forthese groups ranged from 79.5 to 110.2% and 78.7 to 115.1% of the intendeddose for males and 69.9 to 109.8% and 70.4 to 110.2% for females. In allcases, the lowest values in the ranges occurred during the second week ofthe study before dose levels were adjusted with regard to body weight andfood consumption trends. The intended LAP concentrations (in ppm) are
presented in Appendix C, along with the required analyses of the dietpreparations. The last analyses were performed on the diets prepared forWeeks 95 and 96; five additional diets were prepared during the finalquarter of the study.
Food Consumption
The average weekly food consumption values (g/day) for male and femalerats are presented in Appendices D and E, respectively. These data aregraphically presented in Figures 4 and 5 for male and female rats, respec-tively, based on percent change in food consumption relative to controls.Males in the mid- and high-dose groups showed a consistent, generallysignificantly, low food consumption compared with the controls, beginningin the first week on test. With the lower LAP content in the diet,beginning with Week 13, food consumption in the high-dose males began to
increase, but it remained significantly lower than controls through Week16. Beginning at Week 17, food consumption of the high-dose males exceededthat of the controls. This trend continued through Week 33 (significantlyso during Weeks 28 through 31), when all high-dose males were terminated.Food consumption for the mid-dose males also began to increase andoccasionally exceeded control values beginning at Week 24. A statisticallysignificant increase in food consumption was seen in the mid-dose malesduring Weeks 39 through 45; this trend continued throughout the study andwas significant at times. Food consumption in the low-dose males wasgenerally lower than in controls throughout the study; this trend wassignificant only during Week 15.
Beginning with the first week on test, high-dose females consumedsignificantly less than controls through Week 10. From Week 13, when thedose level was reduced, food consumption in the high-dose females began toexceed that of the controls; from Week 23 and continuing until the groupwas terminated (Week 34), this increase was often significant. Foodconsumption was significantly low in the mid-dose females from Weeks 4
42
I
Table 11
AVERAGE DOSE OF LAP, TNT, AND RDX RECEIVED BYMALE AND FEMALE RATS TREATED WITH LAP FOR UP TO 2 YEARS
Treatment Level LAP* TNT RDX(mg/kg/day) (mg/kg/day) (mg/kg/day) (mg/kg/day)
Males
12.5 12.40 7.63 4.77(9.94-13.77)** (6.12-8.47) (3.82-5.30)
50 50.11 30.84 19.27(39.33-57.53) (24.20-35.40) (15.13-22.13)
200/lOOt 135.90 83.63 52.27(86.77-232.58) (53.40-143.13) (33.37-89.45)
Females
12.5 12.32 7.58 4.74(8.74-13.72) (5.38-8.44) (3.36-5.28)
50 49.83 30.66 19.17(35.19-55.10) (21.66-33.91) (13.53-21.19)
200/100t 135.71 83.51 52.20(97.27-233.33) (59.86-143.59) (37.41-89.74)
I
S* Values given assume that lOOX of the compound was available in the diet;
based on a TNT/RDX ratio of 1.6 to 1.
I ** Parentheses indicate the range of dose received.
t The 200-mg/kg/day dose was reduced to 100 mg/kg/day after 12 weeks;all animals at this dose level were terminated after 33 weeks on test.
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Ithrough 10. Throughout the study, the mid-dose females occasionallyconsumed more than the controls, significantly so only during Week 102.While food consumption of the low-dose females was generally lower thanthat of the controls throughout the study, this difference was rarely3 significant.
Food consumption in terms of grams of food consumed per kilogram ofbody weight are presented in Appendices F and G for males and females,respectively. After I week of acclimation to the diet, food consumption inthe high-dose males increased compared with controls, even when foodconsumed (g/day) was significantly low (Weeks 1-16). From Week 18, foodconsumption based on body weight in the mid-dose males exceeded controlvalues; during Weeks 26 through 104, this difference was oftensignificant. Although food consumption per body weight in the low-dosemales was usually less than in controls throughout the study, thedifferences were not significant.
Food consumption based on body weight for the females showed no con-sistent trends during the first 33 weeks of the study. Females at allthree dose levels occasionally showed a statistically significant
difference from control values; however, these differences were not con-sistently in the same direction. At approximately the same time that thehigh-dose females were sacrificed (Week 34), food consumption based on bodyweight for the mid-dose females began to increase over that of controls,usually to a significant degree throughout the rest of the study.Beginning with Week 40, an increase was also seen in the low-dose group,although a significant difference was much less frequent.
3 Body Weights
The average weekly body weights for males and females are presented inIAppendices H and I, respectively; these data are illustrated in Figures 6and 7 in terms of percent change in body weight relative to controls.Because the rats were weight-sorted into cages, and cages were randomizedinto test groups, there is no explanation for the somewhat low initialaverage body weights for high-dose males compared with controls. After 1week on test, body weights for the mid- and low-dose groups were also
decreased corpared with controls; in the mid-dose males these differenceswere significant. A consistent, statistically significant decrease wasalso seen in the low-dose males beginning at Week 13. A dose-relatedresponse was seen in the body weights for all three dose levels; however,during the last few weeks before the high-dose groups were terminated, theaverage body weight of the high-dose males began to increase at a faster
rate than that of the mid-dose group, exceeding it during Weeks 32 and33. This increased average weight was due in part to the death of thelighter rats in the high-dose group during this period.
In the females, average body weights showed a dose-related decreasethat was significant beginning at Weeks 1, 2, and 5 for the high-, mid-,and low-dose groups, respectively. When the high-dose level was reducedfrom 200 to 100 mg/kg/day (Week 13), the rats immediately began to gainweight, exceeding first the mid-dose and then the low-dose values. FromWeek 23 until their termination after 33 weeks on test, the high-dose
females weighed significantly more than controls. The low- and mid-dose
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females continued to weigh significantly less than controls throughout thestudy.
3 Weekly body weight gains for males and females are presented in Appen-dices J and K, respectively. A dramatic decrease in weight gain in thehigh-dose animals compared with controls was evident through Week 12. Whenthe dose level was reduced from 200 to 100 mg/kg/day, a statisticallysignificant increase in weight gain was seen in these animals compared withcontrols during Weeks 13 through 15. For the low- and mid-dose males, themain differences in body weight gain compared with controls occurred duringthe first few months of the study when growth in the control animals was ata maximum and acclimation of the treated groups to the diet greatly reducedtheir body weight gain. After the first few months, the low-dose animalsbegan to gain weight consistently with the controls; statistically signifi-cant differences in both directions were seen occasionally. The mid-doseanimals continued to gain less weight than the controls, significantly soat times.
In the females, the initial body weight loss in the high-dose groupduring the first week of the study was dramatic. Beginning with Week 6,body weight gain began to exceed that of controls, often significantlyso. The decrease in body weight gain in the low- and mid-dose groupsoccurred mainly during the first few months of acclimation. Weight gainfluctuated at all dose levels, and statistically significant changes
occurred in both directions.
3 Clinical Signs and Mortality
Beginning 1 day after the study was initiated and continuingthroughout the 104 weeks of the test, the urine of all rats receiving LAPhad a reddish coloration. The intensity of the coloration increased withincreasing dose. By Week 7, the high-dose males receiving 200 mg/kg/daybegan to show a low incidence of bloody exudate from the eyes and nose. Bythe ninth week of the study, this toxic response had occurred in approxi-mately 10% of males and females in the high-dose group. At this time,convulsions and heavy salivation lasting 5 to 15 seconds were observed in afew of these rats. Although both sexes were affected, convulsions occurredmore frequently in the males. By the eleventh week of the study, the con-vulsions were more frequent; approximately 20% of the high-dose males and10% of the high-dose females had a bloody exudate from the eyes and nose.These males were noted to be aggressive; approximately 10% had abrasions of
the face and body, possibly the results of fighting and of impact with thecage and feeders during convulsions. High-dose females appeared to be moreexcitable than controls or rats at the other levels. By Week 12, sevenmales at the high-dose level had died or were observed to be moribund andwere necropsied, and the high dose was reduced to 100 mg/kg/day for bothsexes.
At 100 mg/kg/day, both males and females continued to have convulsionsand exhibit aggressive behavior. The incidence of males with abrasionsalso continued to increase. Thirty percent of the males were affected inthis manner by Week 15, and 80% had abrasions by Week 30.
49
ITables 12 and 13 present the mortality data for males and females,
respectively; these data are illustrated in Figures 8 and 9. Of the high-dose males, 50% had died after 26 weeks on test. Because of the excessivetoxicity and mortality in the high-dose males, all surviving rats at thisdose level were necropsied after 33 weeks. Of the initial 70 rats per sex
fin the high-dose groups, 19 males and 69 females survived to this termina-tion date.
During Week 29, one male from the mid-dose group was observed to havea convulsion. By Week 30, approximately 30% of the mid-dose males hadabrasions on their faces and bodies and were considered to be more aggres-sive than the controls; the females appeared more excitable than controlswhen handled. Beginning Week 41, the females in the mid-dose group beganto exhibit a low incidence of convulsions. The mid-dose males continued toshow abrasions, convulsions, and aggressive behavior throughout the
Astudy. An increased mortality rate in the mid-dose males became apparentearly in the second year of the study (Table 12, Figure 8). Only 11 of theoriginal males at this level survived the 104 weeks of the study. FromWeek 89, approximately 70% of the mid-dose males appeared slightly humpedand depressed. Mortality in the mid-dose females was not increasedcompared with the controls; 78% of the rats in this group survived to studytermination (Table 13, Figure 9).
Male and female rats at the low-dose level showed no evidence of theconvulsions or increased aggression noted in the mid- and high-dose groups,and mortality rates were comparable to controls. Seventy-two and 80% ofthe control and low-dose males and 75 and 68% of the control and low-dosefemales, respectively, survived the 104 weeks of study.
Ophthalmic Examination and Histopathology
Initially, the eyes of all rats used in this study were determined tobe normal. Table 14 presents a summary of ocular lesions found after 1year on test. Low incidences of serous or reddish discharge; corneal,lenticular, or lens opacity; and hyperemic scleral or decreased retinalvessels were observed. It is likely that the reddish ocular discharge(chromodacryorrhea), noted more frequently in the females, representschanges in the lacrimal glands rather in the globe itself. These "redtears" may be caused by a variety of factors in rats. Because of theaggressive and excitable behavior of the mid-dose rats, these animals wereheld more tightly for examination. The effects of physical restraint ofthe rats during the eye examination is difficult to determine; however,blood flow to the head may have been reduced in the mid-dose rats, therebyaffecting the diameter of the retinal vessels. The incidence of all ocularlesions found was too low to determine any dose response.
Tables 15 and 16 present a summary of the results of ocular exami-nations of male and female rats, respectively, immediately before terminalsacrifice. Corneal dystrophy, anterior Y sutural vacuoles, posteriorcortical vacuoles, and conjunctivitis are spontaneous changes that occur inaging rats. Posterior cortical opacity (focal) is a congenital defectassociated with the hyaloid membrane.
50
II
Table 123 MORTALITY OF MALE RATS TREATED WITH LAP
Control 12.5 mg/kg/day 50 ag/kg/day 200/100 mg/kg/day*3 Weeks on Study (65)** (60) (60) (60)
0-4 0 0 0 0
5-8 0 0 0 1
9-12 0 0 0 6
13-16 0 0 0 2
- 17-20 0 0 0 9
21-24 0 0 0 9
25-28 0 0 0 16
I 29-32 0 0 0 6
33-36 0 0 1 25 37-40 0 0 4
41-44 0 0 1
45-48 1 0 4
49-52 0 0 2
53-56 0 0 2
i 57-60 0 0 4
61-64 1 0 2
65-68 2 0 0
69-72 1 0 3
73-76 0 0 7
6 77-80 0 1 4
81-84 1 1 1
85-88 0 1 3
89-92 3 0 293-96 2 1 0
3 97-100 3 2 3
101-104 4 5 3
Total Mortality 18 11 46 51I (27.7%) (18.3%) (76.7%) (72.9%)
* Terminated after 33 weeks on test.
** Number of rats in each group (excluding those scheduled for the 12-monthinterim evaluation).
3 51
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I
U Table 13
g MORTALITY OF FEMALE RATS TREATED WITH LAP
Control 12.5 mg/kg/day 50 mg/kg/day 200/100 mg/kg/day*Weeks on Study (64)** (60) (60) (60)
0-4 0 0 0 0
5-8 0 0 0 0
9-12 0 0 0 1
13-16 0 0 0 0
17-20 0 0 0 0
21-24 0 0 0 0
25-28 0 0 0 0
29-32 0 0 0 0
33-36 0 0 0 0
37-40 0 0 0
41-44 0 0 0
45-48 0 1 0
49-52 0 0 0
53-56 1 0 0
57-60 0 0 0
61-64 0 0 0
65-68 0 1 0
69-72 0 0 2
73-76 2 0 0
77-80 1 1 0
81-84 0 2 2
85-88 1 1 0
89-92 0 0 4
93-96 2 7 1
97-100 4 3 1
101-104 4 2 3
Total Mortality 15 18 13 1(23.4%) (30.0%) (21.7%) (1.42)
* Terminated after 33 weeks on test.
