J-A168 754
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IITRI Project No. L6116-Study No. 11
DETERMINATION OF THE CHRONIC MAMMALIAN
TOXICOLOGICAL EFFECTS OF TNT
(Twenty-four Month Chronic Toxlclty/Carclnogenlcity
Study of Trinitrotoluene (TNT) in the B6C3F1 Hybrid Mouse)
LJ
<
F I NAL REPORT--PHASE
VOLUME I
E. Mar I anna Fured
Barry S. Levine
John W . Sagartz
Vladislava S. Rac
Paul M. LIs h
December 1964
Supported by
U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND
Fort Detrlck, Frederick, Maryland 21701-5012
Contract No. DAMD 17-79-C-9120
I V
DTIC
:lecte«
JUN 1 6 1986 3
D
NT Research Institute
10 West 35th Street
Chicago, Illinois 60616
Project Officer: Jesse J. Barkley, Jr.
U.S. Army Medical Bioengineering and
Research Development Laboratory
Fort Detrlck, Frederick, Maryland 21701-5012
Approved for public release; distribution unlimited
The findings In this report are not to be construed
as an official Department of the Army position unless
so designated by other authorized documents.
THIS DOCUMENT IS BEST
QUALITY AVAILABLE. THE COPY
FURNISHED TO DTIC CONTAINED
A SIGNIFICANT NUMBER OF
PAGES WHICH DO NOT
REPRODUCE LEGIBLY.
la" RE=OR' SEC-RTy C-ASS-CATiluN
Unclassified _
2 a SECcR.ir C_aSS.f.CA'iON AUTHOR.~Y
2d DEC-ASSiF^CAT,ON , DOWNGRADING SChEDUl
4 PERFORM,NG ORGANIZATION 3E S CRT NUMBERS;
L 6 1 16 Study No . n
6a NAME C e PERFORMING ORGAN.2AT;ON 6o CF = .CE S v MSO-
(If applicable)
I IT Research Institute
6c ADDRESS :Cf>, State, and ZIP Coat)
10 West 35th Street
Chicago , IL 6061 6
8a NAME OF FUNDING. SPONSORING 8 d GRACE SYMBC-
ORGanizat.o.n U.S. Army Medical (if applicable)
Research £. Development Command
8c ADDRESS (City, State, and ZIP Code)
For Detrick
Frederick, Maryland 21701-5C12
D-rtlt*7S
report documentation page
‘0 RESTn,
3 0IS*?..B-*}CN/A^A.^A3‘L:TY Of REPORT
Approved for public release;
distribution unlimited
ORGAN.ZAT,ON REPORT \ 0 M3
OF MONITORING ORGAN.ZAT.ON
7b ADDRESS (City. State, and ZIP Code)
9 PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER
DAMD17-79-C-9120
'0 SOURCE OF FUNDING NUMBERS
PROGRAM
PROJECT
TASK
ELEMENT NO.
no 3 E 1 -
NO.
62720A
62720A835
AA
WORK UNI'
ACCESSION NO
11 TiTlE (include Security Classification) D ET r Ry | n;, T | ; Qr THE CHRONIC MAMMALIAN TOXICOLOGICAL EFFECTS
OF TNT (Twenty-four Month Chronic Toxicity/Carcinoaenicity Study of Trinitrotoluene (TNT)
in the B 6 C R ^ 1 Hvbrid Mouse__
1Z PERSONA. AuTiROR(S)
E.M. Furedi, B.S. Levine, J.W. Saca'tz, V. S ._ Rsc and P. M. List- _
13a TY°E OF REPORT 13b TIME COVERED 74 DATE OF RE = ORT (Year, Month. Day)
Phase IV Final Reboot from_tq__ 1 9 8 A December
■7 COSA". CODE! 18 Si-3,EC* TERMS . Continue on reverse if necessary and identify by bio ck number)
.3 GROUP Su3-GRQUP TNT CAS Reg. No. 118-96-7
06 15 _ B 6 C 3 F 1 hybrid mouse Chronic toxic ty
19 I 01 Trinitrotoluene _
19 A8STRAC* (Continue on reverse if necessary and identify by bioct number)
This study was conducted to evaluate the toxicity o ( the munitions compound 2,A, 6 -trinitrc-
toluene (TNT) CAS Reg. No. 118-96-?) In B6C3F1 hybr id mice when administered in their diet
for uc to 2A months. Groups o ( 75 mice ne- sex received TNT at doses of 0, 1 .5, 10, o" 7C
mq/kg/day. Ten mice/sex/dcse we r e sacrificed following 6 and 12 months on test with sur¬
viving animals sacrificed afte r 2 A months of treatment. Toxicologic endpoints includt
clinical signs, bodv weights, food consumption, hematology, clinical chemistry, ophthal¬
mology, organ weights, and gross anc tissue morphology.
The major toxic effects observed du r inq the administration of 70 mq/kg/day of TNT to B 6 C 3 r1
mice for up to 2A months included ane~ia and probable hepatotoxicity. In addition, peri¬
pheral 1ymphocytosis and 1eukr i s/~a 1 i q"an; lymphoma in spleen were apparent. A s i oc1e
instance o f lymphocytosis was evident for males at the 10 rrtg/kg/day dose level. These
animals also demonstrated a 5"7 reduction in body weight gain throughout the treatment
period. This dose therefore appeals to be minimally toxic, and the no-eftect level unde"
the conditions of the present studs is 1.5 mq/kg/day. __
20 Distribution / availability OF abs*rac~
□ uNC.ASS F ED UNL’M.’ED £ SAVE AS
ZZa NAVE jr 3ES~ONS<8IE NO
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DO FORM 1473. 34 VAR 32-
21 ABSTRACT SECURITY CLASSIFICATION
CZu'r ,se 3 s Unclassified___
22b TE.Ep-ONE (Include Area Code/ 22c OFF CE SYMBOL
( 301) 663-7315_SGRD-Rms_
a * -'' p F-"7 e« r 4u:nec SEC-' 0 CLASSjF CAT ON Of -- f £AQ j_
-e-ectosaie-.psoe-.e UNCLASS I HEP
Contract No. DAMD 17 - 79-C-9120
IITRI Project No. L6116
Study No. 11
DETERMINATION OF THE CHRONIC MAMMALIAN TOXICOLOGICAL EFFECTS OF TNT
(Twenty-four Month Chronic Tox1c1ty/Carc 1 nogen Ic1ty
Study of Trinitrotoluene (TNT) In the B6C3F1 Hybrid Mouse)
FINAL REPORT
Prepared by
E. Marianna Furedl
Barry S. Levine
John W. Sagartz
Vladislava S. Rac
Paul M. LIs h
NT Research Institute
10 West 35th Street
Chicago, Illinois 60616
Supported by
U.S. ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND
Fort Detrick, Frederick, Maryland 21701-5012
Project Officer: Jesse J. Barkley, Jr.
■ TIuvClaC - JjUE^ksxxJr
-Paul M . L I s h
Scientific AdvIsor
Life Sciences Research
£. Marianna FuredI-Machacek
Staff Toxicologist
Life Sciences Research
Alan M. Shefnej
Associate Director
Life Sciences Research
E£££iiim. iUMMARI
This study was conducted to evaluate the toxicity of the
munitions compound trinitrotoluene (TNT; CAS Reg. No.
118-96-71 In B6C3F1 hybrid mice when administered In their diet
for up to 24 months. Groups of 75 mice per sex received TNT at
doses of 0, 1.5, 10, or 70 mg/kg/day. Ten mlce/sex/dose were
sacrificed following 6 and 12 months on test with surviving
animals sacrificed after 24 months of treatment. Toxicologic
endpoints Included clinical signs, body weights, food
consumption, hematology, clinical chemistry, ophthalmology, orgar
weights, and gross and tissue morphology.
The dose levels for the chronic toxIcIty/carc I nogen I c 11y
study of TNT In B6C3F1 hybrid mice were selected on the basis of
results of a four-week oral (diet) range-finding study. In this
range-f I nd I ng study groups of 10 mice per sex received TNT at
doses of 0, 0.3, 2, 14, 100 or 700 mg/kg/day. Toxicologic
endpoints Included clinical signs, body weights, food
consumption, hematology, clinical chemistry, organ weights, and
gross and tissue morphology.
Doses of up to 700 mg/kg/day failed to result In death. The
only clinical findings were reductions In body weight gain at 100
mg/kg/day and slight body weight loss at the 7 00 mg/kg/day dose-
level. Major toxic effects Included hemolysis of red cells as
suggested by hyperbilirubinemia and splenic hemosiderosis.
Additional toxic effects of TNT seen primarily at the 7CC
mg/kg/day dose level Included leukopenia without a differential
shift, thrombocytosis, slight hepatomegaly, marginal decreasec
testes weights, and elevated renal weights. None of these orga r
weight changes was accompanied by treatment-related histologic
alterations. Minimal toxic effects were seen at the ICC
mg/kg/day dose level. Thus, the appparent no-effect level under
♦he conditions of this range-finding study was 14 mg/kg/day.
The chronic administration of TNT to male and female B6C3F1
mice at doses up to 7C mg/kg/day did not alter survival rates.
Clinically, reductions in body weight gain (15-20?) were seen fcr
animals of both sexes given 70 mg/kg/day; a 5-7? reduction
throughout the treatment period also occurred for males at the 1C
mg/kg/day dose level. By contrast, increases In food consuir.pt lc r
were seen at the high dose level.
Anemia consisting of reduced hematocrit, hemoglobin and REC
count was observed for males and females receiving 70 mg/kg/day.
The effect was mild, and compensatory physiologic responses,
l.e.. Increased production of reticulocytes and larger
erythrocytes were not apparent. in addition, splenic lesions
previously seen for TM-treated rats and dogs which were
considered secondary to a hemolytic anemic state, i.e, sinusoidal
congestion, extramedullary hematopoiesis, and hemosiderosis, were
not observed in this study. This was probably a function of the
limited extent of anemia seen at the doses tested.
Additional toxic effects of TNT to B6C3F1 mice were seer-
only at the 7C mg/kg/day dose level. These included potential
liver Injury observed as reduced serum triglyceride levels,
reduced serum globublln levels (A/G ratios were unaffected), and
Increased brain, liver size without microscopic alterations.
Sporadic Increases for kidneys, spleen, heart and testecular
weights were also seen, but were also not supported
histological ly.
Increased lymphocyte counts were observed In animals
administered 70 and, to a lesser extent, 10 mg/kg/day. For
females given the high dose. Increased Incidence of leukemia and
malignant lymphoma, with a positive dose-response was observed in
the spleen. This increase was not seen for males and no other
tumcrigenlc effect of TNT was apparent In this study.
In summary, the major toxic effects observed during the
administration of 70 mg/kg/day of TNT to B6C3F1 mice for up to 2 4
months included anemia and probable hepatotoxicity. In addition,
peripheral lymphocytosis and leukemia/malignant lymphoma in
spleen was apparent. A single Instance of lymphocytosis was
evident for males at the 10 mg/kg/day dose level. These animals
also demonstrated a 5-7? reduction In body weight gain throughout
the treatment period* This dose therefore appears to be
minimally toxic, and the no-effect level under the conditions of
the present study is 1 .5 mg/kg/day.
Acce-
ion For
--1
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NT IS
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one
7
1
LO£LWQR£
The G.S. Army Medical Bioengineering Research end
Cevelcpment Laboratory (USAM6RCL), Fort Detrick, Frederick, MG,
has ceen conducting a research program since 1973 tor the purpose
of developing the scientific data base necessary for recommending
»ate r quality criteria for compounds unique to the munitions
industry. A water quality criterion (as defined by the amended
Clean Water Act, 1977) Is a qualitative or quantitative estimate
c* tne concertratic of a pollutant In ambient waters that, when
net exceeded, will ensure a water quality sufficient to protect a
specified water use. The criterion Is a scientific entity based
solely on data and scientific judgement. It does not reflect
considerations of economic or technological feasibility.
Currently, a water quality criterion consists of two separate
numerical limits, one for the protection of human health and the
other for the protection of aquatic organisms. These numbers,
when translated by the appropriate regulatory agency, can be the
basis o f enforceable discharge or effluent limitations In a pol r t
source discharge p e r m I + Issued under the Clean Water Act.
Since a water quality criterion Is to protect designated
water uses, a diverse, multidisciplinary research program was
developed by USAMBRDL that Includes "effects" studies or
laboratory and domestic animals, wildlife species, aquatic
orcarlsm?, plants, and economically important crops. In
adcition, extensive chemical and biological fate and persistence
ies f s are conducted to provide Information on the behavior of a
pollutant In the aqueous environment. These kinds of cate ere
especially useful for making site-specific translation of
criteria Into enforceable discharge limits.
This report represents a portion of the mam mi a I Ian tex I c c I oc \
data base being developed by USAMBRDL on trinitrotoluene.
In conducting the research described In this report, the
l'-vost Igator(s) adhered to the "Guide for the Care ard Use c +
baberaterv Animals," prepared by the Committee on Care and Lise c*
Laboratory Animals In the Institute of Laboratory Animal
Resources, National Research Council (DHEW Publication No.
(N i H ) 7 6- 2 3 . Revised 1978).
LQ1 iNQWLLt£M£Nl
This report was prepared at NT Research Institute, 10 West
35th Street, Chicago, Illinois, 60616, under U.S. Department of
Army Contract No. DAMD 17-79-C-9120 ( l ITRI Project No. L06116)
entitled "Determ I n a t I o n of the Chronic Mammalian Toxicological
Effects of TNT". Mr. Jesse J. Barkley, Jr., Environmental
Protection Research Division, USAMbRDL, served as the Contract
Officer's Technical Representative for this program.
The work reported herein was conducted In the Toxicology and
Pharmacology Section of the Life Sciences Department and
represents a portion of the overall effort of the above named
research program. Paul M. LIsh, Ph.D., Scientific Advisor,
served as Principal Investigator. Eva M. Fured I-Machacek, DVM,
served as study director and study toxicologist, and was
responsible for the overal I conduct of the study, supervision of
the technical support personnel, and final report generation.
Vladislava S. Rac, DVM, M.S., and Carol A. Thompson, DVM, M.S.,
Senior Veterinary Pathologists, consecutively served as Study
Pathologists and were responsible for supervision of gross
necropsies, tabulation of the gross necropsy data and
histological tissue-processing. John Sagartz, DVM, Consultant,
Veterinary Pathology, was responsible for tabulation and
evaluation of hlstopathology data. Barry S. Levine, D.Sc.,
Senior Toxicologist, served as head of the clinical pathology
laboratory and was also responsible for generation of the final
report. Don Reltman, Samuel Terese, B.S., (ASCP-MT), and Debbie
L. Sava, B.S., (ASCP-MT), were responsible for generation of
clinical pathology date. Joseph B. Harder, DVM, served as
cllncal veterinarian and supervised animal care personnel. Joann
M. H I n z, B.S., and Robert M. Renaud, B.S., were responsible for
the collection of test date. Dorothy Davis, (ASCP-HT), was
responsible for preparation of histology slides. C. Susan West,
DVM, performed the ophthalmic examinations. Josephine M. Reed,
M.M., M.S., Quality Assurance, was responsible for the quality
assurance program. Robert Remaly, B.S., Senior Engineer, was
responsible for preparation of the test article premixes. Hugh
J. O’Neill, Ph.D., Manager, Analytical Chemistry, and Walter C.
Elsenberg, Ph.D., Senior Chemist, were responsible for chemical
analyses of test articles, test article premixes and test diets,
.ean Graf pro viced the particle size analyses. Klrlt Parlkh
B.S., was responsible for the Quality Assurance program of
chemical analyses. Robert Gltbons, Ph.D., provldeo statistical
and computational assistance.
fiU&Lill £££U£AN££ £IAT£M£N1
Biciogloa! laboratory Inspections were perforn.tc on
September 29 and November 11 and 24, 1 981; January 16, February
2, March 5, April 15, June 2, July 8, August 12 and 24, October
7 , November 10, 1982; and January 26, March 17, May 19, August
2, September 25 and October 12 and 13, 1983; and February 21,
1964. Cata audits were performed on January 18, August 2, and
October 2? 1o November 4, 1982; January 19, February 2 to 7, anc
June 21 to 23, 1983; and March 21 to 23, May 15 to 22, November
11 to 15, and Dece mi per 4, 1984. The final draft report was
audited May 12 and 15, 1986. Inspections and audits were
performed tv Josephine
the study was found
performed tv Josephine Reed, Julie McPhlllps, and KIrIt Parlkh.
the study was found to meet Life Sciences Quality Assurance
criteria. Specimens and raw data generated during the study will
be retained In the IITRI Life Sciences Arc fives as specified in
standard operating procedures.
// Josephine M. Reed
/ Quality Assurance
tfitilLIilS.