•* Number of rats in each group (excluding those scheduled for the 12-monthinterim evaluation). One female was also excluded when discovered to bepregnant.
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Table 14
RESULTS OF OCULAR EXAMINATION OF RATSTREATED WITH LAP FOR ONE YEAR
Number of Rats Observed with Ocular LesionsControl 12.5 mg/kg/day 50 mg/kg/day
Finding Right Left Right Left Right Left
Males: (64)* (60) (47)
Reddish ocular discharge 0 0 0 0 1 1
Lenticular opacity 0 0 0 0 0 2
Hyperemic scleral vessels 0 0 0 0 1 0
Retinal vessels decreasedin diameter 0 0 1 0 2 1
Females: (64) (59) (60)
Serous ocular discharge 2 1 0 0 0 0
Reddish ocular discharge 2 5 1 3 1 1
Corneal opacity 0 2 0 0 0 0
Lenticular opacity 1 0 1 1 1 1
Lens opacity 0 0 0 1 0 0
Retinal vessels decreasedin diameter 0 0 0 0 2 0
*Number of rats examined.
55
I
I Table 15
RESULTS OF OCULAR EXAMINATION OF MALE RATSTREATED WITH LAP FOR TWO YEARS
Number of Rats Observed with Ocular LesionsControl 12.5 mg/kg/day 50 mgfkg/day
Finding Right Left Right Left Right Left(47) * - (49) (13T-
Normal 2 10 4 3 1 0
Corneal dystrophy 35 28 37 41 6 9
Anterior Y sutural vacuoles 32 22 23 33 0 2
Posterior cortical vacuoles 12 6 8 5 1 1
Conjunctivitis 2 1 3 1 1 1
Anterior cortical opacity 1 1 1 0 1 1
Posterior cortical opacity 4 3 3 1 0 2
Anterior and posteriorcortical opacity 1 2 1 1 4 4
Deep anterior chamber 0 1 0 0 3 2
Lens resolution or lensluxation 0 1 0 0 2 1
Complete cataract 1 1 2 0 2 1
Retina blurred 1 1 0 0 0 0
Retina (fundus) blurred 0 0 0 0 1 1
I Fundus, cannot visualize 2 2 2 0 5 2
Hyphemia1 1 0 1 0 0 0
Phthisis2 0 0 0 0 1 0
I Lid defect3 1 0 0 0 1 2
Keratits 4 0 0 0 0 0 2
* Number of rats examined.Anterior chamber filled with blood.
2 Small, missing or dead globe
3V-shaped defect in lid margin.4Vascular proliferation into the cornea.
56
U Table 16
RESULTS OF OCULAR EXAMINATION OF FEMALE RATSTREATED WITH LAP FOR TWO YEARS
Number of Rats Observed with Ocular LesionsControl 12.5 mg/kg/day 50 mg/kg/day
Finding Right Left Right Left Right Left(49) (42) (477
U Normal 4 6 3 4 0 0
Corneal dystrophy 37 36 30 34 18 173 Anterior Y sutural vacuoles 15 9 17 14 5 5
Posterior cortical vacuoles 6 3 7 4 0 0
Conjunctivitis 7 3 3 2 5 2
Anterfr-': cortical opacity 1 2 1 1 4 3
Posterior cortical opacity 6 6 4 4 5 5
Anterior and posteriorcortical opacity 6 5 6 4 10 8
Deep anterior chamber 1 0 0 0 26 26
Lens resolution or lensluxation 1 0 0 0 17 18
Complete cataract 2 1 0 1 9 9
Retina blurred 2 2 0 2 2 1
Retina (fundus) blurred 1 1 2 0 1 3
Fundus, cannot visualize 1 1 0 0 14 11
Complete corneal opacity 0 0 0 0 1 1
Extensive posterior corticalopacity 0 2 0 0 0 0
Hyphemia1 0 0 0 0 0 2
Phthisis 2 0 0 0 0 0 1
Lid defect3 0 0 1 0 0 0
Keratitis 4 0 0 0 0 2 1
Sublux medial 5 0 0 0 0 1 0
Sublux laternal6 0 0 0 0 1 1
* Number of rats examined.1 Anterior chamber filled with blood.2 Small, missing, or dead globe.
3 V-shaped defect in lid margin.4 Vascular proliferation into the cornea.5 Displacement of lens in medial direction.6 Displacement of lens in lateral direction.
57
L
UThe most consistent ocular defect observed occurred in the lens.
Occurrences of anterior cortical opacity, posterior cortical opacity,anterior and posterior cortical opacity, complete cataract, deep anteriorchamber, and lens luxation or resolution were observed in both treated andcontrol groups. In all groups (including controls), the occurrence of lenschanges was greater in the females. The incidences of these findings wereno greater in the low-dose animals than in controls of either sex. Theincidence of these ocular defects in the mid-dose animals was 5.2 timeshigher than in controls in the males and 4.7 times higher in the females.
Tables 17 and 18 summarize the ocular lesions seen during histo-pathologic evaluation in the male and female rats, respectively, that diedduring the second year on test or were terminated after completion of 104weeks. Phthisis bulbi (shrinkage and wasting of the eyeball) was seen inthree mid-dose males. This may be a sequela to chemically related eyedamage, but it may also be due to an unrelated injury such as trauma. Allother biologically significant changes in the mid-dose males--acute inflam-mation in the anterior chamber, acute inflammation and neovascularizationof the cornea, moderate/marked lens degeneration and multifocal/diffuseretinal degeneration--were treatment-related.36,7 Age-relatedchanges,38, 39 such as mild focal lens degeneration and mild peripheralretinal degeneration, common in control and low-dose males were usuallyobliterated in the mid-dose group by more severe toxic changes and earlymortality. The decreased incidence of scleral mineralization seen in mid-dose males may be due to early mortality.
In the mid-dose females, the neovascularization of the cornea,moderate/marked lens degeneration, lens mineralization, andmultifocal/diffuse retinal degeneration were treatment-related and werestatistically and biologically significant.36,3 7 Age-related changes
3 8 ,39
were common in control and low-dose females but were obliterated by moresevere toxic changes in the mid-dose females. The cause and biologicalsignificance of decreased focal scleral mineralization is not known.
5HematologyTables 19 and 20 summarize the hematology and clinical chemistry
analyses for animals terminated after one year. In both sexes, a signi-ficant increase in WBC and a decrease in RBC, Hgb, and Hct were seen at themid-dose level (Table 19). The low-dose level showed no effect for theseparameters and the differential count did not appear to be affected at any
* dose level.
Numerous clinical chemistry parameters were also significantly
affected in animals at the mid-dose level (Table 20). Phosphorus, however,was the only parameter that was consistently altered (increased) in bothsexes at this dose level. Some parameters were significantly affected in
both sexes at the low-dose level; however, the direction of the effects andthe parameters affected were not consistent.
Summaries of hematology and clinical chemistry analyses for theterminal necropsy are presented in Tables 21 and 22. Numerousstatistically significant changes in hematologic parameters in both sexeswere seen at the mid-dose level, including an increase in the WBC and a
58
I
3 Table 17
SUMMARY OF MICROSCOPIC OCULAR LESIONS IN MALE RATS[Dead or Moribund (13-24 Months), and Terminal Necropsy]
3 Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Location Lesion Percent* Percent Percent(T10)W (9T) (38
Phthisis bulbi (shrinkage and 0 0 3twastage of the eyeball) 0 0 8
Anterior Hemorrhage 0 0 1Chamber 0 0 3
Inflammation, acute 0 0 3t0 0 8
Cornea Inflammation, acute 0 0 3t0 0 8
Inflammation, chronic 0 2 20 2 5
Hyperplasia, epithelial, focal 1 1 03 1 1 0
Mineralization, focal 1 0 23 1 0 5
Neovascularization 2 0 5t2 0 13
Lens Degeneration, focal mild 52 50 2249 52 58
Degeneration, focal moderate/ 8 1 lotmarked 8 1 26
Degeneration, diffuse mild 0 0 10 0 3
* * Percent based on number of eyes examined.
** Number of eyes examined.3 t Statistically significant, p < 0.05.
1 59
.1
U
3 Table 17 (concluded)
3 Number of Lesions12.5 50
Control mg/kg/d mg/kg/dLocation Lesion Percent* Percent Percent
103** (967 (T)
Lens Degeneration, diffuse moderate/ 1 0 3marked 1 0 8
Mineralization 6 2 66 2 16
Posterior Hemorrhage 2 0 0Chamber 2 0 0
Retina Degeneration, peripheral 59 44 1456 46 37
Degeneration, multifocal/ 6 5 9t
diffuse 6 5 24
Sclera Hemorrhage, focal 1 0 01. 0 0
Inflammation, chronic focal 0 0 10 0 3
Mineralization, focal mild 33 30 4t31 31 11
6
U
I
a3 60
I* Table 18
SUMMARY OF MICROSCOPIC OCULAR LESIONS IN FEMALE RATS[Dead or Moribund (13-24 Months) and Terminal Necropsy]
Number of Lesions
12.5 50control mLkd mgk~
Location Lesion Percent* Percent PercentT7iY4w* 7
Anterior Inflammation, acute focal 0 0 1Chamber 0 0 0
Inflammation, chronic focal 1 0 01 0 0
Proteinaceous debris 0 1 3
0 1 3
Cornea Degeneration, focal 0 0 1o 0 1
Hyperplasia, epithelial, focal 1 0 01 0 0
Inflammation, acute 1 0 1
1 0 1
Inflammation, chronic 1 0 3
1 0 3
Neovascularization 1 0 lit1 0 10
Iris Inflammation, chronic 0 0 13 0 0 1
Hyperplasia, focal mild 0 0 15 0 0 1
Lens Degeneration, focal mild 49 42 19t43 46 18
Degeneration, focal moderate/ 7 12 53tmarked 6 13 49
* Percent based on number of eyes examined.**Number of eyes examined.
t Statistically significant, p < 0.05.
5 61
I
5 Table 18 (concluded)
U Number of Lesions12.5 50
Control mg/kg/d mg/kg/d
Location Lesion Percent* Percent Percent(114)* (- (108)
Lens Degeneration, diffuse, mild 0 1 00 1 0
Degeneration, diffuse, moderate 3 7 26t
to marked 3 8 24
Hyperplasia, subcapsule 0 0 2
epithelium 0 0 2
Mineralization 3 3 55t3 3 51
Optic Nerve Gliosis 0 0 30 0 3
Retina Degeneration, peripheral 71 58 35t62 63 32
Degeneration, multifocal/ 21 21 66tdiffuse 18 23 61
Inflammation, acute 1 0 01 0 0
Leukemia 1 1 01 1 0
Posterior Hemorrhage 0 0 1Chamber 0 0 1
Sclera Mineralization, focal 12 6 it11 7 1
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Idecrease in the RBC in females (seen in both sexes at the one-year sacri-fice) and decreases in Hgb, Hct, MCH, and MCHC in both sexes. Females atthe low-dose level had slightly less pronounced decreases in the RBC, Hgb,and Hct; no effects were observed at this level in the males. Thedifferential counts in mid-dose males showed a significantly increasedpercentage of PMNs and a decreased percentage of lymphocytes; females in
the mid- and low-dose groups had a decreased percentage of eosinophils andan increased percentage of reticulocytes, respectively.
Few statistically significant differences were seen in the clinicalchemistry parameters of treated males. A significant increase inphosphorus and decreases in LDH, total iron, albumin, and the A/G ratiowere seen in the mid-dose group. Relatively large increases in a few otherparameters such as BUN, creatinine, and potassium reflected kidney damagein the mid-dose males; however, they were not statistically cited becauseof one rat in the mid-dose group with extensive kidney damage, whichgreatly inflated the variance in these parameters. Decreases in LDH,electrolyte balance, and triglycerides were seen in males at the low-doselevel. This strengthens the significance of the decrease in theseparameters in males at the mid-dose level at the one-year necropsy.
Unlike the males, numerous statistically significant changes were seenin low- and mid-dose females at the terminal necropsy. Glucose, uric acid,potassium, triglycerides, LDH, total iron, albumin, and the A/G ratio weresignificantly decreased at both dose levels, Na+ and CO2 were increased.Phosphorus levels were significantly high at the mid-dose level only;albumin was decreased. The total bilirubin was increased only at the low-
* dose level.
In summary, results of the analyses of the hematology parameters indi-cated that LAP had an effect on both the myelocytic and erythrocytic seriesin both males and females at the 50-mg/kg level, and a slight effect(decrease) on the red blood cell parameters in females treated with 12.5mg/kg. Clinical chemistry parameters for the males at the 50 mg/kg levelreflect kidney lesions seen histolopically, but do not accurately expressthe degree of tissue damage.3 8 ,40, 4 In the males, only the LDH showed adose-response, affecting the 12.5 mg/kg level as well as the high dose; aclear dose-response was seen in numerous parameters for the females.
Organ Weights
Tables 23 through 25 present the final average body and organ weightsand organ weight ratios for male rats at the one-year necropsy, terminalnecropsy, and for those found dead or moribund, respectively. Table 26presents average body and organ weights and organ weight ratios for thehigh-dose males and females terminated after 33 weeks on test; these datawere not statistically compared with any other group because of the earlynecropsy date. A statistically significant treatment-related decrease inbody weights was seen at the mid-dose level for the one-year necropsy, andat both the low- and mid-dose levels for the terminal necropsy and for ratsfound dead or moribund. The excessively low body weight of the high dosemales that were found dead or moribund was believed to be influenced by thelarge number of very early deaths in that group. At the one-year necropsy,the kidney weight of the low-dose group was significantly increased
67
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Icompared with controls. A significant treatment-related increase in kidneyweight was also seen in both the low- and mid-dose males at the terminalnecropsy and at the mid-dose level for males found dead or moribund. Atthe terminal necropsy, the mid-dose group also showed a small but sIgni-ficant decrease in brain weight and an increase in liver weight. Theincreased liver weight may reflect the induction of liver enzymes tometabolize LAP.4 2 In addition to the increased kidney weight in mid-dosemales found dead or moribund, this group also showed a significant decreasein spleen weight. Several factors may have contributed to this, including(1) the large number of young, lighter weight, leukemia-free, mid-doseanimals that died early in the study, and (2) a lymphoid depletion seenmicroscopically.
The significant increases in the organ-to-body weight ratios of thevarious organs measured in at least one of the treated groups--the brain,I heart, liver, spleen and kidney-at the one-year necropsy, the brain,heart, liver, kidney, and testes at the terminal necropsy, and the brain,heart, spleen, and kidney in males found dead or moribund-may be due tothe treatment-related decrease in body weight in these groups. The treat-ment-related kidney and liver weight increases discussed previously add tothe increase in organ-to-body weight ratio for these organs. The decreasedspleen-to-body weight ratio seen in the mid- and high-dose males found deador moribund is also partly a reflection of the decreased spleen weight inthose groups, as previously discussed.
LAt the 1-year necropsy (Table 23), the only significant change in theorgan-to-brain weight ratios was a decrease in the testes-to-brain weightratio at the mid-dose level. This appears to have resulted from a slightlyincreased brain weight and a slightly decreased testes weight. At theterminal necropsy (Table 24), a significantly increased organ-to-brainweight ratio was seen for the liver (mid-dose only) and kidneys (low- andmid-doses), probably reflecting an induction of liver enzymes to metabolizeLAP and treatment-related kidney damage, as previously discussed. Theincreased kidney-to-brain weight ratio was also apparent in the mid-dosemales found dead or moribund (Table 25). A significantly decreased spleen-to-brain weight ratio was seen at the mid-dose level in rats found dead ormoribund, reflecting the decreased spleen weight in this group previouslydiscussed. The significant decrease in organ-to-brain weight ratios forthe liver, spleen, and testes seen in the high-dose males that were founddead or moribund reflects a comparison between this group of animals thatdied within the first 33 weeks of the study, and the death of controls latein the study with a higher incidence of both leukemia and testicular tumorscommon to aging Fisher-344 rats.
4 3 ,44
Tables 27 through 29 present the final average body and organ weightsand organ weight ratios for female rats at the one-year necropsy, terminalnecropsy, and for those found dead or moribund, respectively. Average body
and organ weights and organ weight ratios for the high-dose females termi-nated after 33 weeks on test were presented in Table 26. A statisticallysignificant, treatment-related decrease in body weight in the low- and mid-dose levels were seen in females throughout all stages of the study.Females at the mid-dose level at the one-year necropsy showed increasedbrain and liver weights compared with controls. The increased liver weight(also seen in the males) may have resulted from the induction of liver
52 72
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enzymes to metabolize LAP. A small increase in brain weight was also seenat the mid-dose level in females at the terminal necropsy; conversely, asmall decrease in brain weight was seen in the mid-dose males at terminalnecropsy. A small but statistically significant decrease was seen in theheart weight of the mid-dose females found dead or moribund.
Significantly increased organ-to-body weight ratios were seen in atleast one treated female group for the brain, heart, liver, spleen, andkidney at the one-year necropsy, and for these same organs except thespleen at the terminal necropsy and in rats found dead or moribund. Thisis mainly due to the treatment-related decrease in body weight, exceptperhaps in the cases of the increased brain and liver weights previouslydiscussed.
A significant increase in the liver-to-brain weight ratio was seen inthe mid-dose females at the one-year necropsy (Table 27), reflecting theincreased liver weight in this group. A significant decrease in the liver-to-brain weight ratio was seen in the low-dose group resulting from aslight decrease in the liver weight, and a small decrease in the spleen-to-brain weight ratio, reflecting the small increase in brain weight andUdecreased spleen weight in this group. No changes were seen in the organ-to-brain weight ratios for females at the terminal necropsy (Table 28). Asignificant decrease in the heart-to-brain weight ratio was seen in themid-dose females that were found dead or moribund, reflecting the decreasedheart weight previously discussed (Table 29).
In summary, significant findings reflecting treatment-related changesI in body weights and relative organ weights included (1) a decrease in body
weight with increasing dose in both sexes, (2) an increase in liver weightwith increasing dose in males for all three time periods, and in mid-dosefemales at the terminal necropsy, thought to be influenced by the inductionof hepatic enzymes to metabolize LAP, and (3) an increase in kidney weightwith increasing dose in males, reflecting treatment-related kidneydamage. Although no statistical comparisons were made, examination of theresults of the data from the high-dose females terminated at 34 weeks ontest (Table 26), which is most appropriately compared with the one-yearnecropsy data, generally appears to support these conclusions.
Histopathology
Tables 30 and 31 summarize the incidence of tumors with a frequency ofat least 5% in each group for all male and female rats examined histo-logically. No tumors were found in rats at the high-dose level; however,these animals were terminated after only 33 weeks on test. The decreasedincidence of anterior pituitary adenomas and interstitial cell tumors ofthe testes in the mid-dose males is attributable to the early deaths inthis group, since the incidence of these tumors usually increases withage.4 4 6 In the females, a decreased incidence of mammary gland fibro-adenomas and pituitary adenomas was seen at both the low- and mid-doselevels and is believed to be treatment-related. (Anterior pituitary tumorsare associated with the occurrence of fibroadenomas of the mammary gland
3 6
since the former often produce mammotropic hormones.4 5)
76
ITable 30
INCIDENCE OF TUMORS IN MALE RATS TREATED WITH LAP
3 Number of Tumors*12.5 50
Control mg/kg/d mg/kg/dOrgan Tumor Percent Percent Percent
(75yW*T 7- -Tg 7
Adrenal, medulla Adenoma 4 7 25 10 6
Bone marrow Leukemia 10 5 313 7 5
Heart Leukemia 4 2 0
5 53Kidney Leukemia 4 1 2
I 5 1 3
Liver Leukemia 9 2 412 3 6
Lung Leukemia 7 4 19 6 2
I Lymph nodes, mesenteric Leukemia 5 1 17 1 2
Preputial gland Adenocarcinoma 1 3 21 5 3
Pituitary, anterior Adenoma 16 18 3*21 26 5
Carcinoma 3 5 14 7 2
I* Includes only tumors occurring with a frequency of 5% or less in each
group. No tumors were found at the 200/100-mg/kg/day dose level, whichwas terminated after 33 weeks on test.
** Number of animals in group.
t The tissues of 7 rats at the 50 mg/kg/day dose level were lost tocannibalization.
Significantly less than control (p < 0.05).
S .77
i
Table 30 (Concluded)
3 Number of Tumors12.5 50
Control mg/ky/d ma/kg/dOrgan Tumor Percent Percent Percent
(75) (70) (63)
Spleen Leukemia 12 9 516 13 8
Stomach, glandular Leukemia 4 0 05
Testes Interstitial 53 56 25tcell tumor 71 80 40
Thymus Leukemia 4 2 25 3 3
Thyroid Carcinoma, 5 9 2C-cell 7 13 3
I
I|
i
IL
Table 31
INCIDENCE OF TUMORS IN FEMALE RATS TREATED WITH LAP
Number of Tumors*12.5 50
Control mg/kg/d mg/kg/dOrgan Tumor Percent Percent Percent
Adrenal Leukemia 4 3 15 4 1
Bone marrow Leukemia 5 9 67 13 9
Kidney Leukemia 4 1 25 1 3
Liver Leukemia 7 7 39 10 4
Lung Leukemia 4 6 25 9 3
Lymph nodes, mesenteric Leukemia 3 5 14 7 1
Mammary gland Fibroadenoma 20 6* 2*27 9 3
Pituitary Adenoma 31 10N 15*42 14 21
Carcinoma 4 4 05 6
Includes only tumors occurring with a frequency of 5% or less in eachgroup. No tumors were found at the 200/100-mg/kg/day dose level, whichwas terminated after 33 weeks on test.
** Number of animals in group.
t One control rat was removed from the study when discovered to bepregnant.
Significantly less than control (p < 0.05).
79
I -
I
Table 31 (Concluded)
U Number of Tumors12.5 50
Control mg/kg/d mg/kg/d
Organ Tumor Percent Percent Percent(74) (70) (70)
Spleen Leukemia 14 19 1019 27 14
Thyroid Carcinoma, 3 3 5
C-cell 4 4 7
Uterus Stromal polyp 21 14 1728 20 24
Stromal 2 4 3
sarcoma 3 6 4
8
~80
iTable 32
SUMMARY OF MICROSCOPIC LESIONS IN HIGH-DOSE (200/100 mg/kg/d) MALE RATS3 (Dead or Moribund, 1-33 Weeks)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent
Adrenal
Cortex Degeneration 413
Aorta Mineralization, focal 39
I Bone No lesions observed
Bone Marrow No lesions observed
Brain Hemorrhage, focal 13
Diaphragm Inflammation, acute focal 26
Inflammation, chronic focal 13
Esophagus Hemorrhage, focal 13
Heart Inflammation, chronic focal 39
Inflammation, purulent 1: : 3
Mineralization, focal 26
Necrosis, focal 13
INumber of animals in group.
81
U
Table 32 (continued)
UNumber of Lesions
200/100mg/kg/d
Organ Lesion Percent
Intestine
Cecum Izflammation, acute 13
Mineralization, focal 13
Necrosis, focal 13
Parasitism I
3
Colon Inflammation, chronic 1
* 3
Parasitism 516
Duodenum No lesions observed
Ileum No lesions observed
Jejunum No lesions observed
Kidney Inflammation, chronic 516
Inflammation, acute 2
Mineralization, focal 11
34
Necrosis, cortical 413
Necrosis, papillary 722
82
Table 32 (continued)INumber of Lesions
200/100mg/kg/d
Organ Lesion Percent~(32)*
Liver Congestion 516
Inflammation, acute focal I3
1Z Necrosis, focal 516
Lung Congestion 722
Atelectasis I3
Inflammation, chronic focal 2991
Mineralization, vascular I3
Lymph Node
Cervical Lymphoid depletion 26
Mesenteric Lymphoid depletion 26
Mammary Gland No lesions observed
Nerve Inflammation, chronic focal 1
3
Pancreas Inflammation, chronic focal I
Parathyroid No lesions observed
83
Table 32 (continued)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent
Pituitary No lesions observed
Prostate Inflammation, acute 619
Inflammation, chronic 1238
Salivary Gland No lesions observed
Seminal Vesicle Inflammation, acute 26
Inflammation, chronic 516
Mineralization, focal 13
Skeletal Muscle Inflammation, acute focal 26
Necrosis, focal 26
Skin Inflammation, acute 13
Spinal Cord Vacuolization, gray matter 516
Spleen Congestion 1an 3
Lymphoid depletion 2269
Stomach Mineralization, focal 13
.n84
Bi
Table 32 (concluded)
UNumber of Lesions
200/100mg/kg/d
Organ Lesion Percent
Testes Abscess 1
3
Mineralization, focal 4
13
Thymus Hemorrhage 26
Lymphoid depletion 2063
Thyroid Hyperplasia, C-cell 13
5 Trachea Inflammation, chronic focal 13
Urinary Bladder Hemorrhage 722
Inflammation, acute 722
Inflammation, chronic 39
83 8
ITable 33
SUMMARY OF MICROSCOPIC LESIONS IN HIGH-DOSE (200/100 mg/kg/d) MALE RATS(Terminal Necropsy, Week 34)
Number of LesionsI 200/100mg/kgld
Organ Lesion Percent(3w4)*
Adrenal
Cortex No lesions observed
Hemorrhage, periadrenal 13
Bone No lesions observed
Bone Marrow Hyperplasia, myeloid 39
Hyperplasia, erythroid 39
C Brain Glioma 13
Inflammation, purulent 13
Esophagus No lesions observed
Heart Inflammation, chronic focal 6* 18
Mineralization, focal 2* 6
Mineralization, vascular focal 515
Necrosis, focal 26
Fibrosis 26
* Number of animals in group.