1 Efiaa Ha
I. INTRODUCTION. 16
II. MATERIALS AND METHODS. 16
A. Test Article. 16
6. Test Diets. .. 17
C. Animals. 19
D. Experimental Design. 20
E. Statistical Analysis. 25
III. RESULTS. 25
A. Test Diets. 25
6. Food and Water Contaminants. 26
C. Mcrta I I ty/C I I n I c a I Observations. 26
D. Body Weight.......... 26
E. Food Consumption.. 26
F. Hematology. 27
G. Clinical Chemistry. 27
H. Ophthalmology. 27
I. Organ Weights. 27
J. Pathology. 26
IV. DISCUSSION. 26
V. REFERENCES. 30
APPENDIX I TEST ARTICLE ANALYSIS. 97
APPENDIX II 5002 CERTIFICATION PROF I LE/ANALYS I S. 119
APPENDIX III TEI ANALYTICAL CHEMISTRY METHODS. 123
APPENDIX IV FOUR WEEK SUBCHRONIC (EXPLOR ATORY/RANGE-
FINDING) ORAL (DIET) TOXICITY STUDY
OF TRINITROTOLUENE (TNT) IN THE B6C3F1
HYBR 1C MOUSE. 125
APPENDIX V HEMATOLOGY METHODOLOGY. 173
APPENDIX VI CLINICAL CHEMISTRY METHODOLOGY. 177
APPENDIX VII INDIVIDUAL ANIMAL DATA. 161
APPENDIX VIII CHLORTETRACYCL I NE CONTENT OF 5002 . 3 1 1
APPENDIX IX NITRATE, NITRITE AND MERCURY CONTENT
OF 5002 . 313
APPENDIX X CHICAGO WATER CHEMICAL ANALYSIS. 315
APPENDIX XI OPHTHALMOLOGY NARRATIVE REPORT. 317
APPENDIX XII PATHOLOGY NARRATIVE REPORT. 325
DISTRIBUTION LIST
336
1A6LL CL C.QHILLIL iQ.QLLLUC.LLl
VO.LLLL ill Li££ fcta
APPEND I)” X ' A OPh T HALMOLOGY REPORT (COMPLETE). 337
VQkQME. llli
APPENDIX X i IA PATHOLOGY REPORT (COMPLETE) . 537
* Requests for Volumes II
Effects Research Division,
and Development Laboratory
21701-5012 .
and III shoulG be directed to Healtf
U.S. Army Medical Bioengineering Researc
, Fort Detrlck, Frederick, Maryland
mi ql mm
Male
F ema
Test
Mean
Male
Male
F ema
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Male
F ema
Stat
for
F ema
Actual Doses Received. 32
e Actual Doses Received. 36
Diet Concentration of TNT..... 40
Survival Time. 41
Body Weight Values. 42
Body Weight Gain Values.. 45
e Body Weight Values... 46
e Body Weight Gain Values. 51
Food Consumption Values... 54
e Food Consumption Values..... 57
Hematology Values - Week 14. 60
e Hematology Values - Week
Hematology Values - Week 27
e Hematology Values - Week
Hematology Values - Week 52
e Hematology Values - Week
Hematology Values - Week 79
e Hematology Values - Week
Hematology Values - Week 10
e Hematology Values - Week
Clinical Chemistry Values -
e Clinical Chemistry Values
Clinical Chemistry Values -
e Clinical Chemistry Values
- Week 14.
Week 27.
- Week 27.
Week 52........
- Week 5 2.
Week 7 9.
- Week 7 9.
Week 105.
- Week 105.
alues - Week 14
- Week 14,
Week 27 . .
- Week 27
Week 52..
- Week 52
Week 79..
Clinical Chemistry Values - Week 52.........
le Cl. leal Chemistry Values - Week 52.
Clinical Chemistry Values - Week 79.
le Clinical Chemistry Values - Week 79.......
Clinical Chemistry Values - Week 105.
le Clinical Chemistry Values - Week 105.
Relative Organ Weight Values - Week 27.
le Relative Organ Weight Values - Week 27....
Relative Organ Weight Values - Week 52.
le Relative Organ Weight Values - Week 52....
Relative Organ Weight Values - Week 105.
le Relative Organ Weight Values - Week 105...
Organ Weight Values - Week 27 ...
le Organ Weight Values - Week 27.
Organ Weight Values - Week 52....
le Organ Weight Values - Week 52.....
Organ Weight Values - Week 105...
le Organ Weight Values - Week 105.
Istical Evaluation of Histopathologic Lesions
the 12-24 Month MS/SD and Terminal Sacrificed
kill QL IAfikki LSLkNkkkkkkl
lakle tit
IV.1 Body Weight Measurements of Male Mice. 13 8
' V . 2 Body Weight Measurements of Female Mice. 139
I V.3 Body Weight Measurements of Male Mice, Change
from Week -IB... 1 4C
iV.4 Body Weight Measurements of Female Mice, Change
from Week -IE.. 141
IV.5 Food Consumption Measurements of Male Mice. 142
IV.6 Food Consumption Measurements of Female Mice. 143
■ V .7 Hematology Measurements of Male Mice. 144
IV. 8 Hematology Measurements of Female Mice. 145
iv.9 Clinical Chemistry Measurements of Male Mice. 146
t V.1C Clinical Chemistry Measurements of Female Mice. 147
lV.11 Organ Weight Measurements of Female Mice, Percent
Terminal Body Weight... 148
i V , 1 2 Organ Weight Measurements of Male Mice, Percent
Terminal Body Weight. 149
IV.13 Organ Weight Measurements of Male Mice.. 150
IV.14 Organ Weight Measurements of Female Mice... 151
V I I . 1 Survival Rate Data. 182
Vi 1.2 Individual Body Weight Measurements (g). 187
v I I .3 Individual Food Consumption Measurements (g/day). 232
V! I .4a Individual Hematology Values - Test Week 14. 277
Vi 1.4b Individual Hematology Values - Test Week 27 . 27 9
VI 1.4c Individual Hematology Values - Test Week 52. 281
VI I . 4 d Individual Hematology Values - Test Week 79. 263
VII. 4e Individual Hematology Values - Test Week 1 05 . 285
VII.5a Individual Clinical Chemistry Values - Test Week 14.... 287
VII.5b Individual Clinical Chemistry Values - Test Week 27 .... 289
VII.5c Individual Clinical Chemistry Values - Test Week 52.... 291
VII. 5d Individual Clinical Chemistry Values - Test Week 79.... 293
YM.5e Individual Clinical Chemistry Values - Test Week 105... 295
VII.6a Individual Organ Weight Values (g) - Test Week 27 . 297
VI 1.6b Individual Organ Weight Values (g) - Test Week 52. 299
V I i.6c Individual Organ Weight Values (g) - Test Week 105. 301
l£iIR2£iOlijN
The U . S . Army Medical Research and Development Comm, and 9
(L'SAMRDu) has teen directed to evaluate the potential hazards to
living systems. of wastewater discharges from munitions
facilities. Of primary concern are the toxicologic effects to
mammalia" systems of trinitrotoluene (TNT; CAS Reg. No.
118-96-7). This high explosive Is routinely used In filling
shells and bombs. Wastewaters resulting from the loading of this
explosive Into shells are discharged Into the environment without
significant treatment and are subject to limitations Imposed t>
governmental regulatory agencies. Evaluation of the potential
hazards of these wastewaters to human health Is therefore c
necessary portion of the data-base required to establish
comprehensive environmental criteria.
Thie present study was conducted to aid in this evaluation ty
assessing the chronic toxicity and potential carcinogenicity cf
TNT in E6C3F1 hybrid mice when administered In the diet for at
least 194 weeks. Information ultimately derived from t h is
comprehensive long-term toxicology study will aid USAMRDC i r
developing criteria for the establishment of effluent standards
and in defining levels of treatment for Its pollution abatement
program.
The study reported herein was conducted In accordance with
the IITRI Quality Assurance Program designed to comply with FDA
Good Laboratory Practice Regulations (1). Thus, all terms usee
In this report, e.g. test article, raw data, specimens, etc.,
are In agreement with the definitions set fort hi ir, the
aforementioned document.
'1 • H£I£R1AL£ £N£ *
A • Ifeit fULiialfe
One hundred pounds of trinitrotoluene (TNT; CAS Reg. Nc.
I 18-96-7), Batch No. VOL 11-011, were made available for this
study from stocks at the IITR 1 Kingsbury Ordnance Plant (KOPr 1
Explosive Facility, La Porte, IN. The test article was stored at
the facility in the dark and at ambient room temperature a n d
relative humidity. Upon Initiation and at termination of the
treatment phase of the study, 30 g samples were taken anc stored
under conditions similar to those for the batch No. VOL 11-011.
The purity of the test article was determined by high
performance liquid chromatography, as described In Appendix I A
(page ), with analytical standards provided by the Sponsor.
TNT purity was analyzed three times during this study and tht
resuits were as follows: May 1962 (99.43 + 2.89?), Marc( 196;
(99.09 + 4.11?) and October 1963 (96.86 + 2.01?). \
1 A
»
Particle size analyses were done In November 1975 and
November 1961 by the Fine Particles Research Section of the
Chemistry and Chemical Engineering Depart mi ent of NT Research
Institute. The results were as follows:
C-fllfi. 1212 . N£L*£[[ifc.£L 12£1
ili£ LkCl
NkCLkL
1
Q-ii-Udw-l J. i.
klLffib.£L
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<22
1 87
3 6.4
36.4
6 8
1 3 .6
1 3.6
22-44
1 C 1
2 0.2
5 8.6
1 43
26.7
42.3
44-66
6 9
14.2
72.8
87
17.4
59.7
6 6-110
57
11.7
84.5
86
17.2
76.9
110-220
44
9.0
52.5
73
1 4.6
91.5
220-330
i e
3 .7
97 .2
20
4.0
95.5
330-440
9
1 .9
99.1
1 4
2.6
96.3
>440
2
0 .4
99.4
6
1 .6
99.9
B . IfiS.1 C.l£l£
Premixes for the test article, approximately 10? In Purina
Certified Rodent Chow No. 5002 (Ralston Purina Co., St. Louis,
MC.), hereafter r eferred to as 5002, were prepared on a monthly
basis Ir 4 kg quantities at the K0P by Chemistry Department
personnel. The 10? premixes and test diets were stored at
approximately 4°C. Undiluted TNT was handled In accordance with
procedures for explosive and fire hazards. The test article was
ball milled with equal parts of 5002 and subsequently diluted
with additional 5002 In a twin shell blender to yield approximate
1C? premixes. On and fol lowing Test Week 32, the 5002 used In
this procedure was passed through a No. 45 (350 urn) sieve prior
to balI milling with TNT. The procedure was changed because
homogeneity tests of some TNT diets showed a large relative
standard deviation.
Each TNT premix was tested for accuracy, homogeneity,
potency and recovery of the test article. Homogeneity testing
consisted of analyzing for test article concentration In each
batch of premix taken from six random locations of Its container.
Premix stability was established for a period of seven weeks and
later for a period of nine weeks by conducting homogeneity tests
at the Initial and the terminal points of the 7, as previously
reported (2), or 9 week period (see below). Recovery tests for
the premix consisted of adding a known quantity of test article
to an aliquot of the extracted premix. The spiked sample
subsequently underwent the Identical analytical procedure as the
actual premix.
T oxlcology Section personnel received the test article as
approximate 10$ prefixes In 5002. These prefixes posed little
explosive or fire hazard as previously demonstrated (2). Results
of premix analyses were as follows:
LOT NO.
DATE PREPARED
DATE ANALYZED
$ TNT 4
; S
.D .*
162-4
9-11-81
9-18-81
9.54
4
0.56
162-5
10-1S-81
10-23-81
9.94
4
0.46
162-6
11-12-81
11-20-81
9.70
4
0 .25
162-7
12-18-81
12-22-81
9.68
4
0.49
i 62-9
1-29-82
2-C2-e2
9.73
4
0.50
1 62-1 C
2-12-82
2-22-82
9.5 1
4
0.64
162-1 1
3-23-82
3-31-82
9.91
4
0 .27
162-12
4-26-82
5-06-82
9.99
4
0.11
162-17
6-02-82
6-07-82
9.88
4
0.23
162-17**
6-C2-82
8-18-82
9.47
4
0.43
162-19
7-13-82
7-19-62
9.04
4
0.33
162-20
8-16-62
8-19-82
9.58
4
0.29
162-21
9-10-82
9-16-82
9.87
4
0.20
162-22
10-18-82
10-22-82
9.85
4
0.56
162-23
11-22-82
12-01-82
9.47
4
0.68
16 2-24
12-2C-82
12-30-82
9.93
4
0.21
162-25
1-20-83
1-25-83
9.7 8
4
0.37
216-01
2-24-83
3-01-83
9.84
4
0.37
216-03
3-23-83
4-01-83
9.52
4
C .1 6
* Six sampling locations
* * Stability study
Following chemical analysis of the premixes to determine
test article concentration (Appendix IB), sufficient quantities
were diluted with 5002 In a twin shell blender by toxicology
personnel to achieve the concentrations of the test article
necessary to administer the required dose levels on a mg/kg/day
basis. the previous weeks’ body weight and food consumption
measurements for each test group, by sex, were used to calculate
the predicted weight gain and food consumption and afterward, the
desired dietary coneentrat Ions of the test article. Eight kg of
each test diet were prepared on an approximately weekly basis.
Unused portions of 10$ premixes were returned to KOP for disposal
In accordance with instructions for safe disposal of explosives.
Surplus and uneaten portions of test diets were Incinerated.
Thirty-six test diets (2 diets/sampling week) used In Test
Weeks 1, 8, 14, 20, 26, 32, 38, 44, 50, 57, 61, 67, 74, 79, 65,
91, 9 7 , and 104 were analyzed for concentration and homogeneity.
I r- addition, one test diet was monitored for stability under
animal cage conditions for one week. First, It was sampled the
day it was placed in the animals' cages and again one week later
from the uneaten portion of the diet. Recovery studies of test
diets consisted of adding a known quantity of test article
(spiking) to a weighed quantity of untreated 5002 In a measured
volume of acetonitrile (the solvent used in the extraction
procedure). The spiked samples subsequently underwent the
Identical analytical procedures as the actual diets.
One sample of 5002, Lot No. March 24 82 G, was analyzed
during the course of the study by Trace Elements, Inc., Park
Ridge, IL (TEI) for those contaminants listed In the 5002
certification profile shown in Appendix It. The analytical
results are also shown In Appendix II except for the
chIcrtetracycI I ne levels shown in Appendix VII. The references
to the procedures used by TEI are In Appendix III. On the basis
of the analytical results for ch I ortetracyc I I ne content, aliquots
from this and three additional reserve samples of 5002 were sent
to TEI for analysis. In addition, aliquots from these four
reserve samples were sent to Scientific Associates, Inc., St.
Louis, MO, Woodson-Tenent Laboratories, Inc., Memphis, TN, and
Harris Laboratories, Inc., Lincoln, NE for chIortetracycI I ne
analysis. Samples of each 5002 lot used In the study were also
analyzed for nitrate, nitrite and mercury content by TEI.
c • dulniflis.
B6C3F1 mice obtained from Charles River Breeding
Labonator I es, Wilmington, Mass., Portage, Ml facility were used
for this study. Three-hundred fifty eight males and 352 females
were received in good condition on September 16, 1981. They were
3 to 4 weeks old upor arrival and mean random body weights
recorded within three days of receipt were 14.9 + 3.4 g (males)
and 13.1 + 2.4 g (females).
The shipment was housed In two quarantine rooms, one for
each sex. The animal room conditions during quarantine, pretest
and test periods were as follows; 21-25 C° ambient relative
humidity (3C-7C?) and 12 hour llght/12 hour dark cycle. No other
test animals were In the rooms. The animals were housed five per
polycarbonate cage (16.5"x 8"; 8" height) with Ab-sorb-drl
bedding (Ap-sorb-drl Inc., Rochelle Park, N.J.) from arrival
until their termination. Animals were transferred to clean cages
twice weekly. Each animal was Identified during the quarantine
period by a combination of cage number and tall mark. Test
animal selection was done at the onset of Test Week -2 (2 weeks
prior to Initiation of treatment). Animals placed on test
received a study-unique test animal number (N=600) which appeared
as a combination ear punch and toe clip. The Identifying ears
and paws were Included with necropsy specimens. This number
appeared on 1 h e cage card that also contained the study number,
dose level and sex. In addition, the cage cards were color coded
as to the dose level and sex.
Upon arrival at the IITRI animal facility, the animals were
held In quarantine for 13 days. During this period, they were
cbseived for signs of disease, general unthr I ft I ness , poor coat,
discharge from body openings, abnormal feces, etc. Any animals
found to be unhealthy were eliminated from the test animal
selection process. At the end of the quarantine period, five
animals of each sex were sacrificed. Extensive gross necropsies
were performed under the supervision of the pathologist. Blood
samples were collected for measurments of hematology and cllncal
chemistry parameters (see section II.D.) Results of pretreatment
health screen were within limits for the mice of this strain and
age. Microbiological examination of the digestive and
respiratory system for pathogens, molds, yeasts, parasites and
Mycgp I flsmp pjilnifinis. was also performed for the above mice with
negative results. Serum antibody titer was determined for the
following diseases: GD-VI1 virus, K virus. Mouse Adenovirus,
Sendai virus, Reovlrus 3, Pneumonia virus of mice. Lymphocytic
Choriomeningitis, Polyoma virus. Minute virus of mice. Mouse
Hepatitis and Ectomella. These antibody titers were negative as
measured by Microbiological Associates, Bethesda MD.
Animals received 5002 rodent chow from arrival urtll their
termination, except during a 2 to 5 hour fast prior to blood
collection and/or scheduled sacrifice. The food was available
from powdered diet feeders (Model LC-207/C, Wahman Mfg. Co.).
Tap water was available && lib.lluJD from glass or plastic bottles.
Bimonthly analytical results of drinking water of the City of
Chicago were Included In the monthly or bimonthly technical
reports and an example is shown in Appendix X, page 316.
d . iACLfiLlmaatal Q.e.s_LflJi
Following the quarantine period, test-eligible animals were
assigned to four treatment groups by a stratified randomization
procedure (blocked by body weight). Following assignment to
treatment groups, all animals were randomly assigned test animal
numbers as shown below. The animal cages were assigned permanent
randomized location on racks without regard to dose level or sex.
Mean body weight values at randomization were 19.1 + 2.3 g for
males and 15.8 ± 1.8 g for females. This procedure was performed
at the onset of Test Week -2. The animals were approximately 6-7
weeks old upon Initiation of treatment and body weight ranges
recorded during Test Week -1 (the most recent data prior to
Initiation of treatment) were 20.8-26.8 g for males and 15.0-17.9
g for females. The first day of exposure to the test article was
October 14, 1981. Dietary administration continued until Test
Week 106 (October 25, 1983).
lL£^i!ii£ai £lq.u.& iilatailan:
Treatment
An I ma
Dose Level Test Anlma
An I me I
Group
Treatment
per Sex
(mg/kg/day)
No. (males)
No . ( f err a 1
1 .
-
75
0 .0
1- 75
76-1 50
1 1 .
TNT
75
1 .5
151-225
226-300
1 1 .
TNT
7 5
1 0.0
301-375
376-450
1 V .
TNT
7 5
7 C . 0
451-525
526-600
The dose levels for this study were selected on the basis of
results of the "four Week Subchronic (Exploratory/Range-Flndlng)
Oral (Diet) Toxicity Study of Trinitrotoluene (TNT) In the B6C3F1
Hybrid Mouse," performed by IITRI under Contract No.
DAMD17-79-C-9120. The report of this study Is Included In
Appendix IV, page 127.
The appropriate test diets were available to tne test
animals lltilu.IL from Test Day 1 until their termination,
except during a 2 to 5 hour fast prior to either blood collection
in Test Weeks 14, 27, 52, 79 and 105 or scheduled sacrifice in
Test Weeks 27, 52 and 105-106. Thus, all animals received the
appropriate test diet until approximately 2-5 hrs prior to their
scheduled sacrifice. Weekly test diets were prepared for each
treatment group, by sex, on the basis of the projected body
weight and food consumption data.
Commencing with Test Week -1 until their termination, all
animals were observed once dally In the morning for any
pharmacologic and/or toxicologic signs. Afternoon mortality
checks were Initiated on Test Day 1. The presence or absence of
red bedding |n animals' cages was recorded weekly from Test Week
1 until termination. Physical examinations, which Included
palpations for masses, were conducted weekly from Test Week -1
until Test Week 15 and then biweekly until Test Week 104. Food
consumption was measured weekly for each cage of test a nIm a Is
commencing with Test Week -2 through Test Week 13 and biweekly
until Test Week 104. Mean dally food consumption per animal was
calculated from these data. Body weight values were recorded
ween Iy starting In Test Week -2 until Test Week 13, and biweekly
thereafter until termination.
All surviving animals were subjected to ophthalmic
examinations during Test Weeks -2, 25, 51, 77 and 103. The
examination consisted of Indirect ophthalmoscopy and
biomicroscopy. Only animals found to be free of clinically
apparent lesions In the pretest examination were used In the
study .
blood 5£T r its *ere pe^ 'cc kc I collected for measurements
of hematology and clinical chemistry p a r a me t er s for 10 randomly
selected rr'ce/sev/cose level. During Test Weeks 27, 52, and 10 5,
ihe selected mice were sacrificed and blood was collected prlcr
to necropsy. OuMrg Test Weeks 14 and 79, one set of 1C
mice/sex/dose level was randomly selected for hematology tests
anri a second set of mice was selected for measurements of
clinical c n em Is tr y parameters. Approx I mate Iy 0.5—1 • w ml of blood
*a.s co 1 lectec from each animal via the orbital sinus. The
sarnies were collected and analyzed In a randomized order over a
7 oi 5 consecutive day pertod.
T r. e following parameters were measured:
tifemaifiiftttildi.
Hema toe r 11 (he t)
HemogIob I n (Hgb 1
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Hemoglobin Concentration (MCHC)
Methemoglobln (METHGbT
Erythrocyte count (RBCs)
Plateiet count !PLT)
Leukocyte count (WBC), total and differential
Reticulocyte count (RETlC)
RBCs with Howell-Jolly bodies (qualitative) (HOW J 0 L)
RECs with Heinz bodies (qualitative)
SLLlalCLfll C.iu*ails.lE.ii
GIucose (GLU)
Blood urea nitrogen (BUN 1
Serum glutamic-pyruvic transamlnase (SGPT)
Bilirubin, total and direct (T-BIL and D-BIL)
Triglycerides (TRIG)
Total cholesterol (CHOL)
Total protein (PRO)
Albumin (ALB)
Globulin (calculated value) (GLOB)
A/G ratio (calculated value) (ALB/GLOb)
Methods used to measure the above parameters are listed I r
appendix V (Hematology) and Appendix VI (Clinical Chemistry).
Ail animals which were sacrificed In a morltund state or
died on test were necropsIed regardless of autolytlc state. Ten
randomly selected an ImaI s/s ex/dose level, after exclusion of
animals designated for serial blood collection, were sacrificed
duing Test Weeks 27 and 52. Two hundred seventy surviving test
an're Is were sacrificed and necropsled Ir random order during
Test Weeks 105 and 106 (October 12 to 25, 1983). Terminal body
weights were recorded immediately prior to sacrifice following a
2 to 5 hour fast. Euthanasia was accomplished with carbon
dioxide anesthesia followed by exsanguInat I on from the orbital
sinus or abdominal aorta. The necropsy procedure was a thorough
and systematic examination of the animal viscera and carcass with
collection and fixation of the following tissues:
Adrenals
Bone marrow smear
•Brain
Cecum
Colon
Costochondral junction, rib
Duodenum
EpId Idymes
Esophagus
Eyes and optic nerves
Gall bladder
Gross lesions
•Heart
I 1 e u m
Jejunum
*K I dney s
Larynx
•Liver
Lungs and mainstem bronchi
Lymph nodes (miandibu'ar and mesenteric)
Mammary gland
Muse I e
Nasal turbinates
Ovaries
Pancreas
Pituitary gland
Prostate
Rectum
Salivary gland
Sciatic nerve
Seminal vesicles
Skin, abdominal
Spinal cord (cervical, thoracic, lumbar)
•Sp I een
Sternum, including bone marrow
S tomac h
•Testes
Thymus
Thyroids (parathyroids)
Tissue messes
Trachea
Urinary bladder
Uterus
* These organs were weighed during the scheduled necropsies.
Ail -tissues, except eyes, testes and bone rrarro* smears,
•ere fixed at a thickness not exceeding 0.5 cm in 1 C% neutral
buffered formalin ( N B F 1 w h ' c h was changed 2 4 hours later. Eyes
and tes 4 es were fixed if' 3? aqueous glutaraldehyde anc Bcuir's
Solution, respectively, for 24 hours. They were transferred tc
5Qi e thc.no i for 24 hours, then placed In 70 % ethanol . Bone
marrow smears were prepared from, the femur using the "paint brush
technique". They were air-dried and fixed in absolute methanol,
lungs ano urinary bladder were Inflated with NBF prior tc
immersion in this fixative. The stomach was opened and flattened
on paper prior tc fixation. All tissues examined microscopical I v
were cut at a thickness of 4 to 6 mIcrons and stained wit r
hematoxylin and e o sIn.
Tissues from all control animals and those receiving 7C.C
mg/kg/da, were subjected to comprehensive histopathologic
exam. Inarlon, defined as microscopic examination of the following
tissues and/or organs:
A d r e r> a I s
* 6 r a ! r (3 sections)
Cecum;
Colon
Duodenum
EoIdIdymes
Esophagus
Eyes and optic nerve
G o n a d s
Gross lesions
heart
j e j u n u m
K I c n e y s
L a r y r x
Liver
L u nc s and malnster bronchi
Farr ary gland
he sen ter lc lymph node
r ' c, n c r e a s
f r estate
F'tctun
Seminal vesicles
Sr In, abdominal
i ; 1 r a I cord (cervical, thoracic and lumbar)
S P i t e -
E t e- r n u it Including bone m arrow
C i om ac t
Tissue masses
r r v r c Ids/peratfvrolds
Tissue- masses
Iterus
Urinary bladder
*(1) frontal cortex and basal ganglia; (2) parietal cortex and
t h a I m u s ; and (3) cerebellum and pons.
Tissues from all animals receiving 1.5 and 10.0 mg/kg/day
were subjected to limited histopathologic examination defined as
microscopic examination of at least the following tissues and/or
organs: bralr (section of frontal cortex and basal ganglia;
section of parietal cortex and thalmus and section of cerebel lum
and pons) gonads, heart, liver, kidneys, spleen, spinal cord
(cervical, thoracic and lumbar) and in addition for the females,
urinary bladders and sternal bone marrow.
E • ilails.tlc.al Aaalxaia
Those variables that were repeatedly measured, e.g. body
weight, food consumption, and clinical pathology parameters were
statistically analyzed using a multivariate analysis of variance
fcr repeated measurements model. Variables that were measured a
single time, e.g. organ weights, were analyzed using both
univariate and multivariate analysis of variance procedures. In
the presence of significant ANCVA results, a series of pest-hoc
analyses were conducted. Individual between group comparisons at
each tine-point were performed using Tukey's b test for multiple
comparisons. Frequency data, such as Incidence of mortality,
gross necropsy observations and histopathologic lesions, were
compared using log linear analysis techniques where appropriate.
Time to death data were analyzed using the Kaplan-Meler and Cox
regression analyses. Individual animal data can be f o u n p in
Appendix VII.
'll. Eliilli
A • l£s.l ilelsi
Weekly doses received by test animals based on their bedy
weight end food consumption were very close to the Intended dose
levels. F*ean dose calculations across time were within 9 6? of
anticipated values for all treatment groups (Tables 1 and 2).
Analytically determined concentrations of ThT In test diets
were generally close to their Intended concentratIons. The only
exception occurred at Test Week 20 for the 10 mg/kg/day diets.
The homogeneity (relative standard deviation) of some of the
tested diets was not satisfactory, mostly at the lowest
concentration and In the period between Text Weeks 14 and 26.
-*est diets were sampled one week after being placed |r the
animal room, a si Ight aecrease in TNT concentration occurred
IT at Ie o ) . T he known volatility of TNT may have accounted for
1 h I e c h urge ,
t" • LcfiC flaw waIae tcatfloiiaflais.
The analysis of a 5002 sample for those contaminants listed
in + he 3 00 2 certification profile is shown In Appendix III. The
results of the repeat testing of 5002 samples fcr
chi or tetracycline content are contained In Appendix VIII. Tne
three reference laboratories which reanalyzed the 5002 samples
fcilowirg TEl generally reported negligible quantities of
c h i or te rr ac yc 1 I ne .
A sample from each 5002 let was analyzed for nitrate,
nitrite and mercury content. The results are shown In Appendix
IX. Analytical results obtained from a sample of Chicago water
are contained In Appendix X.
' • (ificiaJ.llxZ.CJ.laic.fll CflAflcxAiicaA
TNT did not Induce lethality at the doses tested In this
s + u c y as mean survival times were similar among control and
treatment groups. In addition, clinical signs related to TNT
a dm I n | s t,-al 1 on were net readily apparent (Table 4).
5- fcflfii DLCifim
Reductions I" body weight gains were seen for male and
female mice acr I rIs+ered 70 mg/kg/day cf TNT. This decrease was
a c proximo tely 10? for both sexes up ttrougr. the first 6-6 months
c* the study, with a further reduction of about 15? for females
a r d 20? fcr males for the remainder of the test period. Although
not statistically significant, an approximate 5-7? reduction |r
body weight gain was apparent for males but not females at the 10
mg/kg/day dose level for the majority of the study. Body weights
were no* a ' t e r e d for mi Ice given 1.5 mg/kg/day (Tables 5-6).
f • Lew;. Cfiaflu.ffiii.llfiu
After an initial statistically significant (p < C.5)
decrease In food consumption for the male mice at the 70
mg/kg/day dose level. Increases up to 30? were evident for the
mice o f both sexes throughout the study. Sporadic Increases and
decreases were observed at the lower dose levels and were not
cons ICerea to be related to TNT-treatment (Tables 9 and 10).
■r- v
*
f . ti£EiiQ.l£ay
Anemia, as evidenced by reductions in hematocrit, hemoglobin
and RBC count, was general ly seen from Test Week 27 through Test
Week 79 for males and females administered 70 mg/kg/day. The
effect was mild and normal physiologic compensatory responses to
the anemic state, e.g. reticulecytosis, macrocytosls, etc., were
not apparent .
Lymphocytosis was observed during the Test Weeks 27 and 52
primarily for the 70 mg/kg/day animals. Males receiving 10
mg/kg/day also had increased lymphocyte counts at Test Week 27.
No other hematologic changes were seen for TNT-treated mice
(Tables 11-20; Figures 1 and 2).
G • 2J.ialC.fll CaflmlS.lL*
Females at Test Week 14 and males at Test Week 52 receiving
70 mg/kg/day were hypotriglycerldemlc. This effect was not seen
at the other sampling points or for the other dose levels.
Occasional decreases In serum total protein (statistically
not significant) and globulin levels were also seen at the high
dose level. This was seen for males at Test Week 52 and for
females at Test Week 105. A/G ratios for + hese animals however
were similar to those of the control animals. No other measured
clinical chemistry parameter was altered by TNT treatment (Tables
21-30 .
h • Cflliiiifllinfllfli*
The ophthalmology narrative report Is contained in Appendix
XI (attached) and In Volume II*, Appendix XIA. All opthalmologic
abnormalities seen occurred In random fashion and were net
considered to be treatment-related.
I • QjLfi.AL Wfl.ifi.iiA
Occasional elevations In brain, heat, kidneys, liver, spleen
and testes relative weights were seen only for mice receiving 70
mg/kg/day. These changes were small although statistically
significant, however a pattern with respect to sex or time was
not observed (Tables 31-42).
* Requests for Volume I I
Research Division, U.S.
Development Laboratory,
217 0 1 -5012 .
should be directed to Health Effects
Army Medical Bioengineering Research end
Fort Cetrick, Frederick, Maryland
0 .
iiflj.li it
The Pathology Narrative Report appears In Appendix XII
(atfacned) ana in Volume III*, Appendix XII A.
Gross lesions observed at the 6 and 12 month interim
sacrifices and microscopic examinations of tissues taken during
these study periods die not reveal lesions which were considered
to be Induced by the adm. inlstraticn of TNT.
Enlarged spleen and lymph nodes for females receiving 7C
rrg/kg/day were observed at the terminal sacrifice necropsy and in
mice that died or were sacrificed moribund between 12 ana 24
norths. Microscopic examination of these animals revealed
leukemia (granulocytic or lymphocytic type) and malignant
lymphoma (histiocytic, lymphocytic or mixed type) In the spleen,
'.eukemla and malignant lymphoma were systemic reticuloendothelial
neopiasias ana die involve other tissues and organs. There was a
dose-related increase of occurrences of leukemia/malignant
lymphoma for the female mice (Group 1, 16.7$; Group 2, 27.8$;
Group 3, 31.5$ and Group 4, 38.9$). However, only for the female
mice at the 70 mg/kg/day dose, this Increase was statistl ally
significant (p < 0.01) (Table 43).
'V. iLLiCUiilQN
The dose levels for the chronic fox i cIty/ca re I nogen i c I ty
study of TNT In B6C3F1 hybrid mice were selected on the basis of
results of a four-week oral (diet) range-f I ndIng study. In this
range-finding study groups of 10 mice per sex received TNT at
doses of 0, 0.3, 2, 14, 100 or 700 mg/kg/day. Toxicologic
endpoints included clinical signs, body weights, food
consumption, hematology, clinical chemistry, organ weights, and
gross and tissue morphology.
T h I s study examined the oral toxicity of TNT following
•jletofy administration to mice for four weeks. Doses of up to
700 mg/kg/day failed to result in death. The only clinical
findings were reductions In body weight gain at 100 mg/kg/day and
slight body weight loss at the 700 mg/kg/day dose level.
* Requests for Volume III should be directed to Health Effects
Research Division, U.E. Army Medical Bioengineering Research anc
Development Laboratory, Fort Cetrick, Frederick, Maryland
21701-5012.
Major toxic effects Included hemolysis of red cells as
suggested by hyperbilirubinemia and splenic hemosiderosis.
Additional toxic effects of TNT seen primarily at the 700
mg/kg/day dose level Included leukopenia without a differential
shift, thrombocytosis, slight hepatomegaly, marginal decreased
testes weights, and elevated renal weights. None of these organ
weight changes was accompanied by treatment-related histologic
alterations. Minimal toxic effects were seen at the 10C
mg/kg/day dose level. Thus, the appparent no-effect level under
the conditions cf this range-finding study was 14 mg/kg/day.