86
I
* Table 33 (continued)
U Number of Lesions200/100mg/kg/d
Organ Lesion Percent
3 Intestines
Cecum Edema 13
Necrosis, focal I
3
Inflammation, purulent 1
3
Colon Parasitism 4
12
Edema 13
B Inflammation, acute 13
Mineralization, focal 13
Necrosis, focal I3
Duodenum No lesions observed
Ileum Parasitism 1
Jejunum No lesions observed
Kidney Nephropathy, chronic focal, mild 927
Inflammation, purulent, parenchyma, 1focal, mild 3
Atrophy, tubular focal, mild 1
*
* 87
------
Table 33 (continued)
i Number of Lesions200/100mg/kg/d
Organ Lesion Percent(34)*
5 Kidney Necrosis, papilla, focal, 6moderate 18
j Mineralization, focal 824
Mineralization, vascular, focal 26
Inflammation, chronic focal 412
Liver Necrosis, focal 26
Mineralization, focal, mild 1
* 3
Lung Hemorrhage, focal 721
Congestion, diffuse, mild 1030
UAtelectasis, focal, mild 618
Atelectasis, diffuse 26
Mineralization, interstitial, 2
focal, mild 6
Mineralization, vascular 3
9
Edema, focal, mild 26
Inflammation, chronic, focal 1
1 3
r88
I I Table 33 (continued)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent(34)*
I Lung Pigmented macrophages, focal 1
3
3Lymph NodeCervical Hyperplasia, lymphoid 11 3
Hyperplasia, plasma cell 1
* 3Mesenteric Congestion i
3
Mineralization, vascular 26
I Mammary Gland Hyperplasia, diffuse, mild 26
Mineralization, vascular 13
Pancreas Lipidosis 13
Inflammation, chronic focal 26
Parathyroid No lesions observed
Pituitary No lesions observed
Prostate Inflammation, chronic 1030
Inflammation, purulent 26
* 89|
I
I Table 33 (continued)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent(34)*
if Salivary Gland Inflammation, chronic focal 13
Skin Necrosis, focal 13
Edema I3
Granulation, tissue 13
Skeletal Muscle Mineralization, focal I3
Necrosis, focal I1 3Degeneration, focal mild I
3
Regeneration, focal mild 26
I Inflammation, acute focal 1
3
Spinal Cord Hemorrhage, focal 13
Spleen Congestion, moderate 3
9
Hyperplasia, lymphoid 1
3
Lymphoid depletion 515
Extramedullary hematopoiesis 6increased 18
i 90
i
ITable 33 (continued)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent
I Stomach Mineralization, focal 26
Mineralization, vascular focal 13
Necrosis, focal 13
Testes Atrophy 13
Mineralization, focal I3
Congestion, focal 113Thymus Mineralization, vessels, focal 1
mild 3
Hemorrhage, focal 412
3Congestion 13
Involution 824
Trachea Inflammation, chronic focal, 4submucosal 12
Hyperplasia, mucosa, focal I3
Inflammation, chronic lumen, 1focal mild 3
I5 91
I .. . '" ' ' I I ' '! 1 '
Table 33 (concluded)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent(34)*
Urinary Bladder Hyperplasia, mucosa 39
Necrosis 4
12
Inflammation, chronic, sub- 1mucosa, focal mild 3
Inflammation, chronic 26
Inflammation, acute 26
Inflammation, purulent 13
Hemorrhage 515
Mineralization, focal 13
9II
.. ... ..I* , .. J7 , " r " , q r v'
Table 34
SUMMARY OF MICROSCOPIC LESIONS IN HIGH-DOSE (200/100 mg/kg/d) FEMALE RATS(Terminal Necropsy, Week 34)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent
Adrenal
Cortex Hyperplasia, focal 12
Degeneration, focal 12
Medulla Hyperplasia, focal 23
Inflammation, chronic, mild 12
Bone No lesions observed
Bone Marrow Hyperplasia, erythroid 23
Brain Hemorrhage 23
Gliosis, focal 23
Esophagus No lesions observed
Heart Inflammation, chronic focal 69
Intestines
Cecum Edema 23
Colon Parasitism 58
* Number of animals in group.
93
I
Table 34 (continued)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent(69)*
I Intestine
Duodenum Ectopic pancreatic tissue 1
Iu 1
Ileum No lesions observedJJejunum No lesions observed
Kidney Hyperplasia, transitional 1epithelium, focal mild 1
Mineralization, focal 913
Inflammation, chronic focal 10* 14
Degeneration, tubular 1
Liver Necrosis, focal I
I Mineralization, focal mild 11
Inflammation, chronic focal, 21mild 45
Hyperplasia, bile ductules, 5focal mild 7
Vacuolization, hepatocytic, focal 23
Foci of cellular alteration I1
94
Table 34 (continued) 1Number of Lesions
200/100I mg/kg/d
Organ Lesion Percent(697*
Lung Hemorrhage, focal 1420
Hemorrhage, diffuse 11
Congestion, diffuse, mild 46
Atelectasis, focal mild 11
Atelectasis, diffuse 23
Mineralization, vascular 34
Inflammation, chronic focal 69
Pigmented macrophages, focal 46
Lymph Node
Cervical Hyperplasia, lymphoid 1
1
Mesenteric Hemorrhage 11
Mammary Gland No lesions observed
Ovary Necrosis, focal I
Hemorrhage 23
Congestion
95
D-A187 273 CHRONIC MAMMALIAN TOXICOLOGICAL EFFECTS OF LAP 211URSTEMATER(U) SRI INTERNATIONAL MENLO PARK CAJ M BROWN ET AL JUN 81 DAMD17-79-C-9121
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4l
Table 34 (continued)
Number of Lesions200/100mg/kg/d
Organ Lesion Percent(69)*
Pancreas Lipidosis 23
Inflammation, chronic focal 23
Hyperplasia, ductal, focal 11
Parathyroid No lesions observed
Pituitary Inflammation, chronic focal 11
Necrosis, focal 11
Hyperplasia, focal mild 34
Salivary Gland No lesions observed
Skin Inflammation, chronic focal 11
Necrosis of fat 34
Skeletal Muscle No lesions observed
Spinal Cord Hemorrhage, focal 23
Spleen Congestion, moderate 23
Extramedullary hematopolesis, 2increased 3
Stomach No lesions observed
96
p
Table 34 (concluded)
Number of Lesions200/100mg/kg/d
Organ Lesion Percentw69)*
Thymus No lesions observed
Trachea Inflammation, chronic focal, 19submucosal 28
Hemorrhage, lumen 11
Urinary Bladder Hyperplasia, mucosa 46
Necrosis 11
Inflammation, chronic 69
Uterus Inflammation, purulent 11
Hydrometria 812
Hypertrophy 11
97
II3 Table 35
SUMMARY OF MICROSCOPIC LESIONS IN MALE AND FEMALE RATS(Dead or Moribund, 1-12 Months)
Number of Lesions
Control 12.5 50
Organ Lesion Percent Percent PercentS(1 M)* -1iF) (7 M)
Adrenal
Cortex Degeneration 0 1 10 100 14
Inflammation, chronic focal 0 0 10 0 14
Bone No lesions observedBone Marrow No lesions observed
Brain Infarction 0 0 13 0 0 14
Inflammation, acute 0 0 10 0 14
Leukemiat 0 0 10 0 14
Diaphragm Inflammation, acute 0 1 10 100 14
Retina Inflammation, chronic focal 0 0 10 0 14
Heart Inflammation, chronic focal 0 0 10 0 14
Necrosis, focal 0 0 30 0 43
* Number of animals in group.
t All listings of leukemia in this table are Leukemia, Fischer Rat (mononuclear cell).
5 98
5
I
3 Table 35 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kgldOrgan Lesion Percent Percent PercentIf M)W -T17-F- (7M)
Intestine
Cecum No lesions observed
Colon Parasitism 0 0 130 0 14
Inflammation, chronic 0 0 1
I Duodenum No lesions observed 0 0 14
Ileum Parasitism 0 1 00 100 0
Necrosis, lymphoid 0 0 13 0 0 14
Jejunum No lesions observed
Kidney Necrosis, cortical 0 1 00 100 0
Necrosis, papillary 0 0 10 0 14
Inflammation, chronic focal, 1 1 33 mild 100 100 43
Leukemia 0 0 10 0 14
5 Tubular dilation and atrophy 0 1 70 100 100
9I
1 9
B
Table 35 (continued)
Number of Lesionis
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(1 M)*' (7 7 M
Kidney Inflammation, chronic focal, 0 0 3marked 0 0 43
Mineralization, focal minor 0 1 10 100 14
Tubular casts 1 1 7100 100 100
Liver Hyperplasia, bile ductules 0 0 10 0 14
Dinusoidal dilation 1 0 0100 0 0
Leukemia 0 0 10 0 14
Necrosis, focal 0 0 20 0 29
Congestion 0 0 10 0 14
Lung Inflammation, chronic focal 1 1 6100 100 86
Congestion 0 1 30 100 43
Edema 1 0 1
100 0 14
1 100
II3 Table 35 (continued)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(1 M)* ( F)~ M7 -
Lung Hemorrhage 0 0 1
Lymph Node
0 0 14
3 Cervical No lesions observed
Mesenteric Edema 0 0 10 0 14
3 Mammary Gland Leukemia 0 0 10 0 14
Inflammation, chronic focal 0 0 20 0 29
Nerve No lesions observed
Ovary Inflammation, purulent 0 1 00 100 0
Pancreas Leukemia 0 0 10 0 14
Parathyroid No lesions observed
Pituitary No lesions observed
Prostate Inflammation, chronic 0 0 30 0 43
Hyperplasia, focal 0 0 1
0 0 14
Hemorrhage, focal 0 0 1
0 0 14
Salivary Gland No lesions observed
1
5 101
I
I53 Table 35 (continued)
Number of Lesions
12.5 50Control mgtkg/d mg/kg/d
Organ Lesion Percent Percent PercentII M)* F
Seminal Vesicle Leukemia 0 0 15 0 0 14
Skeletal Muscle No lesions observed
Skin Inflammation, acute 0 0 10 0 14
Spinal Cord Leukemia 0 0 10 0 14
Spleen Leukemia 0 0 15 0 0 14
Necrosis, focal 0 1 10 100 14
Lymphoid depletion 0 0 1
0 0 14
Stomach No lesions observed
Testes No lesions observed
5 Thymus Lymphoid depletion 1 0 0
100 0 0
Inflammation, acute 0 1 00 100 0
Thyroid Leukemia 0 0 11 0 0 14
Cyst 0 0 10 0 14
I Trachea No lesions observed
1I5 102
I
IU3 Table 35 (concluded)
5 Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent( 1Th?3 - F 7M)
Urinary Bladder Inflammation, chronic 0 0 20 0 29
Uterus Leiomyoma 0 1 03 0 100 0
1IIiIIIII
3 103
k
I*
Table 36
SUMMARY OF MICROSCOPIC LESIONS IN MALE RATS(1-Year Necropsy)
Number of Lesions12.5 50Cont eLaLt4 e&L
Organ Lesion Percent Percent Percent
(10 )7 -7TT -
Adrenal
Cortex Degeneration, focal 9 8 990 80 100
Medulla Tumor, ganglioneuroma 0 1 00 10 0
IBone No lesions observed
Bone Marrow No lesions observed
Brain No lesions observed
Diaphragm Lipidosis 1 0 010 0 0
Degeneration, focal 0 1 00 10 0
Esophagus No lesions observed
Heart Focal necrosis, minor 5 6 350 60 33
3 Inflammation, chronic 6 6 260 60 22
Inflammation, acute focal 0 0 10 0 11
Intestine
Cecum No lesions observed
Colon Parasitism 1 1 110 10 I
Duodenum No lesions observed
3 Ileum Parasitism 0 1 00 10 0
3* Number of animals in group.