The chronic administration of TNT to male and female B6C3F1
mice at doses up to 70 mg/kg/day did not alter survival rates.
Clinically, reductions In body weight gain (15-20$) were seen for
animals of both sexes given 70 mg/kg/day; a 5-7$ reduction
throughout the treatment period also occurred for males at the 10
mg/kg/day dose level. By conrast. Increases In food consumption
were seen for the female mice at the high dose level throughout
the study and for the male mice, at this dose level after an
initial decrease In food consumption.
Anemia consisting of reduced hematocrit, hemoglobin and RBC
count was sporadically observed for males and females receiving
70 mg/kg/day. The effect was mild, and compensatory physiologic
responses, l.e. retIcuIocytosIs , macrocytos1s, etc., were net
apparent. In addition, splenic lesions previously seen for
TNT-treated rats and dogs (2,3) which were cons leered secondary
to a hemolytic anemic state, l.e. sinusoidal congestion,
extramedullary hematopoiesis, and hemosiderosis, were net
observed In this study. This was probably a function of tie
limited extent of anemia seen at the doses tested.
Additional toxic effects of TNT to B6C3F1 mice were seen
only at the 70 mg/kg/day dose level. These Included potential
liver injury observed as hypotrIgIycer1demI a, reduced serum
globulin levels ( A / G ratios were unaffected), and hepatomegaly
without microscopic alterations. Sporadic increases for kidneys,
spleen and heart weights were seen but were not supported
histological ly.
Lymphocytosis was apparent for animals administered 70 and
to a lesser extent 10 mg/kg/day. For the female mice an
increased Incidence of Ieukemia/maI I gnant lymphoma, with a
positive dose response, was seen In the spleen. This Increase
was statistically significant (p < 0.01) only for the female mice
at the highest dose level tested. This dose-related Increase In
the Incidence of malignancy was not seen In the male mice. The
Incidences of these neoplasias in the untreated study control
male and female mice (17$ for each sex) were within range of the
historical control values for male and female mice (13 + 7$ and
9
2 7 + 10$, respectively) from the N T P Rodent Control Date base,
Marcn, 1963. No other tumor I genic effect of TNT was apparer.t In
this study.
In summary, the major toxic effects observed during the
administration of 7C mg/kg/day of TNT to B6C3F! mice for up tc 24
montns included anemia and probable hepatotox1cIty. In addition,
peripheral lymphocytosis and leukemia/malignant lymphoma here
apparent In the spleen. A single Instance of lymphocytosis was
evident 'or males at the 10 mg/kg/day dose level. These animals
also demonstrated a 5-7$ reduction In body weight gain throughout
+ he treatment period. This dose, therefore, appears tc be
m|n|ma| jy toxic, and the no-effect level under the conditions of
the present study Is 1.5 mg/kg/day.
V. £JLifL££LNQ.££
! . Good Laboratory Practice Regulations. Fed. Reg. 21
CFR Part 36. 60013-60020, 1976.
2. Levin6, 3.S., Furedl, E.K., Gordon, D.E., Burns, J . M. ,
and Lish, P.M. Thirteen week oral (diet) toxicity study
of trinitrotoluene (TNT), hexahycro-1,3,5-trInItro-1,
3,5-trIczlne (RDX) and TNT/RDX fixtures In the Fischer ; 4 4
rat, 1 I T R I Final Report No. L6116/L6121, Study No. 1 .
3. -evIre, B.S., Rust, J.P., Burns, J.V. and Llsh, F . P .
Twenty-six week subchrcnlc oral toxicity study cf
trinitrotoluene (TNT) In the Beagle dog. I ITRi F i r c I
Report No. 16116, Study No. 5.
roim month chronic toxicity/carcinogf.nicity study of
TRINITR010LUF.NF<TNT) IN 1HF. BBC IF 1 HYBRID MOUSF
MAL.F ACTUA1. DOSF.S RFCFIVF.D < inf. /k y )
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TEST DIET CONCENTRATION OF TNT
'ESI
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%
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I
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Diets stability test after they were one week in the animal c
TABLE 4
TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
MEAN SURVIVAL TIME*
DOSE
MEAN
SURV1 VAL
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No significant differences between control and treatment groups, p<0.05.
I IT RESEARCH INSTITUTE
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOCFNICTTY STUDY OF
TP I N IT ROTOLUF.NE( TNT ) IN THE D6C3F1 HYBRID MOUSE
MAI.E BODY WFICHT VAI.IIES < f. 3
[HEAN AND STANDARD DEVIATION (n)i
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TWENTY FOUR MONTH CHRON IC TOXICITY/CARCINOCENICITY STUDY OF
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56
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
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FEMALE FOOD CONSUMPTION VALUES (p,/day)
[MEAN AND STANDARD DEVIATION (n))
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
TRINITROTOLUENE*TNT) IN THE B6C3F1 HYBRID MOUSE
FEMALE CLINICAL CHEMISTRY VALUES - WEEK 27
[MEAN AND STANDARD DEVIATION (n)J
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOCENICITY STUDY OF
TRINnROIOLUFNElTNT) IN THE C6C3F1 HYBRID MOUSE
FFMAL.F CLINICAL CHEMISTRY VALUES - WEEK S2
[MEAN AND STANDARD DEVIATION (n > J
75
TWENTY FOUR MONTI! CHRONIC TOXICITY/CARC INOC.FNIC ITY STUDY OF
TP IN T TP' 'TOLUENFlTNT) IN THE B6C3F1 HYBRID MOUSE
FEMALE CLINICAL CHEMISTRY VALUES WEEK 7Q
(MEAN AND STANDARD DEVIATION ( i> ) J
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
TRINITROTOLUENE(TNT) IN THE B6C3F1 HYBRID MOUSE
FEMALE RELATIVE ORGAN WEIGHT VALUES - WEEK 105
I(r ORGAN WEIGHT/r BODY WEIGHT) x 100J
[MEAN AND STANDARD DEVIATION (n))
TWENTY FOUR MONTH CHRONIC TOXICITY/CARC INOCENICITY STUDY OF
TRINITROTOLUENE*TNT) IN THE RAC IF! HYBRID MOUSF
FEMALE ORGAN WEIGHT VALUES < p, ) - WEEK lO^
[MEAN AND STANDARD DEVIATION <n)l
Table 43
Twenty-Four Month Chronic ToxicIty/Carc1 nogen 1city Study of
Trinitrotoluene (TNT) in the B6C3F1 Hybrid Mouse.
Statistical Evaluation of HistopathoIogIca I Lesions for the
12-24 Month MS/SD and Terminal Sacrificed Females
DOSE (mg/kg/day)
1*5. io*o. zo^a
H±£LN£i£ - ££Uli£fcilAZfci£ki£MMI LLMEJdQMA
PRESENT 5(9.2$) 11(20.4$) 8(14.8$) 10(18.5$)
ABSENT 49 43 46 44
£EJL££N - LEUH£ElAZU£JJ.£ti&MI LmEUQMA
PRESENT 9(16.7$) 15(27.8$) 17(31.5$) 21*(38.9$)
ABSENT 45 39 37 33
k±V£R - A&£UQMA££Afi£JHGtlA
5(9.2$) 11(20.4$) 8(14.8$) 10(18.5$)
49 43 46 44
PRESENT
ABSENT
FIGURES
93
FIGURE 1
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WEEK
70
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LEGEND- t X
0-0 MG/KG/DA v
10 MG/KG/DA v
>->-->■ 1 . 5 MG 'KG 'D A v
v ^ 70 MG'KG./DA V
• I
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY' STUDY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
MEAN HCT VALUES ( percent ) VS TIME
MALES AND FEMALES COMBINED •!
• I
94
FIGURE 2
• TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY’ STUDY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
MEAN MALE LYM VALUES (X 10 3 /MM 3 ) VS TIME
10.5-
10-0-
V 8-0-
A
S 7.5-
1 7.0-
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WEEK
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APPENDIX I
TEST ARTICLE ANALYSIS
Hi
PREVIOUS PAOF
IS BLANK
97
Vr*. ■.
APPENDIX IA
ANALYSIS OF THE TNT TEST ARTICLE
SCOPE
1.1 The procedure describes the analysis of the TNT test article for
purity.
1.2 This method is recommended for use only by experienced analysts fami-
iar with Hiah Performance Liquid Chromatography (HPLC) or under close
supervision of such qualified person.
INTERFERENCES
2.1 Solvents, reagents, qlassware and other sample processing hardware may
yeild discrete artifacts and/or elevated baselines causing misinter¬
pretation of chromatoarams. All of these materials must be shown to be
free from interferences under the conditions of the analysis by running
method bland.
EQUIPMENT
3.1 Higher Performance Liquid Chromatography
• constant flow, isocratic pumping system
• reverse phase column, 10 p - 3.9 run x 30 cm p-Bondpak C ]B column
• ultraviolet detector capable of monitoring \ = 254 nm
• strip chart recorder and electronic integrator capable of measuring
peak areas and performing an internal standard calculation
REAGENTS
4.1 Benzophenone, an internal standard, Aldrich Chemical Company (Purity 99:.)
4.2 Methanol, Acetronitrile, and Water H p LC Grade or equivalent
4.3 Standard Army Reference Material (S.A.R.M.) 2,4,6-TNT, Supplied by sponsor
(pu rity 99-8 )
CALIBRATION
5.1 Calibration standards were prepared fro r ' stock solutions containing
200 pg TNT, and benzophenone per ml acetonitrile so as to bracket
the workinp ranae of the chronatonraphic system. These concentrations
were: 2 pa/ml, 10 yg/ml, 20 yg/ml, and 40 yg/ml.
5.2 A constant injection volume of 10 yl was employed for all measurements.
5.3 In order to determine the precision of the HPLC system, a series of 6
replicate injections of the 20 ya/ml solution were made.
5.4 Retention times should remain relatively constant (within + 5 % day to
day) with TNT being 5.1 minutes, and benzophenone 8.2 minules under
the specified conditions. If the retention times are not within + 5 %,
supervising chemist should be informed prior to the analysis and cor¬
rective actions should be taken.
QUALITY CONTROL
6.1 Before processing any samples, the analyst should demonstrate through
the analysis of a blank that all glassware and reagents are interfer¬
ence free.
6.2 In a typical sample set, a minimum of one blank and five samples will
be analyzed.
6.3 The analyst will follow each step in an analytical protocol without
deviation or improvisions in order to accurately assess the perfor¬
mance of the method. Prior to making any changes in the procedure,
analyst will consult the supervising chemist and the supervising
chemist and Q.A. officer will review and approve all the changes.
SAMPLE PREPARATION
7.1 The test article will be spread on a sheet of paper, and five samples
will be taken from different areas. Each sample shall have a weight of
%150 mg. The samples will be collected in amber yials and stored at
refrigerator temperatures in the dark until analysis.
7.2 A portion of the sample (100 mg) will be weighed and transferred to a
100 ml volumetric flask. The internal standard will be added and it
will be added and it will be diluted to volume. It will be further
diluted to a concentration of 20 yg/ml and analyzed by high performance
liquid chromatography.
7.3 If the sample is not analyzed immediately it will be stored at refrig¬
erator temperatures in the desk.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
8.1 Each sample was analyzed by reverse phase HPLC usinq the conditions
described below: Column 3.9 nrr x 30.0 cm y-Bondpak Cis; Solvent
System, mentanol:water (70':3 CL . v/v); Flow Rate, 1.0 ml/min;
99
Detection, UV at 254 nm; Sensitivity, 0.1 AUFS. The retention times
of TNT and benzophenone were 5.1 and 8.2 minutes, respectively. The
limit of detection was 2 yg TNT/ml acetonitrile and is defined as 5x
the background noise. The representative chromatogram is Figure IA. 1 .
8.2 The chromatographic system was calibrated daily with a minimum of two
injections of our standard representative of chromatographic range.
8.3 An injection volume of 10.0 yl was used for each sample. If the peak
area exceed the linear range of a sample it was diluted and reanalyzed.
CALCULATIONS
9.1 Determine the concentration of TNT using the formula:
% TNT in Sample = (A*) ( w1s ) * D x 100
(Fx) Ais (Ws)
where
Ax = Area (X) where x is TNT
Ais = Area (internal standard)
p _ Area (X) x weight (is)
Area (is) x weight (Wx)
Wis = Weight of the internal standard
Ws = Weight of the sample
D = The dilution factor
Wx = Wt of component x is TNT
9.2 The results should be reported in percent TNT in the sample. Where
replicate samples are analyzed, all data should be reported. All
results were recorded in standard IITRI logbooks and these plus
chromatograms and data tapes were retained in the Chemistry Division
Q.A. files.
Bill
APPENDIX IB
ANALYSIS OF TNT IN DIETS
SCOPE AND APPLICATION
1.1 This method covers the determination of TNT in diets from the 0.0005% to
0.1% level.
1.2 The sensitivity of this method is dependent on the level of inter¬
ferences present in the samples, rather than the instrumental limitations.
1.3 This method is recommended for use only by experienced analysts familiar
with High Performance Liquid Chromatography (HPLC) or under close super¬
vision of such qualified persons.
SUMMAPJ OF TEE METHOD
2.1 A weighed quantity of the sample was stirred with 50 ml of acetonitrile
for 30 minutes. The suspension was filtered through a porous glass
filter and the filtrate was transferred with washings to a volumetric
flask. Benzophenone, the internal standard was added to the filtrate or
a portion, thereof and this solution was diluted to its final volume.
The samples were analyzed using reverse phase high performance liquid
chromatography. Each was eluted on 3.9 mm x 30.0 cm p-Bondapak Ci 8
column with methanol:water (70%:30%) and the eluant was monitored with
an ultraviolet absorption detector at X = 254 nm.
INTERFERENCES
3.1 Solvents, reagents, glassware, and other sample processing hardware may
yield discrete artifacts and/or elevated baselines causing misinter¬
pretation of chromatograms. All of these materials must be shown to be
free from interferences under the conditions of the analysis by running
method blanks.
3.2 Interferences coextracted from the samples will vary considerably from
source to source, depending on the type of animal feed used in the study
MATERIALS
4.1 Erlenmeyer flasks, 125 ml.
4.2 Filtering apparatus, vacuum flask, 125 ml; fritted glass filters,
porosity M, ASTM 10-20 microns.
EQUIPMENT
5.1 Mettler Grammatic Analytical Balance, No. 1-910
5.2 Corning Hot Plate Stirrers, BC 351
5.3 Buchi Evaporator, Model R
5.4 Sample Clarification Kit, Organic (Water's Associates)
5.5 Higher Performance Liquid Chromatography
• constant flow, isocratic pumping system
• reverse phase column, 10 m - 3.9 mm x 30 cm p-Bondapak Ci# column
• ultraviolet detector capable of monitoring X = 254 nm
• strip chart recorder and electronic integrator capable of measuring
peak areas and performing an internal standard calibration.
REAGENTS
6.1 Benzophenone, an internal standard, Aldrich Chemical Company (Purity 99%)
6.2 Methanol, Acetonitrile, and water, HPLC Grade or equivalent
6.3 S.A.R.M. 2,4,6-TNT, Supplied by sponsor (Purity 99.8%)
CALIBRATION
7.1 Calibration standards were prepared from stock solutions containing
200 pg TNT, and benzopheone per ml acetonitrile so as to bracket
the working range of the chromatographic system. These concentrations
were: 0.5 Mg/ml, 2 ug/ml , 10 Mg/ml, 20 Mg/ml, and 40 Mg/ml.
7.2 A constant injection volume of 10 pi was employed for all measurements.
7.3 In order to determine the precision of the HPLC system, a series of 6
replicate injections of the 20 jjg/ml solution were made.
7.4 Retention times should remain relatively constant (within ±5% day to
day) with TNT being 5.1 minutes, and benzophenone 8.2 minutes under the
specified conditions. If the retention times are not within ±5%, super¬
vising chemist should be informed prior to the analysis and corrective
actions should be taken.
QUALITY CONTROL
8.1 Before processing any samples, the analyst should demonstrate through
the analysis of a blank that all glassware and reagents are interfer¬
ence free. Each time a set of samples is extracted or there is a
change in reagents, a method blank should be processed as a safeguard
against laboratory contamination.
8.2 Standard quality assurance practices were used with this method. A
minimum of six replicate spiked samples were analyzed to validate
the accuracy of the method. If doubt should arise concerning the
identity of the peak on a chromatogram, confirmatory techniques
such as mass spectrometry should be used.
8.3 In a typical sample set, a minimum of one blank and scheduled samples
will be analyzed. A control sample will be prepared by adding a known
concentration of TNT to the sample. The concentration will be in the
working range of chromatographic system as determined by calibration
experiment.
8.4 The analyst will follow each step in an analytical protocol without
deviation or improvisions in order to accurately assess the perfor¬
mance of the method. Prior to making any changes in the procedure,
analyst will consult the supervising chemist and the supervising
chemist and the Q.A. officer will review and approve all the changes.
8.5 The typical analysis will consist of the following samples, one blank
sample, 6 diet samples as is, 3 feed samples spiked for recovery
determination at the diet concentration.
SAMPLE COLLECTION
9.1 Samples are collected and stored prior to analysis according to
SOP 81 sample collection ^TNT and RDX diet samples).
SAMPLE EXTRACTION
10.1 The appropriate amount of sample is weighed into a 125 ml Erlenmeyer
flask using standard operating procedures. The sample amount for the
diet mixture is ten grams. Approximately 50 mis of acetonitrile is
added to the flask and it is stoppered. The sample is extracted by
stirring for 30 minutes only at room temperature.
10.2 Following extraction, the sample was filtered through a medium porosity
fritted glass filter. In this operation the extraction mixture was
swirled to form a uniform suspension and immediately poured into the
glass funnel. A stirring rod was used to drain the last drop of liquid
from the flask.
10.3 The extraction flask was rinsed with three portions of acetonitrile of
approximately three mis each and the rinses are poured into the funnel.
The vacuum is reapplied and the washing process is completed.
10.4 The filtrate is transferred via a short-stem funnel into a volumetric
flask. The filtering flask is rinsed three times, with approximately
5 ml portions of acetonitrile and the rinses are added to the volumetric
flask. The size of the volumetric flask and the subsequent treatment
of the sample depend on the initial TNT concentration in the sample.
The dilution for various sample levels is shown in Table ib.i. Diet
samples will be diluted to a volume that places them in the working range
of the chromatographic system.
10.5 An aliquot (approximately 10 ml) is filtered using a Water's Organic
Sample Clarification Kit using 0.5 pm filter. The sample is now ready
for analysis for HPLC.
TABLE iB.l. DILUTION SCHEME FOR TNT DIET SAMPLES
Diet
Level
(%)
Extract
Volume
(ml)
Extract
Diluted
(ml)
Benzophenone (IS)
Added
Final
Volume
(ml)
0.0005
100
--
1 ml 50 Mg/ml
100
0.0050
100
--
1 ml 500 pg/ml
100
0.010
100
--
1 ml 1000 pg/ml
100
0.050
100
10
1 ml 500 pg/ml
25
0.100
100
10
1 ml 1000 |ig/ml
- 50
STORAGE OF SAMPLES
11.1
All samples including diet and blank feed will be stored in the dark
at refrigerator temperatures.
11.2
If the sample preparation procedure is stopped at any point during the
working day, the samples should be stored in stoppered vessels in the
dark at refrigerator temperatures.
11.3
Samples that are ready for HPLC analysis will be stored In the dark
at refrigerator temperatures.
11.4
TNT andbenzophenone standards and all standard solutions will be
stored in the dark at refrigerator temperatures.
ElOR PERFORMANCE LIQUID CHROMAT0GRAP3I (HPLC)
12.1
12.2
12.3
12 . h
An injection volume of 10.0 pi vas used for each sample
except at or belov the 0.005# level them 25-0 pi vas used.
Each injection at the 0.0005#level vas folloved by lOOpl
of acetonitrile to speed alone the long retaining impurities
If the peak area exceeds the linear range of a sample it vas
diluted and reanalyzed.
For the diets at and below the 0.005# level the retention
times are b . 6 and 9*9 minutes for TNT and .benzophenone
respectively.
12.5
Each sample vas analyzed by reverse phase HFLC using the
conditions described belov: Column, 3-9 mm x 30.0 cm
pBondapak C _ « ; So],vent System, Methanol: Water (7O#:30#, v/v);
Flow Rate, 1.0ml/min ; Dection,UV at 25^ nm'. The retention
times of TNT and benzophenone were 5.1 and 8.2 minutes
respectively. The limit of detection van 0.2 pr, TNT/ml
acetonitrile and is defined as 5x the background noise. The
representative chromatogram is Figure- ib. 1. For levels at and
belov 0.005# TNT, the chromatographic conditions have been
changed. The eluting solvent in these cases is Methanol:Water
(60#;Ii0#, v/v) at a flow rate of 1.5 ml/min.
i &
- * h,f*
m |
The chromatographic system vas calibrated daily vith a minimum
of two injections of one standard representative of the
chromatographic range.- __
m
Following the completion of an analysis or set of analyses
a gradient going from intial solvent to 100# methanol in 15 mi-.fi
will be used to elute nonpolar compounds from the column’.
Elution at 100# methanol vill be continued for at least one houi
CALCULATIONS
13.1 Determine the concentration of TNT using the formula:
% TNT in Sample =
(Ax)(Wis) x D x 100
(Fx) Ais (Ws)
where
Ax = Area (X) where x is TNT
Ais = Area (internal standard)
F _ Area (x) x weight (is)
x Area (is) x weight (Wx)
Wis = Weight of the internal standard
Ws = Weight of the sample
D = The dilution factor
Wx = Wt of component x is TNT
13.2 The results should be reported in percent TNT in the sample. Where
replicate samples are analyzed, all data should be reported. All
results are recorded in standard IITRI logbooks and these plus
chromatograms and data tapes are retained in the Chemistry Division
Q.A. files.
SAFETY
14.1 Safety regulations will be followed at all times especially with regard
to the handling of toxic materials. When the diet samples are being
handled, a lab coat and gloves will be appropriate attire. When
solutions or extracts are being handled, a lab coat and gloves should
be worn when there is the chance of direct contact with these materials.
APPENDIX 1C
ANALYSIS OF TNT IN DIET PREMIXES
SCOPE AND APPLICATION
1.1 This method covers the determination of TNT in diet premixes at 10* and
50* level.
1.2 The sensitivity of this method is usually dependent on the level of inter¬
ferences present in the samples, rather than the instrumental limitations.
1.3 This method is recommended for use only by experienced analysts familiar
with High Performance Liquid Chromatography (HPLC) or under close super¬
vision of such qualified persons.
SIM-1APJ OF THE METHOD
2.1 A weighed quantity of the premix was stirred with 50 ml of acetonitrile
for 30 minutes. The suspension was filtered through a porous glass filter
and the filtrate was transferred with washings to a volumetric flask.
Benzophenone, the internal standard was added to the filtrate or a portion
thereof and this solution was diluted to its final volume. The samples
were analyzed using reverse phase high performance liquid chromatography.
Each was eluted on 3.9 mm x 30.0 cm p-Bondapak C Je column with methanol:
water (70*:30'X) and the eluant was monitored with an ultraviolet
absorption detector at X = 254 nm.
IN. lRFEP.EN CES
3.1 Solvents, reagents, glassware, and other sample processing hardware may
yield discrete artifacts and/or elevated baselines causing misinter¬
pretation of chromatograms. All of these materials must be shown to be
free from interferences under the conditions of the analysis by running
method blanks.
3.2 Interferences coextracted from the samples will vary considerably from
source to source, depending on the type of animal feed used in the study.
MATERIALS
4.1 Erlenmeyer flasks, 125 ml.
4.2 Filtering apparatus, vacuum flask, 125 ml; fritted glass filters,
porosity M, ASTK 10-20 microns.
EQUIPMENT
5.1 Mettler Grammatic Analytical Balance, No. 1-910
5.2 Corning Hot Plate Stirrers, BC 351
5.3 Buchi Evaporator, Model R
5.4 Sample Clarification Kit, Organic (Water's Associates)
5.5 Highe r Performance Liquid Chromatography
• constant flow, isocratic pumping system
• reverse phase column, 10 p - 3.9 mm x 30 cm p-Bondapak C Je column
• ultraviolet detector capable of monitoring X = 254 nm
• strip chart recorder and electronic integrator capable of measuring
peak areas and performing an internal standard calculation.
REAGENTS
6.1 Benzophp^one, an internal standard, Aldrich Chemical Company (Purity 99%)
6.2 Methanol, Acetonitrile, and Water, HPLC Grade or equivalent
6.3 S.A.R.M. 2,4,6-TNT, Supplied by sponsor (Purity 99.8%)
CALIBRATION
7.1 Calibration standards were prepared from stock solutions containing
200 pg TNT, and benzophenone per ml acetonitrile so as to bracket the
working range of the chromatographic system. These concentrations were:
2 pg/ml, 10 pg/ml, 20 pg/ml, and 40 pg/ml.
7.2 A constant injection volume of 10 pi was employed for all measurements.
7.3 In order to determine the precision of the HPLC system, a series of 6
replicate injections of the 20 pg/ml solution were made.
7.4 Retention times should remain relatively constant (within ±5% day to day)
with TNT being 5.1 minutes, and benzophenone 8.2 minutes under the
specified conditions. If the retention times are not within ±5%, the
supervising chemist should be informed prior to the analysis and
corrective actions should be taken.
QUALITY CONTROL
8.1 Before processing any samples, the analyst should demonstrate through the
analysis of a blank that all glassware and reagents are interference
free. Each time a set of samples is extracted or there is a change in
reagents, a method blank should be processed as a safeguard against
laboratory contamination.
8.2 Standard quality assurance practices were used with this method. A
minimum of six replicate spiked samples were analyzed to validate the
accuracy of the method. If doubt should arise concerning the identity
of the peak on a chromatogram, confirmatory techniques such as mass
spectrometry should be used.
8.3 In a typical sample set, a minimum of one blank and scheduled samples
will be analyzed. A control sample will be prepared by adding a known
concentration of TNT to the sample. The concentration will be in the
working range of chromatographic system as determined by calibration
experiments.
8.4 The analyst will follow each step in an analytical protocol without
deviation or improvisions in order to accurately assess the performance
of the method. Prior to making any changes in the procedure, analyst
will consult the supervising chemist and the supervising chemist and
the QA officer will review and approve all the changes.
8.5 The typical analysis will consist of the following samples shown in the
diagram, one blank sample, 6 premix samples as is, 3 spiked samples.
r '
SAMPLE COLLECTION
9.1 Samples are collected and stored prior to analysis according to SOP 81
Sample Collection and Storage (TNT and RDX Premix}.
SAMPLE EXTRACTION
10.1 The appropriate amount of sample is weighed into a 125 ml Erlenmeyer flask
using standard operating procedures. The sample amount for both the
10 percent and 50 percent premix is one gram. Approximately 50 mis of
acetonitrile is added to the flask and it is stoppered. The sample is
extracted by stirring for only 30 minutes at room temperature.
10.2 Following extraction, the sample was filtered through a medium porosity
fritted glass filter. In this operation the extraction mixture was
swirled to form a uniform suspension and immediately poured into the
glass funnel. A stirring rod was used to drain the last drop of liquid
from the flask.
10.3 The extraction flask was rinsed with three portions of acetonitrile of
approximately five mis each and the rinse is poured into the funnel.
The vacuum is reapplied and the washing process is completed.
10.4 The filtrate is transferred via a short-stem funnel into a volumetric
flask. The filtering flask is rinsed three times, with approximately
5 ml portions of acetonitrile and the rinses are added to the volumetric
flask. The size of the volumetric flask and the subsequent treatment of
the sample depend on the initial TNT concentration in the sample. The
dilution for samples is shown in Table IC.1.
10.5 An aliquot (approximately 10 ml) is filtered using a Water's Organic
Sample Clarification Kit using 0.5 pm filter. The sample is now ready
for analysis for HPLC.
TABLE IC.1. DILUTION SCHEME FOR SAMPLE EXTRACTS
Premix Concentration
10%
50%
Original Extract Volume
100 ml
500 ml
Secondary Dilution
1 ml extract plus 1 ml
I.S. to volume of 50 ml
with acetonitrile
1 ml extract plus 1 ml I.S.
to volume of 50 ml with
acetonitrile
1. I.S. solution concentration is 1000 pg/ml.
2. In the case of a sample analyzed by the method of standard addition 1 ml of
the original extract was diluted with 50 ml acetonitrile, and 1 ml of the
extract added to 1 ml of the spiking solution of known concentration was
diluted with acetonitrile as above.
STORAGE OF SAMPLES
11.1 All samples including premixes and blank feed will be stored in the dark
at refrigerator temperatures.
11.2 If the sample preparation procedure is stopped at any point during the
working day, the samples should be stored in stoppered vessels in the
dark at refrigerator temperatures.
11.3 Samples that are ready for HPLC analysis will be stored in the dark at
refrigerator temperatures.
11.4 TNT and benzophenone standards and all standard solutions will be stored
in the dark at refrigerator temperatures.
EIGE PERFORMANCE LIQUID CBBCMATOGRAPHY (HPLC)
12.1 Each sample was analyzed by reverse phase HPLC using the conditions
described below: Column, 3.9 mm x 30.0 cm p-Bondapak Cia; Solvent
System, methanolrwater (70*: 305:, v/v); Flow Rate, 1.0 ml/min;
Detection, UV at 254 nm; Sensitivity, 0.1 AUFS. The retention times of
TNT and benzophenone were 5.1 and 8.2 minutes, respectively. The limit
of detection was 2 pg TNT/ml acetonitrile and is defined as 5x the
background noise. The representative chromatogram is Figure 'C.i.
12.2 The chromatographic system was calibrated daily with a minimum of two
injections of one standard representative of chromatographic range.
12.3 An injection volume of 10.0 pi was used for each sample. If the peak
area exceed the linear range of a sample it was diluted and reanalyzed.
12.4 Following the completion of ar. analysis or set of analyses, a gradient
going from initial solvent conditions to 100* methanol in 15 minutes
will be used to elute polar compounds from the column. Elution at 100%
methanol will be continued for at least 1 hour.
CALCULATIONS
13.1 Determine the concentration of TNT using the formula:
<v tut _ (Ax)(Wis) x D x 100
* TNT Sample - ' Fx) Ais m —
where
Area (x) where x is TNT
Area (internal standard)
Area (X) x weight (is)
Area (is) x weight (Wx)
113
Wis = Weight of the internal standard
Ws = Weight of the sample
D = The dilution factor
Wx = Wt of component x is TNT
13.2 The results should be reported in percent TNT in the sample. Where
replicate samples are analyzed, all data should be reported. All results
were recorded in standard IITRI logbooks and these plus chromatograms
and data tapes were retained in the Chemistry Division Q.A. files.
SAFETY
14.1 Safety regulations will be followed at all times especially with regard
to the handling of toxic materials. When the premix samples are being
handled, a lab coat, gloves, and a mask will be appropriate attire.
When solutions or extracts are being handled, a lab coat and gloves
should be worn when there is the chance of direct contact with these
materials.
fS
APPENDIX ID
.SAMPLE COLLECTION AND STORAGE
(TNT AND/OR RDX PREMIX SAMPLES)
Scope
1.1 This procedure covers the collection and storage of TNT and RDX
premix samples prior to analysis.
Materials and Equipment
2.1 Small scoop
2.2 Powder funnel
2.3 Amber vials with plastic screw cap
Sample Collection
3.1 Personnel of the Life Sciences Division will inform the supervising
chemist and the analyst when they receive TNT or RDX premixes. The analyst
will collect 6 samples from the Velostat bag container, one from each of four corners
and two from the middle. At least 5.0 gram quantities of premix will be
collected in order to permit the extraction and analysis steps to be per¬
formed in duplicate. All samples will be identified according to the
Chemistry Division identification system. All detailed information will
be placed in the sample identification logbook inmediately.
The sampling procedure for the premix will be performed as follows:
One sample is removed from the center of the storage bag with a small
scoop which will permit the removal of a 5.0g quantity. The second
sample will also be removed from the center of the container in the same
manner as the first sample but at a deeper level.
After center sampling, the surface of the premia is restored by leveling and
four additional samples will be removed with a small scoop from each of the
four corners of the bag at gradually increasing depths by lifting the
corners of the bag. The 6 samples will be labeled and placed in amber vials
with plastic screw caps. The label will contain Date Sampled, Sample
Number, Premix Identification, Lot Number and Sampled by Initials.
Sample Storage
4.1 All samples will be stored at refrigerator temperatures in the
dark prior to analysis. This includes feed that will be used for blanks
and control samples. Every three months (from manufacturing date) feed
will be changed. This manufacturing date will be supplied by life Science
Transmittal Record
5.1 Transmitted record will be completed by responsible personnel.
A copy of Test Article Prerr.ix (T.A.P.) and/or T.A.P. Sample Transmittal
(or custody) record is attached (Figure ID.1).
Sample Disposal
6.1 Samples or parts of samples will be returned to the Safety
Officer for disposal.
i i 7
*1 i ft i -k. > !■ i
FIGURE ID.1
TEST ARTICLE PREMIX (T.A.P.) AND/OR T.A.P. SAMPLE TRANSMITTAL .1
(OR CUSTODY) RECORD A
Project No. - Study No(s). _ T.A.P. _
Lot No. _ T.A.P. Prepared (K.O.P.) Date/By: _
Intended Concentration: _ % Quantity (kg): _ 5002 Lot No.:
Logbook No./Page No. _ Storage Conditions of T.A.P. (K.O.P.):_
T.A.P. Received (L.S.R.) Date/By:_
Storage Conditions of T.A.P. in L.S.R.:
Logbook No./Page No.:_
T.A.P, SAMPLING AND ANALYSIS
T.A.P. Sampled Date/By:_ Logbook No./Page No.: _
Witnessed By/Date:_ Storage Conditions of T.A.P. Sample by Chemistry
Personnel: _
Extraction Performed By/Date: _
Analysis Performed By/Date: _
Data Reviewed & Approved By/Date:
Analytical Report Prepared By/Date:
Quality Assurance Check By/Date: _
Analytical Report Received (L.S.R. Supervisor) By/Date: _
T.A.P. First Used By/Date: _ T.A.P. Last Used By/Date: _.