5 104
I
I
* Table 36 (continued)
UNumber of Lesions
12.5 50Control maIka/d m&Zt/d
Organ Lesion Percent Percent Percent(10)* (10) (9
Intestine
Jejunum No lesions observed
Kidney Inflammation, chronic focal 10 10 9100 100 100
3 Tubular regeneration, focal 9 7 5minor 90 70 56
Hyaline casts, focal, minor 9 6 590 60 56
Infarction 1 0 010 0 0
Mineralization, focal 0 0 10 0 11
Liver Hyperplasia, bile ductules, 5 9 0minimal 50 90 0
Inflammation, chronic focal 7 3 35 70 30 33
Necrosis, focal 1 4 01 0 40 0
Vacuolization, hepatocytic, 5 1 0
minimal 50 10 0
I Lung Inflammation, chronic focal, 9 10 9mild 90 100 100
5 Inflammation, chronic focal, 1 0 0moderate 10 0 0
Congestion 0 2 30 20 33
Pigmented macrophages, focal, 0 1 0minimal 0 10 0
3 105
I
I
I Table 36 (continued)
I Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent-u (10) (9)
Lymph Node
Cervical Cyst, solitary minor 1 0 010 0 0
Mesenteric Pigmentation, focal, minimal 0 1 01 0 10 0
Mammary Gland Inflammation, chronic focal 0 0 1
Nerve No lesions observed
Pancreas Inflammation, chronic focal 2 2 020 20 0
Fibrosis, focal 1 0 010 0 0
Parathyroid Hyperplasia 0 1 00 10 0
Pituitary No lesions observed
I Prostate Hyperplasia, focal 0 1 00 10 0
Testes Hyperplasia, interstitial cell 4 2 240 20 22
Salivary Gland Inflammation, chronic focal 0 1 00 10 0
Lipidosis 0 3 00 30 0
Skeletal Muscle No lesions observed
Skin No lesions observed
I3 106
'I
I-_Table 36 (concluded)
Number of Lesions
12.5 50Control mg/kg/d mg/k/d
Organ Lesion Percent Percent Percent(i0)* (10) (9)
Spinal Cord Hypertrophy, vascular focal 1 0 010 0 0
Hemorrhage, focal 0 0 10 0 11
Vacuolization, white matter, 1 0 0focal 10 0 0
Spleen Congestion, diffuse moderate 1 0 110 0 11
Lymphocytic depletion, diffuse 0 0 1moderate 0 0 11
Stomach No lesions observed
Thymus Inflammation, acute 1 0 010 0 0
Lymphocytic depletion, diffuse 1 0 0
slight 10 0 0
Thyroid Inflammation, chronic focal 1 0 010 0 0
Trachea Inflammation, chronic focal, 2 5 2minimal/mild 20 50 22
Ectasia, glands 1 0 010 0 0
Urinary Bladder No lesions observed
107
II Table 37
SUMMARY OF MICROSCOPIC LESIONS IN FEMALE RATS1 (1-Year Sacrifice)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Adrenal
Cortex Degeneration, focal 0 1 00 10 0
Inflammation, chronic focal 0 0 23 0 0 20
Bone No lesions observed
Bone Harrow Focal fibrosis 0 1 0
O 10 0
Brain No lesions observed
Diaphragm Lipidosis 2 0 05 20 0 0
Esophagus No lesions observed
3 Heart Focal necrosis, minor 2 4 320 40 30
Inflammation, chronic 3 7 230 70 20
3IntestineCecum No lesions observed
Colon Parasitism 1 0 010 0 0
Duodenum No lesions observed
Ileum No lesions observed
Jejunum No lesions observed
Kidney Inflammation, chronic focal 6 7 53 60 70 50
* Number of animals in group. 108
I Table 37 (continued)
3 Number of Lesions
12.5 50Control mL
Organ Lesion Percent Percent Percent
Kidney Tubular regeneration, focal, 2 2 1minor 20 20 10
Hyaline casts, focal, minor 2 6 020 60 0
Mineralization, focal 6 8 260 80 20
Liver Hyperplasia, bile ductules, 3 0 3minimal 30 0 30
Inflammation, chronic focal 5 5 750 50 70
Lung Inflammation, chronic focal, 9 10 10mild 90 100 100
5 Inflammation, chronic focal, 1 0 0moderate 10 0 0
Hemorrhage, focal 1 2 010 20 0
Congestion 0 2 00 20 0
Lymph Node
Cervical Pigmentation, focal, minimal 5 4 050 40 0
Mesenteric Pigmentation, focal, minimal 0 1 00 10 0
I Mammary Gland Inflammation, acute focal 0 1 00 10 0
3 Nerve No lesions observed
13 109
I
Table 37 (continued)
I Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Ovary Cyst 1 1 010 10 0
Inflammation, acute 1 0 0
10 0 0
Pancreas Inflammation, chronic focal 0 1 00 10 0
Parathyroid No lesions observed
Pituitary No lesions observed
Salivary Gland Infla-ation, chronic focal 1 0 010 0 0
Skin Tumor, keratoacanthoma 0 0 10 0 10
Skeletal Muscle No lesions observed
Spinal Cord No lesions observed
* Spleen No lesions observed
Stomach No lesions observed
3 Thymus No lesions observed
Thyroid Hyperplasia, C-cell 1 1 010 10 0
Necrosis, focal 0 0 1
3 0 0 10
Trachea Inflammation, chronic focal, 0 1 5minimal/mild 0 10 50
Urinary Bladder Inflammation, chronic focal 1 0 210 0 20
I110
I
I Table 37 (concluded)
3 Number of Lesions12.5 50
Control _/kt/d L aLtOrgan Lesion Percent Percent Percent
710w* (10) (10)
I Uterus Inflammation, acute 0 1 20 10 20
3 Hydrometria 1 0 010 0 0
II
II
IU
II
3 ill
ITable 38
SUMMARY OF MICROSCOPIC LESIONS IN MALE RATS[Dead or Moribund (13-24 Months) and Terminal Necropsy]U
Number of Lesions12.5 50
Control m/kg/d ma/kg/dOrgan Lesion Percent Percent Percent
Adrenal Hematopoiesis, extramedullary 0 lot 10 17 2
3 Leukemia* 3 2 0
5 3 0
U Cortex Cyst 0 1 10 2 2
Degeneration, focal 11 11 817 18 17
Degeneration, diffuse 0 1 00 2 0
Hyperplasia, focal 15 26 9323 43 19
Inflammation, chronic 3 0 13 5 0 2
Thrombosis, focal 0 1 03 0 2 0
Adenoma 2 3 03 5 0
Medulla Hyperplasia, focal 2 7 3
3 12 6
I Inflammation, chronic 0 0 10 0 2
Adenoma 4 7 26 12 4
* Number of animals in group.
t Statistically significant, p < 0.05.* All listings of leukemia in this table are Leukemia, Fischer Rat (mononuclear cell).
3 112
i
II
Table 38 (continued)
UNumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(4)* "(07 T7T
Aorta Mineralization, focal mild 0 0 40 0 9
Bone (Sternum) Degeneration, focal 4 2 16 3 2
3 Bone Marrow Congestion 1 0 0
2 0 0
3 Cyst 0 1 00 2 0
Hypercellularity, NOS 1 2 02 3 0
Hyperplasia, granulocytic 3 2 1
5 3 2
Hyperplasia, myeloid 5 1 48 2 9
Pigmentation, focal 1 0 0
3 2 0 0
Leukemia 10 5 3
3 16 8 6
Brain Congestion 1 0 12 0 2
Cyst, focal 0 0 10 0 2
3 Hemorrhage, focal 2 7 03 12 0
Inflammation, chronic focal 1 1 0
2 2 0
Inflammation, purulent focal 1 0 02 0 0
5 113
I
Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
T(6) 707) T7TBrain Mineralization, focal 1 1 0
2 2 0
Necrosis, focal 1 0 0
2 0 0
I Carcinoma, metastatic 2 1 03 2 0
Granular cell tumor 0 0 1
0 0 2
Leukemia 1 1 02 2 0
Diaphragm Hemorrhage, focal 0 0 10 0 2
Inflammation, chronic 3 0 35 0 6
Mineralization, focal 0 0 13 0 0 2
Pigmentation, focal 0 0 10 0 2
Leukemia 1 0 02 0 0
Esophagus Inflammation, acute focal 1 1 22 2 4
Inflammation, chronic focal 0 1 10 2 2
I Heart Cardiomyopathy, mild 41 46 2464 77 51
13 114
II
Table 38 (continued)UNumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent6m)* -To T"T
Heart Cardiomyopathy, moderate/marked 10 11 716 18 15
Hemorrhage, focal 0 2 0
0 3 0
Inflammation, chronic focal 1 3 12 5 2
m Mineralization, focal mild 1 0 20t
2 0 43
Necrosis, focal 1 2 22 3 4
Thrombosis, atrial 1 1 0
2 2 0
Leukemia 4 2 06 3 0
Neurilemoma 0 0 10 0 2
Sarcoma, metastatic 1 0 0- 2 0 0
Intestine
I Cecum Hemorrhage, focal 0 1 00 2 0
Inflammation, purulent 1 0 02 0 0
Inflammation, chronic 0 1 1I 0 2 2
I3 115
ITable 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
Intestine
Cecum Mineralization, focal 0 0 10 0 2
Parasitism 1 0 02 0 0
Colon Edema 1 0 02 0 0
Mineralization, focal 0 0 40 0 9
Parasitism 10 10 416 17 9
Pigmentation, focal 0 0 10 0 2
Fibrosarcoma, metastatic 1 0 02 0 0
Neurilemoma 0 1 00 2 0
Duodenum Mineralization, focal 0 0 10 0 2
Pigmentation, focal 0 0 10 0 2
Leiomyosarcoma 1 0 02 0 0
1
3 116
I
I Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
I Intestine
Ileum Cyst 0 2 00 3 0
Hyperplasia, lymphoid 1 0 02 0 0
Inflammation, chronic 0 1 10 2 2
Necrosis, lymphoid 1 0 02 0 0
Parasitism 2 0 03 0 0
I Pigmentation, focal 0 0 10 0 2
Leukemia 1 0 02 0 0
Jejunum Parasitism 1 0 0
2 0 0
Pigmentation, focal 0 0 1II 0 0 2
I Kidney Congestion 3 0 05 0 0
Cyst 0 1 20 2 4
Hemorrhage, focal 2 1 03 3 2 0
1I 117
II
Table 38 (continued)UNumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent PercentTGZ7 -TOY- (047)-
Kidney Hydronephrosis 0 0 8tO 0 17
Hyperplasia, transitional 3 19t 8epithelium, mild 5 32 17
U Hyperplasia, transitional 0 3 4epithelium, moderate 0 5 9
Inflammation, acute papillary 0 0 10 0 2
Inflammation, chronic 2 1 73 2 15
Inflammation, purulent focal 1 0 02 0 0
Infarction, focal 3 2 05 3 0
Mineralization, focal 0 0 18t1 0 0 38
Necrosis, cortical 2 0 13 0 2
Necrosis, papillary 0 0 15tO 0 32
Nephropathy, mild 19 14 1830 23 38
I Nephropathy, moderate 40 44 2463 73 51
Nephropathy, marked 1 0 52 0 11
13118
i
Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
Kidney Pigmentation, focal 0 1 00 2 0
Leukemia 4 1 16 2 2
Liver Congestion 0 0 9t0 0 19
Cyst, focal 5 0 18 0 2
Fibrosis, focal 2 0 13 0 2
Foci of cellular alteration 24 34 5t38 57 11
Hyperplasia, hepatocytic, focal 0 5 2O 8 4
Hyperplasia, bile ductules 61 59 4095 98 85
Rematopoiesis, extramedullary 1 0 02 0 0
Hepatocyte, atypia, diffuse 0 1 0O 2 0
Inflammation, chronic focal 6 8 39 13 6
Necrosis, focal 6 8 09 13 0
Necrosis, diffuse 0 0 10 0 2
119
I
Table 38 (continued)
INumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(64)* Toy-
Liver Sinusoidal dilation, focal 0 5 00 8 0
Pigmentation, focal 1 0 02 0 0
Degeneration, hepatocytic, 0 1 1mild 0 2 2
Vacuolization, hepatocytic, 16 25 4
mild 25 42 9
Vacuolization, hepatocytic, 2 1 1moderate 3 2 2
Hepatocellular carcinoma 0 0 10 0 2
Sarcoma, metastatic 2 0 0S 3 0 0