Excess T.A.P. Submitted to K.O.P. Personnel for Disposal by Burning By/Date:
_ Quantity (kg) _
Excess T.A.P. Received By/Date: _
Key
K.O.P. = Kingsbury Ordinance Plant, La Porte, IN.
5002 = Purina Certified Rodent Chow 5002
Logbook No./Page No.:
Logbook No./Page No.:
Checked By/Date:
Certified
Rodent
Chow®
*5002
Cenifieo Rooent Chow is a controlled
constant nutrient rodent diet recommenced
lor Me cyc'e feeding of rats, mice and
hamsters A sample of this product has
been assayed to* certain environmental
contaminants Maximum diet control *s
achieved Oy pre-analysis monitoring of key
nutr.ems and cenarn contaminating
suDstances D>et control he^ps minimize
variaoies in research studies
Guaranteed Analysis
Cajae pfotein. mr. 2C C%
Cruoe ta; mm a s%
C'uOe fibe-, max 6 0%
Asn max 8 C%
Aaaec mme-ait max 2 £%
Certification Profile
Based on analysis of a composite samp.e.
each package contains no: more man
these maximum concentrations of me
following substances
Heavy Maximum
Metals Concentration
Arsenic .10 ppm
Cadmium 5 ppm
Lead i 5 ppm
Mercury 2 ppm
Aflatoxin 1C ppb
Chlorinated Hydrocarbons and PCB
Aid'm 05 PPm
D'e'd'*n 05 ppm
£nd r m 05 ppm
Hep'ach'or 05 ppm
Heptach'or Epoxide . 05 ppm
Lnaane 05 ppm
Cn.o f dane 05 ppm
DDT ne’a’eo Substances 15 ppm
PCB . "5 ppm
Organophosphr.tes
Thime; S ppm
D'azmon . 5 ppm
DisuHaton . .5 ppm
Mefhyf Parathion . 5 ppm
Maiathion 5 ppm
Parathion . . . ..5 ppm
Thioaan . . 5 ppm
Ethion.5 ppm
Tnfhion .5 ppm
Drugs and Estrogens — This product is
manufactured m a plant wne^e antibiotics
anc synthetic estrogens are strictly
P'Ohibited Routine monitoring lor Ove r a
decade nas not shown any aetectaD-e
levets o* these substances No drugs or
synthetic est'ogens are permitted in
manufactunng storage or warehousing to
avoid any contamination of Lab Chows
diets
Other Contaminants — H additional
contaminants assays are needed these
can be obtained by oroenng such ana.yses
prior to manufacture. Cost o* these
additiona 1 assays will be charged based on
current analyses rates at time of assay
Ingredients:
Ground ext'udeo com. soybean mea’
g'ound cat groats d'*ec bee* pu c w'-eat
germ mea fish mea' brewe's d'>ed yeast,
oehydra'ed a“a ! fa mea' cane mousses
C'-ed mr- products meat and bone mea'
wheat middlings animal ia^ preserved with
Eha ca'cijm carbonate d'caicum
pncsphate sa't an.ma? live-' mea’ ca<c-jrr.
locate vitamin 6u supplement, methionine
hydroxy analogue caiogm calcium
pantothenate. c hoi me ch'onde. fc«c acd.
nboftavn supplement, thiamin. macm.
pvridoxme hydrocntoride. ferrous su u ate.
vitamin A supplement D activated animal
S'e'O* vi’amir £ supplement. i'on Oxde.
manganous oxide ccoa : carbonate,
ccppe' cxde zmc oxde
Chemical Composition’
Nutrients**
Protoin % 20 0
A'g nme % .113
Cystine % 27
G ycme X ..... . 86
H-st.dme X . 49
Iso'eucne % * i 03
LeuC.ne % 1 58
Lvsme X . 118
Meth.-cnme % ... . 43
Pne'-y.a a^-ne % B8
Threon ne X 70
Tryc conan % 24
Va ^-e % i 05
Fst % 4 5
Flbtr X 4 6
TON X 77 0
NFE (by aifererce) %"* 55 i
Gross Ene f gy KCa gm 4 i
Ash X 5 8
Calcium X 90
Phosphorus X 70
Potassium % 86
Magnesium X 2 1
Soo'urr, X 30
Chionne % 4 7
Fiuonne. ppm —.
Iron, ppm . 180 0
Zmc. ppm . 52 4
Manganese ppm 63 0
Copper ppm 1 3 3
Cobaft. ppm 6
Iodine, ppm 12
Vitamins
Carotene, ppm 5 6
Menadione (added) ppm —
Thiamin, ppm. 13 3
R.bofiavm ppm 8 0
N.acm. ppm . 60 0
Pantothenic Ace ppm 17 o
Choime ppm xi00 18 0
Folic Acid ppm 4 0
Pyridoxme ppm 6 0
BiOtm. ppm 13
6-12. mcgitb . 9 0
Vitamm A. lU'gm 17 6
Vitamin D lU'gm 2 2
Alpha-tocopherol ILMb . 30 0
Ascorbic Acid mg/gm . —
Feeding Directions
Feed ad iibitjm to rodents Plenty of fresh,
clean wate r should be available to the
animals at an times
Rats — Acuft r ats will eat 12 to 15 g-ams
o’ diet pe r Cay Feeders m rat cages
snouid be oes^gnec to hold two to three
days supply of feed at one time
Mice — Adult mice win eat 4 tc 5 grams o'
pe'ieted ration aarly Some of the larger
strams may eat as much as 8 grams per
day pe r animai ^eed snou'd be avaiiab'e
on. a free choice bas s m wire feece r s
above the floor o‘ the cage
Hamsters — AduUs will eat 10-14 grams
per day
LabChows .
The Control rac:jr.
'Basec ty a ts rg'fCfi i*Awt <S nee mi ccf^x&rcr e>< 'u.v* ><a «i lurvt
CV »ccc»-^vv
I _5 _t_ !
• ^ TEI ANALYTICAL, INC.
460 SOUTH NORTH WIST HIGHWAY * PARK RIDGE. ILLINOIS . 6006* • *12/696-2070
•
October 29, 1982
LABORATORY REPORT
#9166
Page 1 of 2 pages
•
Dr. Marianna Furedl
IIT Research Institute
10 West 35th Street
Chicago, Illinois 60616
P.0. #16092
Sample received
June 9. 1982
•
[TE1-14080] Rodent Chow #5002 - March 24-822G
Result In pur
* Method
Nitrate Nitrogen
19.0
7.030
<*
Nitrite Nitrogen
0.24
7.D30
Mercury
< 0.05
25.103
Arsenic
0.014
JAOAC 60-813
Cadmiurr.
< 0.05
25.02C
m
Lead
0.61
25.058
Penicillin
< 10 Snell & Snell,
Colorimetric Methods
of Analysis
Vol IVAAA, p. 221
•
BHT
< 1.0
JftOAC 60,505
BKA
< 1.0
JAOAC 60,505
Total Estrogen
not detected
39.000
c
Clilortetracycllne
to be reported at a later
date
Aflatoxln Bj
< 0.005
26.003
A.flatoxln Ej
0.01 - 0.02
26.003
Aflatoxln Cj
< 0.005
26.003
*■
Aflatoxln G a
< 0.005
1 26.002
Dleldrin
< o.om
29.000
Endrin
< 0.001
29.000
Aldrin
< o.oor
29.000
*
Heptachlor Epoxide
< 0.001‘
29.000
BHC
< 0.001
29.000
L TEI ANALYTICAL, INC.
«0 EOUTW NORTHWEST HIGHWAY . PARK RIDGE. ILLINOIS • 600C8 • 3l2/£% 207C
October 29, 1982
LABORATORY REPORT
Page 2 of 2 paaes
#9166
'/ >• . \
• • ' * ' I.
•' : n 7
Dr. Marianna Furedi
III Research Institute
10 West 35th Street
Chicago, Illinois 60616
P.0. #16092
Sample received
June 9, 1982
[TEI-140803 Rodent Chow #5002 - March 24-8226
Result in ppm
* Method
Lindane
< 0.001
29.000
DDT Total
< 0.001
29.000
1.ethoxychlor
< 0.001
29.000
Chlordane
< 0.001
29.000
Hi rex
< 0.001
29.000
Toxaphene
< 0.001
29.000
Strobane
< 0.001
29.000
HC6
< 0.001
29.000
PCE
< 0.001
29.000
Polychlorinated Dioxins
< o.ooe
28.128
Parathlon
< 0.001
29.000
I'ethyl Parathion
< 0.001
29.000
Enthion
< 0.001
29.000
Carbophenothion
< 0.001
29.000
Kalathion
< 0.001
29.000
konnel
< 0.001
29.000
Dlazinon
< 0.001
29.000
Disulfeton
< 0.001
29.000
Piiora te
< 0.001
29.000
•Official Methods of Analysis of
the Association of
Official
Analytical Chenlsts.
7 /
ANALYTICAL PROCEDURES USED BY TEI ANALYTICAL, INC. PARK RIDGE, IL
TO ANALYZE PURINA CERTIFIED RODENT CHOW NO. 5002 FOR IMPURITIES
fuCLCfiiuiie
Limit of
D elect a b I I Ity
Chlorinated Pesticide Screen
Phosphated Pesticide Screen
Polychlorinated Biphenyls (PCBs)
Hexa-, hepta-, octachIorodIbenzo-p-dioxin
Heavy Metals
Arsenic
Ca dIum
Lead
Mercury
NItrates
Nitrites
AfIatoxI ns
Penicillin
ChI ortetracycI Ine
Butylated hydroxytoluene
Butylated hydroxyanI sole
Estrooens
10.0 ppm
1.0 ppm
1.0 ppm
fi£±er_e rices
10 ppb
A.O.A.C.
29.000
50 ppb
A.O.A.C.
29.000
100 ppb
A.O.A.C.
29.000
100 ppb
A.O.A.C.
28.128
1.0 ppb
•
o
•
<
•
o
•
<
•
60.813
10 ppb
A.O.A.C.
25.026
10 ppb
A. 0. A. C.
25.058
< 1 ppb
A.O.A.C.
25.103
<1.0 ppm
A.O.A.C.
7.030
<1.0 ppm
A.O.A.C.
7.030
2.0 ppb
A.O.A.C.
26.003
<2.0 ppm
Snell and
1 Snell,
of Analysls Vol IV
AAA, po. 221
Snell and Snell,
Colorimetric Methods
of Analysls Vol IV
AAA, po. 184
J.A.O.A.C. 60.505
J.A.O.A.C. 60.505
A.O.A.C. 39.000
A.O.A.C. - Official methods of analysis of the Association of Official
Analytical Chemists.
2
Contract No. DAMD17-79-C-9120
IITRI Project No. L6 I 16
Study No. 8
DETERMINATION OF THE CHRONIC MAMMALIAN TOXICOLOGICAL EFFECTS OF TNT
FOUR WEEK CHRONIC (EXPLORATORY/RANGE-FINDING) ORAL (DIET) TOXICITY
STUDY OF TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
FINAL REPORT
Prepared by
Barry S. Levine
E. Marianna Fured!
John M. Burns
Paul M. Llsh
NT Research Institute
10 West 35th Street
Chicago, Illinois 60616
Supported by
U.S. Army Medical Research
and Development Command
Fort Detrick, Frederick, Maryland 21701
Project Officer: Jesse J. Barkley, Jr.
Paul M. Llsh Barry S. Levine
Scientific Advisor Senior Toxicologist
Life Sciences Research Life Sciences Research
Alan M.
Assoc I ate DI rector
Life Sciences Research
IAfilE. QL tSmiEMS
Esafi tta*
I . SUMMARY. 129
I I . INTRODUCTION. 129
III. MATERIALS AND METHODS. 129
A. Test Article...... 129
B. Animals. 130
C. Experimental Design. 131
D. Statistical Ana I ys I s . • • • .. 133
IV. RESULTS. 133
A. Morta I I ty/C I I n lea I Observations. 133
B. Body Weight. 133
C. Food Consumption. 134
D. Hematology............... 134
E. Clinical Chemistry. 134
F. Organ Weights. 134
G . Pathology. 134
V. DISCUSSION. 135
VI. RECOMMENDATIONS. 135
VII. ACKNOWLEDGEMENT. 136
VIII. REFERENCES. 137
Tables IV.1 to IV.14 .138-151
H I stopatho I ogy Report...... 152
1 28
I • SWUUABI
This study was conducted to evaluate the toxicity of the munitions
compound tr1nftrotoIune (TNT; CAS Reg. No. 118-96-7) in B6C3F 1
hybrid mice when administered In their diet for four weeks.
Groups of 10 mice per sex received TNT at doses of 0, 0.3, 2, 14,
100 or 700 mg/kg/day. Toxicologic endpoints Included clinical
signs, body weights, food consumption, hematology, clinical
chemistry, organ weights, and gross and tissue morphology.
Administration of 700 mg/kg/day of TNT to B6C3F1 hybrid mice for
four weeks resulted In toxic effects of the liver, kidneys,
testes, and bone marrow and/or Its circulating cellular
constituents. As minimal effects were seen at the 100 mg/kg/day
dose level, the apparent no-effect level under the conditions of
the present study Is 14 mg/kg/day.
I I . JJiIR£>[LU£Lm
The purpose of this study was to assess the toxicity of TNT In
B6C3F1 hybrid mice when administered In the diet for at least four
weeks. The data derived were also to be used to select dose
levels for a chronic fox IcIty/carc I nogen IcIty study of TNT. All
methods and procedures were conducted In accordance with the IITRI
Quality Assurance program designed to conform with FDA
kaLficaiflny. ELl£c.±J.c.£ Rfeau-laliaas. <21 cfr, Part 58).
III. fclAIk&MkS. RM UklhQRk
a . Is.s.1 ALtlalfi
Trinitrotoluene (TNT), Batch No. VOL. 11-011, grade one flake,
100 pounds, was made available for this study from stocks at the
IITRI Kingsbury Ordnance Plant Explosive Facility, LaPorte, IN.
The purity of neat TNT, 99.1 + 0.4$, was determined by high
performance liquid chromatography as described In Appendix I using
analytical standards provided by the Sponsor. TNT was stored at
the Kingsbury facility at ambient room temperature and relative
humidity, and In the dark. Upon availability of the test article
and at termination of the treatment phase of the study, 30 g
samples were taken and stored under conditions Identical to those
for the batch.
TNT premixes, approximately 10$ In Purina Certified Rodent Chow
No. 5002 (5002), Ralston, Purina Co., St. Louis, MO., were
prepared on a monthly basis at the Kingsbury facility by Chemistry
personnel. Undiluted TNT was handled in accordance with
procedures for explosive and fire hazards. The test article was
ball milled with equal parts of 5002 and subsequently diluted with
additional 5002 In a twin shell blender to yield 3 kg of an
approximate 1C$ premix.
Toxicology personnel received TNT as an approximate 10$ premix
which posed little explosive or fire hazard. Previous studies
have shown that the approximate 10$ TNT premix In 5002 rat chow Is
stable for at least 7 weeks (1). Fol lowing the determination of
TNT coneentrat 1 on In the premixes, sufficient quantities were
subsequently diluted with 5002 In a twin shell blender to achieve
the desired concentrations of the test article necessary to
administer the required dose levels. The previous weeks body
weight and most recent food consumption measurements for each test
group by sex were used to calculate the desired dietary
concentrations of the test article, and 2 kg of each test diet
were prepared weekly.
B. Ani mal s
Hybrid mice, B6C3F1 strain, were used for this study. Eighty-four
mice of each sex were received from Charles River Breeding
Laboratories Inc., Wilmington, MA on January 14, 1981. They were
3-4 weeks old upon arrival and random body weights recorded within
three days were 17.6 + 2.2 g (males) and 14.1 +1.5 g (females).
The animals were housed In an air conditioned room (24-26°C) at
ambient relative humidity, 40-60$, and on a 12 hour IIght/12 hour
dark cycle. The room served as a quarantine and test room, and no
other test animals were In the room. The mice were housed three
per polycarbonate cage with Ab-sorb-drl bedding (Ab-sorb-dr I ,
Inc., Rochelle Park, New Jersey) from arrival until test animal
selection at the onset of Test Week -2. At that time, they were
housed two per cage. The animals were randomly assigned permanent
shelf location without sex differentiation. The cage size
conformed to the upper weight range recommended In the S_u.ld.fi IfiL
life CjiLfi find. Ufifi fil LfidfiLfilfiLi: Animals.» dhew, nih no. 78.23.
Animals were transferred to clean cages twice weekly. Each animal
was Identified during the quarantine period by a combination of
cage number and ear punch. Animals placed on test received a
study unique test animal number (N=120) which appeared as an ear
punch/toe clip.
Upon arrival at the IITRI animal facility, the animals were held
In quarantine for two weeks. During this period, they were
observed for signs of disease, general unthr I ft I ness , poor coat,
dlschages from body openings, abnormal feces, etc. Any animals
found to be unhealthy were eliminated from the test animal
selection process.