Leukemia 9 2 3U 14 3 6
Lung Alveolar histiocytosis, focal 6 7 89 12 17
Atelectasis, focal 1 1 2
2 2 4
I Congestion 13 12 21t20 20 45
3 Edema 3 0 55 0 11
Emphysema, focal 0 0 10 0 2
Foreign material, focal 1 0 12 0 2
3 120
I
I
Table 38 (continued)
Number of Lesions12.5 50
Control mg/kgfd mg/kg/dOrgan Lesion Percent Percent Percent
W(64)* (60) T473
Lung Hemorrhage 5 8 68 13 13
Hyperplasia, bronchial 0 1 0epithelium, focal 0 2 0
3 Inflammation, chronic focal 19 24 830 40 17
Inflammation, interstitial 2 3 23 5 4
Mineralization, focal vascular 12 21 1219 35 26
Mineralization, interstitial 0 0 lit0 0 23
Pigmented macrophages, focal, 8 21t 7minimal/mild 13 35 15
Pulmonary adenomatosis 0 2 00 3 0
Adenoma 0 1 00 2 0
Carcinoma 0 2 10 3 2
Adenocarcinoma, metastatic I 1 02 2 0
Leukemia 7 4 111 7 2
121
I
I Table 38 (continued)
INumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Lymph Nodes
Brachial Leukemia 1 0 02 0 0
Cervical Cyst, focal 3 9 05 15 0
Edema 0 2 00 3 0
Fibrosis 0 1 00 2 0
Hyperplasia, lymphoid 1 4 22 7 4
Hyperplasia, plasma cell 1 2 12 3 2
Inflammation, chronic 1 0 02 0 0
I Leukemia 0 0 20 0 4
Hepatic Leukemia 2 0 03 0 0
Mesenteric Congestion 0 0 20 0 4
Cyst, focal 0 2 00 3 0
Edema 0 2 20 3 4
15 122
5
I
ITable 38 (continued)
I Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent PercentW(64)* 60)
I Lymph Nodes
Mesenteric Hemorrhage 1 1 02 2 0
Hyperplasia, lymphoid 1 5 11 2 8 2
Inflammation, chronic 0 1 00 2 0
Necrosis, focal 0 1 0
0 2 0
Necrosis, adjacent fat 1 0 0
2 0 0
5 Mesothelioma, external surface 0 0 1
0 0 2
Leukemia 5 1 18 2 2
Pancreatic Leukemia 1 0 02 0 0
Thymic Edema 0 1 00 2 0
Hemorrhage 1 3 12 5 2
Hemosiderosis, mild 0 2 00 3 0
Hyperplasia, lymphoid 0 1 00 2 0
Hyperplasia, plasma cell 0 2 00 3 0
3 123
0
I
I Table 38 (continued)
INumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
C ;an Lesion Percent Percent Percent
I Lymph Node
Thymic Inflammation, chronic focal 0 1 00 2 0
Adenocarcinoma, metastatic 1 0 02 0 0
Mammary Gland Cyst 1 4 02 7 0
Ectasia, ductular, mild/ 10 14 2moderate 16 23 4
Ectasia, ductular, marked 1 0 2(Galactocele) 2 0 4
I Fibrosis, focal moderate 1 1 02 2 0
Hyperplasia, mild 0 1 10 2 2
3 Inflammation, chronic 1 0 02 0 0
Pigmentation 0 2 00 3 0
Thrombosis, focal 0 0 1O 0 2
Adenoma 1 0 01 2 0 0
Adenocarcinoma 0 1 01 0 2 0
9124
Table 38 (continued)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Mammary Gland Fibroadenoma 2 0 03 0 0
Leukemia 0 0 1O 0 2
Masses
Abdominal Lipoma 1 0 0Cavity 2 0 0
Mesothelioma 0 0 1O 0 2
Necrosis of fat 2 1 23 2 4
Neurilemoma 1 0 02 0 0
Foot Osteoma 0 0 10 0 2
Preputial Adenoma 0 2 0Gland 0 3 0
Adenocarcinoma 1 3 22 5 4
Subcutaneous Fibroma 2 2 23 3 4
Hemangiopericytoma 1 0 02 0 0
Hemangiosarcoma 0 1 00 2 0
Myxoma 1 0 02 0 0
125
Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
3 Masses
Subcutaneous Necrosis of fat 1 0 02 0 0
Sarcoma 1 0 02 0 0
Zymbal's Carcinoma 0 0 1Gland 0 0 2
Muscle Degeneration, focal 0 0 10 0 2
Edema, focal 1 0 02 0 0
3 Inflammation, chronic 0 1 10 2 2
1 Mineralization, focal 0 0 20 0 4
Leukemia 1 1 02 2 0
Pancreas Atrophy, focal 24 22 5t38 37 11
Atrophy, diffuse 1 1 01 2 2 0
Cyst, focal 0 2 00 3 0
Degeneration, focal 0 1 00 2 0
Hyperplasia, acinar cell, 1 4 0focal 2 7 0
13 126
Table 38 (continued)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(64w) -Tor
Pancreas Hyperplasia, ductular, focal 2 2 03 3 0
Hyperplasia, islet cell 15 10 it23 17 2
Fibrosis, focal 0 0 1O 0 2
Inflammation, chronic focal 19 28 930 47 19
Lipidosis 24 31 838 52 17
Mineralization, focal 0 1 5tO 2 11
Vacuolization 3 0 55 0 11
Necrosis of fat, adjacent 0 0 10 0 2
Adenoma, islet cell 2 2 03 3 0
Adenocarcinoma 1 0 02 0 0
Fibrosarcoma, metastatic 1 0 02 0 0
Mesothelioma, surface 0 0 10 0 2
Leukemia 3 0 15 0 2
127
Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
W64)* 67) (47)
Parathyroid Hyperplasia 3 2 9t
5 3 19
Pituitary
Anterior Congestion 7 Ot 411 0 9
Cyst 0 2 10 3 2
Hyperplasia 22 27 1234 45 26
Vacuolization 0 0 10 0 2
Adenoma 16 18 3t25 30 6
Carcinoma 3 5 15 8 2
Leukemia 1 0 02 0 0
Posterior Gliosis 0 2 20 3 4
Prostate Congestion 0 0 10 0 2
Hyperplasia, focal, minimal/ 30 29 7tmild 47 48 15
Hyperplasia, focal, moderate 0 1 10 2 2
128
II
Table 38 (continued)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(64)* 607) (T7T
Prostate Inflammation, chronic focal 23 20 1336 33 28
Inflammation, diffuse 0 2 15t
O 3 32
Inflammation, purulent 0 0 10 0 2
Edema 0 1 00 2 0
Mesothelioma, surface 0 0 10 0 2
Leukemia 0 0 10 0 2
I Salivary Gland Atrophy 0 2 00 3 0
Congestion 1 0 052 0 0
Ectasia, ductular 0 0 1
10 0 2Hemorrhage, focal 0 2 0
0 3 0
Hyperplasia, focal mild 3 5 25 8 4
I Hyperplasia, ductular, focal 2 3 02 5 0
Hyperplasia, ductular mucosa, 0 5 1focal 0 8 2
129
3 Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
(Mg)* (60) -T"TT-
U Salivary Gland Inflammation, chronic focal 3 4 25 7 4
5 Lipidosis, mild 39 39 6t61 65 13
Vacuolization, acinar 0 1 20 2 4
Carcinoma 0 1 00 2 0
Fibrosarcoma 0 1 0O 2 0
Sarcoma 0 1 05 0 2 0
Skin Keratoacanthoma 1 0 15 2 0 2
Squamous cell carcinoma 1 1 02 2 0
Inflammation, chronic focal 0 0 2O 0 4
Inclusion cyst, epidermal 0 0 1O 0 2
3 Scrotal Sac Inflammation, chronic diffuse 0 0 10 0 2
Necrosis 0 0 10 0 2
Seminal Vesicle Atrophy 18 28 4t28 47 9
Cyst 0 1 00 2 0
5130
3
3 Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent7647- (6o) T7
Seminal Vesicle Edema 0 00 0 2
3 Inflammation, chronic 0 0 litO 0 23
Mesothelioma, surface 0 0 1
O 0 2
Spinal Cord Gliosis, focal 0 1 1O 2 2
Hemorrhage, focal 8 2 0I13 3 0
Inflammation, purulent 1 0 03 2 0 0
Mineralization, dura, focal 0 1 05 0 2 0
Vacuolization, gray matter, 35 44 lOt
mild focal 55 73 21
Vacuolization, white matter, 43 47 6tmild focal 67 78 13
I Leukemia 1 0 02 0 0
3 Spleen Congestion 5 2 28 3 4
Hematopoiesis, extramedullary, 2 2 0moderate 3 3 0
Lymphoid depletion, mild 0 0 8tI0 0 17
Necrosis, focal 1 2 0
2 3 0
5 131
I
I
U Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
(64)* (60)f (47)-
I Spleen Fibrosarcoma 1 0 02 0 0
Leukemia 12 9 419 15 9
Infarction 0 0 10 0 2
Stomach
Forestomach Hyperplasia, epithelial, mild 0 6t 10 10 2
Ulceration 0 1 00 2 0
Papilloma 0 0 10 0 2
Squamous cell carcinoma 1 0 02 0 0
Glandular Adhesion 0 1 0Stomach 0 2 0
Edema, focal 0 4 60 7 13
Fibrosis 1 0 02 0 0
Glandular dilation and atrophy 14 32t 1122 54 23
Inflammation, chronic focal 5 0 28 0 4
Mineralization, mild 0 0 20t0 0 43
132
.... . .. . , I ' , ' ,,-,----,--,---...
I
Table 38 (continued)
UNumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Stomach
Glandular Mineralization, moderate 0 0 2Stomach 0 0 4
Leukemia 4 0 06 0 0
Sarcoma 1 0 0£ 2 0 0
Fibrosarcoma, metastatic 1 0 0. 2 0 0
Tail Inflammation, chronic 0 0 10 0 2
Necrosis 0 0 10 0 2
Fibrosarcoma 0 1 00 2 0
I Testes Atrophy 48 52 2875 87 60
Hyperplasia, interstitial cell 4 2 66 3 13
Hemorrhage 1 1 02 2 0
Inflammation, chronic 0 5 30 8 6
Mineralization, focal 0 6t 40 10 9
Necrosis, focal 0 0 15 0 0 2
1 133
* Table 38 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(6W (60T (47T
Testes Necrosis of adjacent fat 0 1 0O 2 0
i Mesothelioma, serosa 0 3 10 5 2
Interstitial cell tumor 53 56 25t83 93 53
Thymus Congestion 0 0 30 0 6
Cyst 0 2 00 3 0
Lymphoid depletion 0 0 10 0 2
Hyperplasia, epithelial cell 0 0 13 0 0 2
Inflammation, chronic focal, 0 1 03 mild 0 2 0
Involution 47 48 3173 80 66
Mineralization, focal 0 0 10 0 2
I Leukemia 4 2 26 3 4
U Thyroid Cyst, focal 3 10 25 17 4
Degeneration, focal 0 1 00 2 0
Hemorrhage, focal 0 1 00 2 0
5 134
-
U
UTable 38 (continued)
I Number of Lesions12.5 50
Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(64)* TOT- -TOT-
Thyroid Hyperplasia, follicular cell, 1 1 0focal 2 2 0
Hyperplasia, C-cell 10 13 116 22 2
Adenoma, C-cell 2 1 03 2 0
Carcinoma, C-cell 5 9 28 15 4
Adenocarcinoma 1 3 12 5 2
Inflammation, chronic focal 2 1 01 3 2 0
Trachea Degeneration, mucosa 1 0 02 0 0
Ectasia, submucosal glands 21 23 6t33 38 13
I Foreign body, lumen 0 0 10 0 2
Hyperplasia, mucosa, focal 0 2 10 3 2
Inflammation, chronic focal 6 9 119 15 23
Urinary Bladder Hemorrhage 1 2 lit2 3 23
Hyperplasia, mucosa 2 2 9t3 3 19
Fibroplasia, serosa 0 0 lot
0 0 21
135
LE
I
3 Table 38 (concluded)
U Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(64)* -Ty (47T
Urinary Bladder Inflammation, chronic 2 8 15t3 13 32
Inflammation, acute 0 0 20 0 4
Mineralization 0 0 20 0 4
Necrosis 0 0 llt0 0 23
Papilloma 0 1 10 2 2
Mesothelioma, serosa 0 0 13 0 0 2
Leukemia 0 1 00 2 0
I
U
I5 136
U3 Table 39
SUMMARY OF MICROSCOPIC LESIONS IN FEMALE RATS1 f[Dead or Moribund (13-24 Months) and Terminal Necropsy]
Number of Lesions12.5 50
Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Adrenal Hematopoiesis (extramedullary) 2 0 23 0 3
Leukemia* 4 3 16 5 2
Congestion 0 0 15 0 0 2
Cortex Atrophy 1 0 02 0 0
Cyst 8 6 113 10 2
U Degeneration, focal 28 23 2044 39 33
Hyperplasia, focal 25 19 1339 32 22
Inflammation, chronic focal 2 0 23 0 3
Adenoma 0 2 10 3 2
Carcinoma 0 1 00 2 0
Medulla Cyst 1 0 02 0 0
Hyperplasia, focal 1 2 22 3 3
* Number of animals in group.