All animals received 5002 Rodent Chow from arrival until
termination, except during a 2-6 hour fast prior to scheduled
sacrifice. A sample 5002 lot was analyzed for nitrate and nitrite
content and the results were as fol lows:
Lai Ha
Hljxala iaaml
i. i
HilLilfi.fi lB.fi.ml
< o.i
12-2-80-G
Tap water was available ad. libitu m. Analytical results obtained
from a Chicago water sample are contained In Appendix X.
c. fLxaaaimaiilal Q.s.fiia.0.
Following the quarantine period, test-eligible animals were
randomly assigned, within sex, by a restricted randomization
procedure (stratified by weight; blocked design) Into the
following treatment groups using a table of random numbers.
lL£.alme.al £Lfiy.B. Ailfifialifin.
Treatment
Dose Leve1
Animals/
Test
Animal
i Test
Animal
LLfiu.fi.
lLfi.filmfi.Ill
ims.Z.lLaZ.ifiy.1
£fi&
Hfi-tltifilfifil
Hfi.tLfLfi.mfilfi.fil
1
•
0.0
1 0
1
- 10
1 1
- 20
1 1
TNT
0 .3
1 0
21
- 30
31
- 40
1 1 1
TNT
2 .0
1 0
41
- 50
51
- 60
1 V
TNT
14.0
1 0
61
- 70
7 1
- 80
V
TNT
1 00.0
1 0
81
- 90
91
- 100
V 1
TNT
7 00.0
1 0
101
- 1 10
1 1 1
- 120
Test Day One of exposure to TNT was February 18, 1981. TNT was
administered to test animals as an admixture In 5002. The
appropriate test diets were available ad. I 1 b 1 tu rn except during a
2-6 hour fast prior to routine sacrifice In Test Week 5.
Test animals were observed once dally In the morning for
pharmacologic and/or toxicologic signs commencing with Test Week
-2 until termination. Afternoon mortality checks were Inltitated
at the onset of Test Week 1. Physical examinations which Included
palpations for masses were conducted weekly from Test Week -1
until termination. Food consumption was measured for each animal
on a weekly basis and body weights were determined twice weekly (A
and B) commencing with Test Week -2 through Test Week 4.
Clinical chemistry and hematology tests were performed for all
animals In Test Week 5. Blood samples were collected from the
orbital sinus immediately prior to necropsy. The samples were
collected and analyzed In a randomized order, and sufficient
quantities were routinely obtained to measure the following
parameters .
131
tlkUAIkkGkl
Hematoc r 11
HamogIob I n
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH5
Mean corpuscular hemoglobin
concentration (MCHC)
PI ate Iet count
kkJLHlkAk ktikMlilfil
Bilirubin (total and direct)
GIutamIc-pyruv Ic transaminase (SGPT)
Triglycerides
TotaI ChoiesteroI
Erythrocyte count
Leukocyte count, total and
dIf ferent I a I
Reticulocyte count
MethemmogIobIn
RBCs with Heinz Bodies
RBCs with Howell-Jolly Bodies
Total protein
Albumin
Globulin (total protein minus
albumin)
Methods used to measure the above parameters are listed In
Appendix V (Hematology) and Appendix VI (Clinical Chemistry).
All surviving test animals were routinely sacrificed and
necropsled during Test Week 5. Three to four test
an ImaIs/sex/treatment group, randomly selected, were sacrificed In
random order each day during a three consecutive day period.
Terminal body weights were recorded Immediately prior to sacrifice
following a 2-6 hour fast. Euthanasia was accomplished with
carbon dioxide anesthesia followed by exsanguInat1 on . The
necropsy procedure was a thorough and systematic examination and
dissection of the animal viscera and carcass, and collection and
fixation of the following tissues:
Adrenals
Brain*
Cecum
Colon
Costochondral junction, rib
Duodenum
EpIdIdymes
Esophagus
Liver*
Lungs and malnstem bronchi
Lymph nodes :
Mandibular
MesenterIc
Mammar y gland
Muse Ie, skeI eta I
Nasal turbinate
Ovaries
Pancreas
PItu I tar y
Prostate
Eyes and optic nerves
Gal (bladder
Gross lesions
Heart*
Ileum
Jejunum
KId neys*
Larynx
Seminal vesicles
Sciatic nerve
Skin, abdomInal
Spinal cord
SpIeen*
Sternum, Including marrow
Stomach
Testes*
Thymus
Thyroids ( parathyro I ds )
Tissue masses
Trachea
Rectum
Salivary gland
Uterus
Urinary bladder
Organs marked with an asterisk (*) were weighed at routine
necropsy .
Bone marrow smears were prepared from the femur# alr-drled and
fixed In absolute methanol. AM tissues# except eyes# testes# and
bone marrow smears, were fixed at a thickness not exceeding 0.5 cm
In 10% neutral buffered formalin which was changed 24 hours later.
Eyes and testes were fixed In 3$ aqueous gIutaraIdehyde and Boulns
solution, respectively# for 24 hours. They were transferred to
50$ ethanol for 24 hours, then placed In 70$ ethanol. All tissues
examined microscopically were cut at a thickness of 4-6 u and
stained with hematoxylin and eosln.
Histopathologic examination was
of the following tissues and/or
Brain (3 sections)
Gross lesions
Kidneys
Liver
defined as microscopic examination
organs :
Spinal cord (3 levels)
S p I een
Tissue masses
Testes
D . ilallsJ.ic.al &n.alis.ls.
The analyses of body weight and food consumption data considered
the change relative to Test Week -1. Body weight, food
consumption, clinical chemistry, hematology and absolute and
relative organ weight data were analyzed by Analysis of Variance
tests with Dunnett's t test used If necessary.
I v. filiULIi
A. lifiLlalitxZ.Q.llalsLal ikaaLialioiis.
Neither death nor clinical signs of toxicity were observed In this
study .
b . iialall
Reductions In body weight gains and/or losses In body weight were
seen throughout the treatment pelrod for male and female mice
administered 700 mg/kg/day. At 100 mg/kg/day, occasional slight
decreases In body weight gains for both sexes were seen. No
effect of treatment was seen at lower doses (Tables IV.1-IV.4).
c. LglqA C.£as.ij.inc.ii£a
Food Intake was Increased for males but not females at the 700
mg/kg/day dose level. Food consumption was not altered for the
other treatment groups (Tables IV.5 and IV.6).
0.
A significant decrease In the WBC count was seen for male but not
female mice at the 700 mg/kg/day dose level. The relative
proportion of leucocyte cell types was unaltered. Females but not
males at this high dose also demonstrated thrombocytosis. No
other measured hematology parameter was altered by TNT treatment
(Tables IV.7 and IV.8).
E. CJLinic.al Cie.mls.lLii
Dose-related hyperb I I I rubInem I a was apparent for both sexes.
Increases of approximately 25$ and 50$ were seen at the 100 and
700 mg/kg/day dose levels, respectively. No other measured
clinical chemistry parameter was altered by the administration of
TNT (Tables IV.9 and IV.10) .
F. CLoan. itaifllia
Hepatomegaly may have occurred for TNT-treated males. Increases
of approx I mate Iy 7$ were seen at the 100 (statistically
significant) and 700 mg/kg/day dose levels. Other
treatment-related changes were only apparent at the high dose and
Included reduced testes weights and elevated kidney weights for
females (Tables I V.1 1 - I V . 14) .
g . Eallaiaaii
The Pathology Report appears at the end of this Appendix.
Treatment-related morphologic alterations were confined to the
spleens of males and females receiving either 100 or 700
mg/kg/day. This organ appeared dark red for some of the animals
at the former dose and most of the animals at the latter dose.
Microscopical ly the lesion consisted of a diffuse Increase In the
relative amounts of yellow-brown pigment In the red pulp. The
pigment was indistinguishable from hemosiderin and was present
within the cytoplasm of macrophages. This Increase of splenic
pigment was clearly dose-related; It was of minimal severity In
all affected TNT 100 mg/kg/day mice of both sexes and of mild
severity In all TNT 700 mg/kg/day mice of both sexes. The
distribution of normal pigment In the spleens of TNT 0 mg/kg/day
mice was either focal and trace In amounts or entirely absent.
v. £LL£C.U££1£N
This study examined the oral toxicity of TNT fol lowing dietary
administration to mice for four weeks. Doses of up to 700
mg/kg/day failed to result In death. The only clinical findings
were reductions In body weight gain at 100 mg/kg/day and slight
body weight loss at the 700 mg/kg/day dose level. The
significance of Increased food Intake at this latter dose level Is
not clear.
Previous subchronic studies of TNT In rats and dogs In our
laboratory have demonstrated treatment-re I ated hemolytic anemia
(1,2). Although alterations In red cell parameters were not
detected In the present study, hemolysis was suggested by
hyperbilirubinemia and splenic hemos I deros I s .
Additional toxic effects of TNT seen primarily at the 700
mg/kg/day dose level Included leukopenia without a differential
shift, thrombocytosis, slight hepatomegaly, marginal decreased
testes weights, and elevated renal weights. None of these organ
weight changes was accompanied by treatment-re Iated histologic
alterations.
In summary, adm I nIstrat 1 on of 700 mg/kg/day of TNT to B6C3F1
hybrid mice for four weeks resulted In toxic effects of the liver,
kidneys, testes, and bone marrow and/or Its circulating cellular
constituents. As minimal effects were seen at the 100 mg/kg/day
dose level, the apparent no-effect level under the conditions of
the present study Is 14 mg/kg/day.
V I . Rit&UUEMAIlQtiS
In adcitlon to evaluating the subchronic toxicity of TNT In B6C3F1
hybrid mice, this study was conducted to select dose levels for a
chronic tox I c I ty/carc I nogenIc 11y study. As described In the
preceding Discussion Section, significant toxicity was apparent
primarily for mice administered 700 mg/kg/day. Only marginal
effects occurred at the 100 mg/kg/day dose level.
By the end of the study, a 20-30$ reduction In body gain was
apparent for a r ! m ! s of both sexes given 100 mg/kg/day. Lower
doses did not appear to appreciably alter body weight growth
curves. Slight toxicity as evidenced by hyperbilirubinemia,
hepatomegaly and splenic hemosiderosis was apparent at the 100
mg/kg/day dose level but not at lower doses. Thus, the maximum
tolerated dose (MTC), under the experimental conditions described
herein, appears to be between 14 and 100 mg/kg/day.
On the basis of the above discussion, the following doses were
recommended to achieve significant toxicologic effects at the high
dose level, an apparent no observable effect level at the low
dose, and marginal or no toxicity at the Intermediate dose level.
Treatment TNT
(m g/kg/day )
0 .0
1 .5
1 0.0
70.0
VII. ££KiJ£WL££I£fcl££iI
This report was prepared at NT Research Institute, 10 West 35th
Street, Chicago, Illinois, 60616, under U.S. Department of Army
Contract No. DAMD17-79-C-9120 (IITRI Project No. L 06116)
entitled "Determination of the Chronic Mammalian Toxicological
Effects of TNT". Mr. Jesse J. Barkley, Jr., Environmental
Protection Research Division, USAMBRDL, served as the Contract
Officer's Technical Representative for this program.
The work reported herein was conducted In the Toxicology and
Pharmacology Section of the Life Sciences Department, and
represents a portion of the overall effort of the above named
research program. Pau I M. Lfsb, Ph.D., Scientific Advisor,
served as Principal Investigator. Barry S. Levine, D.Sc., Senior
Toxicologist, served as study director and was responsible for the
overall conduct of the study. Eva M. Fured 1 -Machacek, D.V.M.,
served as study toxicologist and was also responsible for
supervision of the technical support personnel. John M. Burns,
D.V.M., Senior Veterinary Pathologist, was responsible for
supervision of gross necropsies. Donovan E. Gordon, DVM, Ph.D.,
Consultant, Veterinary Pathology, was responsible for
histopathologic evaluation. Don Reitman was responsible for
generation of clinical pathology data. Bobby R. Collins, D.V.M.,
M.S., served as clinical veterinarian and supervised animal care
personnel. Joann M. Hinz, B.S., and Robert M. Renaud, B.S.,
were responsible for the col lection of test data. Dorothy Davis
(ASCP-HT) was responsible for preparation of histology slides.
Josephine M. Reed, M.M., M.S., Quality Assurance, was responsible
for the quality assurance program. Robert Remaly, B.S., Senior
Engineer, was responsible for preparation of the test article
premixes. Hugh J. O'Neill, Ph.D., Manager, Analytical Chemistry,
and Walter C. Eisenberg, Ph.D., Senior Chemist, were responsible
for chemical analyses of test article, test article premixes and
test diets. Robert 0. Gibbons, Ph.D., Consultant, B1ostatIstIcs,
provided statistical and computational assistance.
I I
I I I
I V
I . R££I££N£££
Levine, B.S., Furedl, E.M., Gordon, D.E., Burns, J.M.
and LIsh, P.M. Thirteen week oral (diet) toxicity
study of trinitrotoluene (TNT), hexahydro-1,3,5-tr 1 n I tro
1,3,5-trlazIne (RDX ) and TNT/RD X mixtures In the
Fischer 344 rat. Final Report No. L6116/L6121,
Study No. 1.
Levine, B.S., Rust, J.H., Burns, J. M. and LIsh, P.M.
Twenty-six week subchronic oral toxicity study of
trinitrotoluene (TNT) In the Beagle dog. Final
Report No. L6116, Study No. 5.
SuREMENTS of male mice
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FOUR WEEK SUBCHRONJC (EXPLORA TOW V/RANGE-F1NO INC) ORAL (DIET) TOXICITY STUDY
OF TR1N1TRUTOLUENE ( TNT > IN THE BGC3F 1 HYBRID MOUSE
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L6 116-
FOUR WtfK SUEUMRONIC ( E'PLORAIORV/RANGE-F INOING» ORAI (PIET) TO* IC1 TY GIUPv
OF TRIM TPOtni UENE (IN'!) IN THE RGC3E I FEERRIP MOUSE
16116 -
POUR WFFK SUBCHRONIC (FX
1 G 1 1C
Four Week Subchronic Oral Toxicity Study Of
Trinitrotoluene (TNT) In The B6C3F1 Hybrid Mouse
Histopathology Report
May 31 , 1981
IITRI Project No. L6116
Study Number 8
Donovan E. Gordon
Consultant Histopathologist
Diplomate, American College
of Veterinary Pathologists
IITRI Project No. L6116, Study No. 8
Sponsor Project No. DAMD17-79-C-9120
Report of Histopathologic Findings
In accordance with the experimental protocol, a histopathologic examination
by light microscopy was conducted on hematoxylin-eosin (H&E) stained tissue
sections from 120 (60 male and 60 female; 10 each sex per group) B6C3F1 hybrid
mice for IITRI Project Number L6116, Study Number 8. Each group was fed either
Trinitrotoluene (TNT) as a dietary admixture or a control diet for four continuous
weeks, and then subjected to an extensive necropsy examination upon sacrifice at
the termination of the study. There were no moribund sacrifices or spontaneous
deaths in the study. The necropsy of animals, collection and fixation of tissues,
and preparation of stained tissue sections were conducted by I IT Research Institute.
The experimental design of the study, as it relates to the histopathologic
evaluation, is outlined below:
Treatment
Group
Treatment
Number of
Males
Number of
Females
Dose Level
mq/kq/day
I
...
10
10
0.0
II
TNT
10
10
0.3
III
TNT
10
10
2.0
IV
TNT
10
10
14.0
V
TNT
10
10
100.0
VI
TNT
10
10
700.0
The tissues examined from all animals were:
Brain (3 levels)
Kidneys
Liver
Spinal Cord (3 levels)
Spleen
Testes
Tissue Masses and
Gross Lesions
At the time of the tissue examination, a copy of the 'Organ Check Lists’ on
which necropsy observations and organ weights were recorded for each animal was
available to the histopathologist. A 'Pathology Report' form, accounting for all
tissues examined and the microscopic morphologic observations, was prepared for
each animal and signed by the histopathologist. These forms have been submitted
to the IITRI archives and contain the data on which this report is based.
The 'Pathology Report' forms and appendices which accompany this report
constitute an accountability of tissues examined in accordance with the experi¬
mental protocol. The grading system and abbreviations used in the tables are
as follows:
Grade 1 = minimal severity
Grade 2 = mild severity
Grade 3 = moderate severity
Grade 4 = marked severity
P = present, no grad'
The incidences of microscopic lesions are summarized by group and sex in
Pathology Appendix A. 'Histonathology Incidences Tables' of all findings are
presented by group and sex in Pathology Appendix B.
Treatment-related morphologic alterations were confined to the spleens of
TNT 100 mg/kg/day (8/10 males, 10/10 females) and TNT 700 mg/kg/day
(10/10 males, 10/10 females) mice and consisted of a diffuse increase in the
relative amounts of yellow-brown pigment in the red pulp. The pigment was
indistinguishable from hemosiderin and was present within the cytoplasm of
macrophages. This increase of splenic pigment was clearly dose-related; it was
of minimal severity in all affected TNT 100 mg/kg/day mice of both sexes, and of
mild severity in all TNT 700 mg/kg/day mice of both sexes. The distribution of
normal pigment in the spleens of TNT 0 mg/kg/day mice was either focal and trace
in amounts, or entirely absent.