I All listings of leukemia in this table are Leukemia, Fischer Rat (mononuclear cell).
137
U Table 39 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent-GTTw -37- 767-
I Adrenal
Medulla Inflammation, chronic focal 0 1 00 2 0
Adenoma 0 2 20 3 3
Carcinoma 1 0 02 0 0
Bone (Sternum) Degeneration, focal mild 9 8 314 14 5
Bone Marrow Atrophy 1 1 12 2 2
I Fibrosis, focal 0 1 10 2 2
Hyperplasia, granulocytic 0 1 7t0 2 12
3 Hyperplasia, myeloid 2 2 33 3 5
Leukemia 5 9 68 15 10
Brain Cyst, focal 1 0 02 0 0
Fibrosis (dura), focal 0 0 15 0 0 2
Gliosis, focal 2 1 03 2 0
t Statistically significant, p < 0.05.
3 138
U
I Table 39 (continued)*Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent PercentTW647 (59)T (7O)
Brain Hemorrhage, focal 0 0 40 0 7
Hydrocephalus 1 0 02 0 0
Inflammation, chronic focal 1 1 02 2 0
Mineralization, focal 3 0 15 0 2
Astrocytoma 0 0 10 0 2
Ependymoma 0 1 00 2 0
Glioma 1 0 02 0 0
Carcinoma, metastatic 2 1 0U 3 2 0
Leukemia 0 1 1
0 2 2
Diaphragm Inflammation, chronic focal 2 7 33 12 5
Atrophy 0 0 10 0 2
Fibrosarcoma, metastatic 0 0 10 0 2
Esophagus Hyperplasia, epithelial 0 0 10 0 2
Inflammation, chronic focal 0 0 10 0 2
5 139
I
I Table 39 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
U Esophagus Mineralization, focal 1 0 02 0 0
I Heart Cardiomyopathy, mild 45 45 3970 76 65
Cardiomyopathy, moderate/ 8 2 5marked 13 3 8
Inflammation, chronic focal 6 4 19 7 2
Mineralization, focal 0 2 00 3 0
Necrosis, focal 1 0 02 0 0
Leukemia 2 3 13 3 5 2
Adenocarcinoma, metastatic 0 0 10 0 2
Neurilemoma 0 2 0
0 3 0
Intestine
Cecum Edema 0 0 10 0 2
Inflammation, chronic 0 0 10 0 2
Parasitism 1 0 02 0 0
Leukemia 1 0 02 0 0
140
U Table 39 (continued)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(64* (59) (0)
Intestine
Colon Congestion 0 0 1O 0 2
Inflammation, focal 0 1 00 2 0
Mineralization, focal 0 0 10 0 2
Parasitism 7 6 611 10 10
Fibrosarcoma, metastatic 0 0 10 0 2
i Leukemia 1 0 0
2 0 0
Duodenum No lesions observed
Ileum Atrophy, mild 0 0 13 0 0 2
Mineralization, focal 0 0 20 0 3
Parasitism 1 1 02 2 0
Adenocarcinoma 0 0 10 0 2
Fibrosarcoma, metastatic 0 0 10 0 2
Jejunum No lesions observed
U3141
b
I
3 Table 39 (continued)
I Number of Lesions12.5 50
Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
l Kidney Cyst 2 1 03 2 0
Hydronephrosis 0 0 20 0 3
Hyperplasia, transitional 11 7 12epithelium, mild 17 12 20
Infarction, focal 1 2 22 3 3
Inflammation, chronic focal 1 0 02 0 0
Inflammation, purulent 0 1 00 2 0
Necrosis, cortical 2 0 03 0 0
Necrosis, papillary 0 0 10 0 2
I Nephropathy, mild 55 50 5186 85 85
Nephropathy, moderate 4 4 26 7 3
3 Minera-Azation, mild 8 6 213 10 3
Pigmentation, focal 1 0 02 0 0
Adenoma 0 0 10 0 2
13 142
U
3 Table 39 (continued)
* Number of Lesions12.5 50
Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Kidney Adenocarcinoma 0 0 1
0 0 2
Leukemia 4 1 26 2 3
Liver Congestion 0 1 20 2 3
Cyst, focal 1 0 32 0 5
Degeneration, hepatocyte 0 2 00 3 0
Foci of cellular alteration 45 32 3070 54 50
Hematopolesis (extramedullary) 0 1 10 2 2
Hyperplasia, hepatocyte focal 4 5 36 9 5
Hyperplasia, bile ductules 30 22 2747 37 57
Hemorrhage, focal 1 2 02 3 0
Inflammation, chronic focal 33 37 4152 63 68
Inflammation, chronic diffuse 0 1 00 2 0
Necrosis 2 4 63 .7 10
Pigmentation, focal 0 1 10 2 2
3143
i
U
I Table 39 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
Liver Sinusoidal dilation, focal 0 1 10 2 2
5 Vacuolization, hepatocytic, 8 3 Otfocal 13 5 0
Vacuolization, hepatocytic, 4 2 1diffuse 7 3 2
Adenocarcinoma, metastatic 1 0 12 0 2
Leukemia 7 7 311 12 5
Lung Atelectasis 0 0 13 0 0 2
Congestion 3 3 45 5 7
Edema 1 1 22 2 3
Hemorrhage 7 4 5
11 7 8
U Histiocytosis, alveolar 3 4 35 7 5
Hyperplasia, bronchial 0 2 0epithelium 0 3 0
Inflammation, chronic focal 27 25 2842 42 47
Inflammation, interstitial 1 4 6
2 7 10
35 144
... 5I
I
3 Table 39 (continued)
I Number of Lesions12.5 50
Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Lung Inflammation, acute 1 0 02 0 0
Infarction, focal 0 0 10 0 2
Mineralization, focal vascular 31 25 3248 42 53
Mineralization, interstitial 0 3 00 5 0
Pigmented macrophages, focal 45 32 29t70 54 48
Pulmonary adenomatosis 4 1 26 2 3
Adenocarcinoma, metastatic 0 0 10 0 2
Carcinoma, metastatic 1 1 02 2 0
Carcinoma, bronchioalveolar 0 0 10 0 2
Leukemia 4 6 26 10 3
Sarcoma, metastatic 0 1 00 2 0
Lymph Node
Cervical Cyst 1 1 02 2 0
Congestion 0 3 00 5 0
I5145
I
I
Table 39 (continued)
INumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Lymph Node
Cervical Hemorrhage 0 0 10 0 2
Hyperplasia, lymphoid 1 1 12 2 2
Hyperplasia, plasma cell 2 2 03 3 0
Pigmentation, focal 1 0 02 0 0
Leukemia 1 3 12 5 2
Brachial Leukemia 0 1 00 2 0
Inguinal Leukemia 0 1 0
0 2 0
I Hepatic Leukemia 1 0 02 0 0
Mammary Leukemia 0 1 00 2 0
Mesenteric Congestion 0 0 10 0 2
Edema 0 1 00 2 0
Hemorrhage 1 0 02 0 0
Hyperplasia, lymphoid 5 0 1
8 0 2
1 146
I
I
H Table 39 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
I Lymph Node
Mesenteric Inflammation, chronic 0 1 50 2 8
Pigmentation, focal 0 2 30 3 5
Leukemia 3 5 1U 5 9 2
Adenocarcinoma, metastatic 0 0 10 0 2
Sarcoma, metastatic 0 0 10 0 2
Pancreatic Cyst 1 0 02 0 0
Pigmentation, focal 1 1 02 2 0
Leukemia 0 0 10 0 2
Thymic Congestion 0 1 10 2 2
Hemorrhage 1 0 02 0 0
Hyperplasia, lymphoid 1 0 02 0 0
Pigmentation, focal 1 2 52 3 8
1i 147
I Table 39 (continued)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Lymph Node
Thymic Leukemia 1 0 02 0 0
Mammary Gland Cyst 2 0 03 0 0
Ectasia, ductular, mild/ 43 36 25tmoderate 67 61 42
Ectasia, ductular, marked 23 6t 3t
(Galactocele) 36 10 5
Hyperplasia, mild 0 2 00 3 0
U Inflammation, chronic 0 1 00 2 0
Adenoma 3 0 05 0 0
Adenocarcinoma 1 0 22 0 3
Fibroma 1 0 02 0 0
Fibroadenoma 20 6t 2t31 10 3
Sarcoma 0 0 10 0 2
1
.* 148
I
Table 39 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent*6) -T97 (033
Masses
Abdominal Fat Necrosis of fat 1 3 4
2 5 7
Lipoma 0 1 0R0 2 0Clitoral Adenomna 1 0 2
Gland 2 0 3
Adenocarcinoma 0 1 00 2 0
Ear (Pinna) Neurilemoma 0 1 00 2 0
Foot Hemangiosarcoma 1 0 02 0 0
Jaw Squamous cell carcinoma 1 1 02 2 0
Subcutaneous Adenocarcinoma 0 0 10 0 2
Fibroma 1 1. 12 2 2
Sarcoma 2 0 0I3 0 0
Pelvis Carcinoma, metastatic 0 0 1E 0 2
Vaginal Sarcoma, metastatic 0 1 10 2 2
Zymbal's Adenocarcinoma 1 1 0
Gland 2 2 0
149
I MR .N
ITable 39 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent3 (T4W (T59)T (60)
Muscle Hemorrhage 1 1 02 2 0Ii±.tmmation, chronic 0 1 0
0 2 0
Mineralization, focal 1 0 02 0 0
Sarcoma, metastatic 0 0 10 0 2
Ovary Atrophy 1 1 02 2 0
Congestion 0 0 10 0 2
Ia Cyst 8 8 313 14 5
Inflammation, chronic 0 1 10 2 2
Inflammation, purulent 1 1 02 2 0
Necrosis of fat (paraovarian) 2 0 03 0 0
Pigmentation 0 1 00 2 0
I Vacuolization 1 0 02 0 0
Adenocarcinoma 1 0 02 0 0
150
. RIMWU
Table 39 (continued)
Number of LesionsR12.5 50
___ __ __ __ __ ___ __ __ Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
(6) DS'7 -
UOvary Adenocarcinoma, metastatic 0 01O 0 2
5Carcinoma 0 0 10 0 2
ifGranulosa, theca cell tumor 1 0 02 0 0
Leukemia 1 1 0I2 2 0
Sarcoma 0 0 20 0 3
Pancreas Atrophy, focal 11 14 1117 24 18
Cyst 1 0 02 0 0
Hyperplasia, acinar cell, 0 0 13focal 0 0 2
Hyperplasia, islet cell, focal 2 1 13 2 2
Hyperplasia, ductular 0 0 10 0 2
Inflammation, chronic focal 13 12 2220 20 37
Inflammation, acute focal 0 1 00 2 0
Lipidosis 30 21 1747 36 28
151
I
Table 39 (continued)
INumber of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent PercentT(64)W (5T -TOY
Pancreas Adenoma, Islet cell 1 0 02 0 0
Adenocarcinoma, metastatic 0 0 10 0 2
j Leukemia 3 -0 05 0 0
Fibrosarcoma, metastatic 0 0 1
0 0 2
Sarcoma, metastatic 0 0 10 0 2
Parathyroid Hyperplasia 2 2 03 3 0
Leukemia 0 1 01 0 2 0
Pituitary Cyst 12 20 1919 34 32
Hyperplasia, focal 14 13 1922 22 32
Mineralization 0 1 00 2 0
Pigmentation 0 2 00 3 0
Adenoma 31 lot 15t48 17 25
Carcinoma 4 4 06 7 0
1~152
I
I Table 39 (continued)
Number of Lesions
12.5 50, Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent(64)* W597 7071
Salivary Gland Atrophy 0 2 10 3 2
Ectasia, ductular 3 3 05 5 0
Hyperplasia, focal mild 3 1 35 2 5
Hyperplasia, ductular focal 0 0 10 0 2
Hyperplasia, ductular mucosa, 4 1 1
focal 6 2 2
Inflammation, chronic focal 3 2 45 3 7
Lipidosis 5 11 138 19 22
Skin Hyperplasia, epithelial focal 0 0 2O 0 3
Hyperplasia, sebaceous gland, 1 0 0
focal 2 0 0
5 Hemorrhage, focal 1 0 0
2 0 0
Inflammation, chronic 1 1 02 2 0
IKeratoacanthoma 1 0 02 0 0
Spinal Cord Gliosis, focal 0 0 10 0 2
Hemorrhage, focal 0 1 00 2 0
j 153
I
I Table 39 (continued)
Number of Lesions12.5 50
Control mg/kg/d mg/kg/dOrgan Lesion Percent Percent Percent
(64)* (59) (60)
I Spinal Cord Vacuolization, gray matter, 50 36 34tfocal 78 61 57
Vacuolization, white matter, 54 45 48focal 84 76 80
Spleen Hematopoiesis, extramedullary, 7 7 6moderate 11 12 10
Hyperplasia, granulocytic 0 1 00 2 0
Hyperplasia, plasma cell 0 2 00 3 0
Inflammation, chronic focal 0 1 00 2 0
Necrosis, focal 1 1 02 2 0
Leukemia 14 19 10
Stomach 22 32 17
Forestomach Edema 0 1 00 2 0
Hyperplasia, epithelial, mild 5 3 88 5 13
Papilloma 1 0 12 0 2
Glandular Glandular dilation and atrophy 40 36 4763 61 78
Inflammation, chronic 1 1 13 2 2 2
154
i Table 39 (continued)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
U Stomach
Glandular Mineralization, focal 0 1 00 2 0
Necrosis, focal 0 1 00 2 0
Adenocarcinoma 0 0 11 0 0 2
Leiomyosarcoma 0 0 10 0 2
Leukemia 1 0 02 0 0
I Thymus Congestion 0 1 10 2 2
I Cyst, focal 0 0 10 0 2
Hemorrhage 1 1 0
2 2 0
Involution 52 47 4881 80 80
Leukemia 0 1 00 2 0
Thymoma 0 0 10 0 2
Thyroid Cyst, focal 1 0 12 0 2
Hyperplasia, C-cell 13 14 620 24 10
155
i Table 39 (continued)
Number of Lesions
12.5 50Control mg/kg/d mg/kg/d
Organ Lesion Percent Percent Percent
Thyroid Adenoma, C-cell 3 3 15 5 2
Carcinoma, C-cell 3 3 5
5 5 8
Leukemia 0 1 00 2 0
Sarcoma, metastatic 0 0 10 0 2
Trachea Atrophy, mucosa 0 0 10 0 2
Ectasia, glands 16 17 18£ 25 29 30
Inflammation, chronic focal, 7 9 16mild 11 15 27
Sarcoma, metastatic 0 0 10 0 2
Urinary Bladder Hyperplasia, mucosa 11 7 16
17 12 27
Inflammation, chronic 0 5 6t0 8 10
i Papilloma 0 2 20 3 3
Uterus Cyst 11 14 2117 24 35
Hemorrhage 0 0 10 0 2
Hydrometrist 6 17t 739 29 12
156
I
Table 39 (concluded)
UNumber of Lesions
12.5 50
Control mg/kg/d mg/kgfdOrgan Lesion Percent Percent PercentS(64)* M5) 7 T
Uterus Hyperplasla, mucosal 0 3 20 5 3
Inflammation, chronic 0 0 60 0 10
Inflammation, purulent 0 2 10 3 2
Adenoma 0 1 10 2 2
Adenocarcinoma 1 0 1I 2 0 2
Adenocarcinoma, metastatic 1 0 1
2 0 2
Fibroma 0 0 10 0 2
Myxoma 1 0 01 2 0 0
Hemangioma 0 1 10 2 2
Stromal polyp 21 14 1733 24 28
I Stromal sarcoma 2 4 33 8 5
f Tumor, NOS, necrotic 0 1 00 2 0
Leukemia 1 0 02 0 0
13 157
Ialthough it was not statistically significant. An increased incidence of
marked chronic nephropathy (generalized degenerative change) was also seenin males at the mid-dose level, although it was not statistically signi-
* ficant.
An increased incidence of congestion was seen in the lungs of mid-dosemales. This is thought to reflect the increased incidence of early deathsin this group. Increased mild focal mineralization was also seen in thisgroup and is thought to be secondary to renal failure. The minimal to mildincrease in pigmented macrophages in the low-dose group is not ofbiological significance, and probably represents metabolic breakdownproducts and minute inhaled particulates and is not unusual in aged rats.
In males at the mid-dose level, the decreased incidence of focalatrophy and islet cell hyperplasia of the pancreas, changes due to
age,38, 4,P 5 reflects the large number of early deaths in this group. Theincreased incidence of mineralization and hyperplasia of the arathyroid inmales at this dose level may be secondary to kidney failure. 7
The decreased incidence of congestion of the pituitary in males at thelow-dose level is of uncertain biological significance. In the mid-dosegroup, the decreased incidence of pituitary adenomas is believed to beattributable to the early deaths in this group, since the incidence of thistumor usually increases with age.
4 4 - 6.
The decreased incidence of focal mild hyperplasia of the prostate inthe mid-dose males is believed to be due to the early deaths of many ofthese animals. The increased incidence of inflammation of the prostate atthe mid-dose level may be due to the proximity of this organ to the urinarybladder, which showed extensive toxic changes in this group (see below).
The decreased incidence of lipidosis of the salivary gland in the mid-dose males is not biologically significant and is attributed to the largenumber of early deaths in this group.
Atrophy of the seminal vesicle, an age-related change,4 4 decreased inthe mid-dose group due to the early deaths. The increased incidence ofinflammation in this organ may be related to inflammation of the urinarybladder.
In males at the mid-dose level, the decreased incidence of mild focalvacuolization of the spinal cord, an age-related change of the centralnervous system, is believed to be due to the early deaths in this group.
The increased mild lymphoid depletion in the spleen in mid-dose malesis believed to be a chemically-induced change.
The increased occurrence in low-dose males of mild epithelial cellhyperplasia and glandular dilation and atrophy of the stomach (usual age-related changes ) may be compound-related but the changes were not severeenough to be biologically significant. The mineralization present in malesat the mid-dose level is believed to be secondary to kidney damage.
4 '
158
IIn the low-dose males, the increased incidence of mild focal minerali-
zation of the testes, often an aging change,3 6 may be compound-related butis not believed severe enough to be biologically significant. Thedecreased incidence of interstitial cell tumors in the mid-dose groupreflects early mortality.
4 3 ,44
In mid-dose males, the decreased occurrence of ectasia of submucosalglands of the trachea, an aging change,I reflects early mortality.
The increased incidence of hemorrhage, mucosal hyperplasia, serosalfibroplasia, inflammation, and necrosis of the urinary bladder in the mid-dose males is biologically significant and appears compound-related. It isbelieved that these changes, coupled with those in the kidney, wereresponsible for the many deaths in this group.
Table 39 summarizes microscopic lesions in the females that were founddead on test or were sacrificed when moribund during the second year of thestudy or that survived until the terminal sacrifice. The statisticallyincreased incidence of granulocytic hyperplasia of the bone marrow in mid-dose females may in part be a response to inflammatory changes present in3 the urinary bladder in this group.
A decreased incidence of focal hepatocytic cytoplasmic vacuolizationof the liver, an aging or metabolic change, was seen in females at the mid-dose level. The reason for this is not apparent but it may be treatment-related.
Females at the mid-dose level showed decreased numbers of small fociof, at times, pigment-laden macrophages in the lung. The cause of thisdecrease is not readily apparent but it is not biologicallysignificant.
44 ,5
The decreased incidences of ductular ectasia and fibroadenoma in themammary gland in the low- and mid-dose groups appear to be treatment-related, presumably resulting from a pituitary gland hormonal influence.
A decreased incidence of anterior pituitary adenomas was seen in thelow- and mid-dose groups and is believed to be treatment-related. Thisdecreased incidence is also reflected in mammary gland changes. Aspreviously mentioned, rat pituitary tumors often produce mammotropichormones,' and there is an association between anterior pituitary tumorsand ectatic ducts and fibroadenomas of the mammary gland. 8
A decreased incidence of mild focal gray matter vacuole formation inthe spinal cord, an aging change,44 04 8 was seen in mid-dose females. Thecause of this decrease is not apparent. Because signs of spinal cord dys-function were not seen clinically, this finding is not believed to bebiologically significant.
The increased incidence of chronic inflammation of the urinary bladderin the low- and mid-dose groups is treatment-related. However, this
increase was statistically significant in females only at the mid-doselevel.
159
i
IAn increased incidence of hydrometria was seen in the low-dose group,
although the biological significance of this uterine condition isuncertain.
In summary, the principal biologically significant, LAP-induced toxiclesions observed in Fischer-344 rats were degenerative changes in the eyeand urinary system and decreased cellularity of lymphoid tissue. Thesechanges were generally restricted to the mid- and high-dose groups and wereusually more pronounced in male animals.
The histologic changes present in the eye were most prominent in thelens and retina. The lens changes were focal to diffuse subcapsularvacuolization, granularity, mineralization, disruption of lens fibers, andoccasional focal proliferation of subcapsular epithelial cells. Theselesions are consistent with clinical cataract formation3 6,3 / and were con-sistently more severe and extensive than the typical age-related changes3 8seen in control animals.
Retinal lesions consisted of multifocal to diffuse selective destruc-tion of the photoreceptor layers (rods, cones, and outer nuclear layer),with the resulting displacement of the remaining layers. These changeswere much more extensive than the mild peripheral retinal degeneration thatwas present in control and low-dose animals and that are commonly found inold Fischer-344 rats. 39 Other changes present in the eye were inflammationand new blood vessel formation in the cornea and lessened focal minerali-zation of the sclera.
Lesions present in the kidneys of treated rats consisted of papillarynecrosis, hydronephrosis, hyperplasia of the pelvic epithelium, andincreased mineralization of the parenchyma. Chronic nephropathy was alsomore severe in the mid-dose group.
The urinary bladder was extensively damaged in the mid- and high-dosemale rats. In addition to necrosis, hemorrhage, and inflammation of alllayers of the bladder wall, mucosal hyperplasia and serosal fibroplasiawere present. The inflammation also commonly extended to the adjacentprostate and seminal vesicle.
Lymphoid depletion was commonly seen in the hematopoietic system,especially the spleen and thymus, in high-dose male rats that died duringthe course of this experiment. This condition was occasionally present toa lesser degree and extent in high-dose males that were sacrificed and inother groups.
A physiological change typically observed in the livers of treatedanimals was a slight expansion and lighter histologic staining of hepato-cyte cytoplasm. This is believed to reflect subcellular adaptation formetabolism of LAP.42,49
Water Quality Criteria
From the results of the chronic study, an Acceptable Daily Intake(ADI) for LAP was estimated according to the National Research Councilrecommendations for chronic nonhuman toxicity data with no indication of
5160
I
carcinogenicity.5 0 The ADI was calculated using a no-observed-adverse-effect-level (NOAEL) of 12.5 mg/kg/day, a safety factor of 100, and anaverage body weight of 70 kg, as follows:
ADI . 12.5 mg/kg x 70 kg = 8.75 mg100
To provide an estimated level of human health protection from thetoxic properties of LAP ingested through water or contaminated aquaticorganisms, a water quality criterion (C) was determined. Standard dailyexposure assumptions of 2 liters of water and 6.5 grams of edible aquaticproducts were used, based on a weighted average of 3% lipids for freshwaterand estuarine fish and shellfish in the average diet.5 1 A bioconcentrationfactor (BCF) was used to relate pollutant residues in aquatic organisms tothe pollutant concentration in ambient waters in which they reside. ForLAP, a BCF of 20.5 L/kg has been estimated for aquatic organisms containingapproximately 7.6% lipids.' 2 The weighted average BCF was calculated asfollows:
20.5 L/kg x 3.0% - 8.1 L/kg7.6%
Based on these data and assumptions, the ambient water qualitycriterion for LAP was calculated as follows:
C W - 8.75 mg = 4.26 mg/L2 L + (0.0065 kg x 8.1 L/kg)
It should be noted that this calculation does not provide an estimatefor exposure to LAP through non-fish dietary intake or throughinhalation. Therefore, it would be inappropriate to use this value as amaximum concentration level. It does, however, provide an estimate if asafe level of exposure to LAP for an adult male through ingestion ot wateror aquatic organisms.
1R
, 161
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