With respect to the testes, a previously established target organ with
TNT in this species, no treatment related lesions were evident in this study.
However, there were solitary, subcapsular foci of seminiferous tubule de¬
generation among both control and test groups with a comparable incidence
and severity. The lesion involved one or both gonads and was restricted to
only a few (2-5) tubules adjacent to the rete testis and/or proximal segment
of the ducti efferentes. Histologically, affected tubules revealed an
absence of spermatogenesis, loss of the germinal epithelium with residual
sertoli cells and atrophy. The lesions were always immediately beneath the
capsule and were classified as naturally occurring. In contrast, TNT induced
testicular lesions usually occur throughout the parenchyma, are more numerous
and usually reveal more stages of germinal cell degeneration.
The remainder of the lesions observed and tabulated among the control and
test mice were regarded as incidental findings ascribed to naturally occurring
diseases or the method of sacrifice. These lesions were present, in most
instances, in both control and treated animals with a comparable incidence and
severity. No neoplastic lesions were observed artfong either control or test
animals.
Summary and Conclusions
A treatment-related lesion was present in the spleens of TNT 100 mg/kg/day
and 700 mg/kg/day mice. Based on this histopathological evaluation of selected
tissues, the maximum no-effect level of TNT appears to be 14 mg/kg/day.
54
N = within normal limits
L = lesion present
M = tissue not present
I = tissue section inadequate
COMPARISON TABLE
COMPARISON TABLE
tera1 _J_1_ 2/10
r cell vacuolation, bilateral 211 12 5/10
r regeneration , unilateral _ 0/10
Group I
16116 SN8
D6C3F1 Hybrid Mice
r.NT Four Week Subcbronic
3 3US?lPUI
9FF0-L8
SfrFO-18
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8880-18
8880-18
1880- 18
0880- 18
6880-18
8280-13
8280-18
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06C3Fl S (lybrid Mice HISTOPATHOLOGY INCIDENCE TABLE
TNT Four Week Subchronic
nfi Urates, focal
HISTOPATHOLOGY INCIDENCE TABLE
subcapsular, bilateral
Group III
B6C3Fl~*Hybrid Mice HISTOPATHOLOGY INCIDENCE TABLE
TNT Four Week Subchrorvic
MALES FEMALES
iesiis _ ^n&nnnKykn*A _
Germinal cell degeneration, focus, _
subcapsular, unilateral _ 2 2 2 2 4/10
Germinal cell d e generation, focus,
subcapsular, bilateral 2 1/10
l>roup IV
B6C3Fl S Hybrid Mice HISTOPATHOLOGY INCIDENCE TABLE
ENT Four Week Subchrom'c
Mononuclear cell infiltrates, focal
HISTOPATHOLOGY INCIDENCE TABLE
Germinal cell degeneration, focus
subcapsular, unilateral
HISTOPATHOLOGY INCIDENCE TABLE
H r STOPATHOLOGY INCIDENCE TABLE
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16116 SN8
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TNI Four Week Subchronic
Cyanme+hemogIobtn method
Coulter Counter Model S System
Mmalcccll
Indirect method; calculated value based on erythrocyte
count and mean corpuscular volume
Coulter Counter Model S System
E rythroc yte CflUfll
Electronic Counting Procedure
Coulter Counter Model S System
Mean Corp uscalar leLume lM£il
Electronic Sizing Procedure
Coulter Counter Model S System
M ean tifiinoalciiiii !M£tLl
Indirect method; calculated value based on erythrocyte
count and hemoglobin
Coulter Counter Model S System
M ean Corpu-S C-ular tLemcsil&kln £flH£finiXA±icn 1M£H£1
Indirect method; calculated value based on hematocrit
and hemoglobin
Coulter Counter Model S System
L eukocy te £sjm±
Electronic Counting Procedure
Coulter Counter Model S System
Leukocyt e DliisEenilal £qiih±
Neutrophils - Immature
Neutroph Ms - Mature
Monocytes
BasophI Is
Lymphocytes
Eos Inoph I Is
Wright stain procedure
Schalm, O.W., Jain, N.C. and Carroll, E.J.
Veterinary Hematology, Color Plates Chapter,
3rd Edition, Lee and Feblger, 1975.
B££.s nt-Llli Hfittfillriclly Belies
Wright stain procedure
Schalm, O.W., Jain, N.C. and Carroll, E.J.
Veterinary Hematology, Color Plates Chapter
3rd Edition, Lee and Feblger, 1975.
REQs Kith tisinz Bedims
Wright stain procedure
Schalm, O.W., Jain, N.C. and Carroll, E.J.
Veterinary Hematology, Color Plates Chapter
3rd Edition, Lee and Feblger, 1975.
BjJ£Ll£a±£ii BB£s
Wright stain procedure
Schalm, O.W., Jain, N.C. and Carroll, E.J.
Veterinary Hematology, Color Plates Chapter
3rd Edition, Lee and Feblger, 1975.
£la±£i£±
DIrect Method
Schalm, O.W., Jain, N.C. and Carroll, E.J.
Veterinary Hematology, p. 69, 3rd Edition,
Lee and Febiger, 1975.
New methylene blue staining procedure
Brecher, G. Am. J. Clin. Path. IB,
895, 1949.
y£±h&mQg±Qbln
CyanomethemogIobIn method
Evelyn, K.A. and Malloy, H.T. J. Biol.
Chem. 12£, 655, 1938.
I
Hexoklnase method
Centrlflchem Centrifugal Analyzer System
Neeley, W.E. Clin. Chem. 11, 509, 1972.
Urea 1EUM1
Modified urease technique
Centrlflchem Centrifugal Analyzer System
Karmen, A. J. Clin. Invest. 14, 131, 1955
GltiiamltrEyoiylc 121EI1
Modified Wroblewskl and LaDue technique
Centrlflchem Centrifugal Analyzer System
Henry, R.J., Chlamorl, N., Golub, O.J., and
Berkman, S. Am. J. Clin. Path. 14, 381,1960.
Ic±ai Prot ein
Biuret technique
Centrlflchem Centrifugal Analyzer System
Falling, I.F., Jr., Buckley, M.W. and Zak, B.
Am. J. Clin. Path. 11, 83, 1960.
Alimmln
Bromocresol green method
Centrlflchem Centrifugal Analyzer System
Rodkey, I.L. Clin. Chem. 11, 478, 1965.
l£JLflly£££ljJfi5
Tetrazollum salt reduction method
Centrlflchem Centrifugal Analyzer System
Klotzsch, S., Serricchlo, M. and Furedl, R.
Advances In Automated Analysis
Vol. 1, Medlad Inc., Tarrytown, N.Y. p 111, 1973
£llQi£S±££fil
Cholesterol esterase-cholesterol oxidase method
Centrlflchem Centrifugal Analyzer System
Roseschlaw, P., Bernt, E. and Gruber, W. Z.F.
Lin. Che. u. Klin. Biochem. 12, 226, 1974.
BJLLiE.ufc.Ln, Iclai
Modified Walters and Gerarde Method
Centrlfichem Centrifugal Analyzer System
• Walters, M. and Gerarde, H. Michrochem. J.
15, 231, 1970.
BiliEUfcin, JQi£££±
Modified Walters and Gerarde Method
® Centrlfichem Centrifugal Analyzer System
Walters, M. and Gerarde, H. Michrochem. J.
15, 231, 1970.
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Table VII.1 (continued)
twenty four month chronic toxicity/carcinogenicity study of
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
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Table V I I .2 (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARC INOGENICITY STUDY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
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Table VI1.2 (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
TRINITROTOLUENE (TNT) IN THE BGC3F1 HYBRID MOUSE
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Table VII.2 (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCI NOGEN ICITY STUDY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
INDIVIDUAL BODY WEIGHT MEASUREMENTS (grams)
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Table V I I .2 (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
TRINITROTOLUENE (TNT) IN THE BGC3F1 HYBRID MOUSE
INDIVIDUAL BODY WEIGHT MEASUREMENTS (gram-,)
AVAILABLE DATA
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TWFNTY FOUR MONT FI CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
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T.ibl v V I I . 2 (cunt i ntietl)
TWENTY fOUR MONTH CHRONIC 10* ICITY/CARC INOGENICITY STUDY OF
TRINI((.(’TOLUENE (TNT) IN THE BGC3FI HYBRID MOUSE
INDIVIDUAL BODY WEIGHT MEASUREMENTS (grams)
NO AVAILABLE DATA
Table V I I .2 (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARC INODENICITY STUDY OF
TRINITROTOLUENE (INT) IN THI B6C3F1 HYBRID MOUSE
INDIVIDUAL BODY WEIGHT MEASUREMENTS (grams)
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Tnble V I I . 2 (continued)
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Table VII. 2 (continued)
TWENTY FOUR MONTH CHRONIC TOXICtTY/CARCtNOGEN 1CITY STUDY OF
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Table V I 1 .2 (continued)
TWENTY FOUR MONTH CHRONIC TOY ICITY/CARC INOGENICITY STUDY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
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T.ihle V I I . 2 (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARC INOGFNICITY STUDY OF
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Table VI1.2 (continued)
1 WENT Y TOUR MONTH CHRONIC TOXICITV/CARC I NOGEN ICITV STUDY OF
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TWEN1Y FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
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T < i b1o V I I .2 (continued)
TWENTY FOUR MONTH CHRONIC tOX IC1tY/CARC INOGENICI tY STUDY OF
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NO AVAILABLE DATA
I,) b I e v I I . l (continued)
TWENTY FOUR month chronic toxicity/carcinogenicity study of
TRINITROTOLUENE (TNT) IN THE RGC3F1 HYBRID MOUSE
INDIVIDUAL BODY WEIGHT MEASUREMENTS (grams)
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I il) I \l I I . 2 (conti r)ued )
HBIJNM in* IT i TY/CARCINOGf NIC I T Y STUDY OF
NO AVAILABLE DATA
!WfN!» M)1J« MONTH CHRONIC TOMC II Y/CAHCI NOM NK'IT ¥ M Ul) V OF
TPIN! F ROIOI IJFNf (TNT) IN 1 ML [if.UI 1 HYBRID MOUSE.
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fable V I I .2 (continued)
1WIN1V FOUR MONTH CHRONIC T OX I C IT V/C ARC I NlUit N I C I T V CHJOY Of
IRINI IRIIIUI IJf Nf (TNI) IN THF B6C.il 1 HYBRID MOUSl
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TWENTY FOUR MONTH CHRONIC T OX IC IT Y/r. ARC I NOGEN IC I T Y STUDY OF
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ABLE DATA
Table V I I .3 (continued)
TWENTY TOUR MONTH CHRONIC T OX I O I I Y/(J ARC I NOOI N I C I T Y S1UOY OF
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TWENIY FOUR MONTH CHRONIC TOX ICIIY/CARC INOGFNICITY STUDY OF
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TWENTY TOUR MONTH CHRONIC TOXICITY/CflRCINOr.f NICI TV STUDY OF
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T.ihlf VI 1.3 (continued)
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T<)ble VI 1.3 (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARC I NOGEN ICITY STUDY OF
TRINIIROlOtUENF (TNT) IN THf K6C3F1 HYRRID MOUSF
INDIVIDUAL FOOD CONSUMPTION ME A SURF MF NTS (g/Uay)
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TWfNrv FOUR MONTH CHRONIC T0AlCItY/CARC 1NOGlNICITV STUDV OF
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INDIVIDUAL FOOD CONSUMPTION Mt ASURF MF NT S (g/Uay)
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TWENTY FOUR MONTH CHRONIC TOx 1CITY/CARC 1NOGlNICI IY STUDY OF
IRINITROTOLUENE (TNT) IN THE H6C3M HYBRID MOUSE
I NO I V IOUA L FOOD CONSUMPTION MEASUREMENTS (g/Uay)
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Table \l I I . 3 (continued)
TWENTY TOUR MONTH CHRONIC I 0 >. I C I I Y/C ARC I NUUt N 1C I T V S1UDY Of
TRINITROTOLUENE (TNT) IN THE BGCIft HYBRID MOUSE
INDIVIDUAL fOOD CUNSUMRI ION* MEASUREMENTS (y/Ua y )
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NO AVAILABLE DATA
Table V I I .3 (continued)
TWENTY FOUR month CHRONIC TOXIC[Iy/carcinogenicity study of
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
INDIVIDUAL FOOD CONSUMPTION MEASUREMENTS Ig/Uay)
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Table VI1.3 (continued)
TWENTY FOUR MONTH CHRONIC TOX ICITY/CARCINOGENICI IY SIUUY OF
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Table V I I .3 (continued)
TWENTY TOUR MONTH CHRONIC TOXICITY/CARC INOGENICITV STUDY OE
TRINITROTOLUENE (TNT) IN THE 66C3F1 HYBRID MOUSE
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Table VII.3 (continued)
TWENTY FOUR MON T FI CHRONIC FOX IC1 I V/CARC INOOtNICITV STUDY OF
TRINITROTOLUENE (INT) IN THE BbC3F1 HYBRID MOUSE
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I able V I I .3 (continued)
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TWENTY FOUR MONTH CHRONIC TOx1C 1TY/CARC I NOGEN IC1TY STODY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
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Table V I I .3 (continued)
TWENTY FOUR MONTH CHRONIC TOxIC1TV/CARC 1NODENIC1 TV STUDV OF
TRINIT ROT OLUFNF (TNT) IN THE BGCJF1 HYBRID MOUSE
INDIVIDUAL FOOD CONSUMPTION MEASUREMENTS (g/day)
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Table VII.3 (continued)
IWfcNIY F OUR MONiH CHRONIC T0A 1C1TY/CARC I NODEN1C11Y STUDY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
INDIVIDUAL FOOD CONSUMPTION MEASUREMENTS (g/day)
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Table \l I I . 3 (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENIC1TY STUDY GT
TRINITROTOLUENE (TNI) IN THE B6C3E 1 HYBRID MOUSE
INDIVIDUAL FOOD CONSUMPTION MEASUREMENTS (g/day)
7 ■ n n n rt i cc coco < co cn cn cn cn
i t r rr rr i co co co * n in in in ^
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7
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Table V I I.3 (continued)
TWENTY F OUR MONTH CHRONIC TOX 1C1TY/CARC INOGENICITY STUOY OF
TR1NITROTOLUENE (TNT) IN THE EGC3F1 HYBRID MOUSE
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Table VI1.3 (continued)
TWENTi I OUR MON T H CHRONIC I Ox I C II i /C ARC I NOL.1 Nl( I T V I UD r Ot
TRINI TROIOl UfNE (INI) IN I Ht E'.GCJII H i HR II) MOUSE
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Table V I I .3 (continued)
T WEN tY FOUR MONTH CHRONIC 10*ICItV/CARC I NOGEN ICITV STUDY OF
TRINITROTOLUENE (TNT) IN THE B6C3F1 HYBRID MOUSE
INDIVIDUAL F000 CONSUMPTION MEASUREMENTS (g/day)
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Table VI I ,5b
ni> i iiiiR muimui chronic r o* i e i i < / c arc i Nnc.f n u: ii •/ r ,iuiw or
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I NO 1 V IIHJAI (I 1 N11 AI CHFMISIRv VAIUFS - IfSI WEEK ?1
NO AVAILABLE DATA
IWINIy FOUR MON III lllkllNII. IOaH I I Y/<AR< INOl.fNICI I i SIUUY 01
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Tdble VI I,6d
TWENTY FOUR MON IH CHRONIC TOKICITY/CARCI NOGEN ICITY STUDY OF
I R1N1 1 ROIOLUENE (INI) IN Htf HGC3F1 FTYBRIO MOUSE
INDIVIDUAL ORGAN WEIGHT VALUES (g) * TEST WEEK 27
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inr^iX^r-cOvX!ttr'r**irr'CCcor^cor^cccOLDCCig>cocooocor^ix>(^r^xocag)cccor^Ln>co
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n’-p-r'X’^GC'CO'krcoOncocNOOODr-OGO — cOfNcocrcNCCCOccmcrccr-irsDcr
» OOGOCOOOCOOOCOOOOOOOOOOOOOOOOOOOCOCOOOOC
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x *r — r'®x'7v."crc^trif^or'nxLn(j'CNixr , ~rN.Lriir r ^cr — ^ cr *r
ir'TTnnnr?rr'^^*?nnr)r)r>crmnninr;rTmr'»7nn'7r'n
5555S55J55JJ555JJ555555555S5555555S5525J
r, ■-» '. r« r, '•. r* r, r, -» !% -j r, r> r-, ,-m r, r* r, r* r, c< > r* r, r% r* rs r% r» :% r* r>, r\ c\
< Z — 5 < - Z
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T.ible V I I . 6 c (continued)
TWFNTr I UUD MONTH CHRONIC TOX ICI TY/CARClNOGFNICI IY STUDY OT
TRINITROTOLUENE (INTI IN THf RfeCOFI HYBRID MOUSE
INDIVIDUAL ORGAN WIGHT VALUES (g) TEST WEEK 105
c c z < c
I lu < a t-
m a < — Z
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<N — — CN CN — <N — — — — CN — CN — CN — •* *- M
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*T*rDnnr“nnTTn^nnr'Tjr'.
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rnnnr'irnm^n^tNn'jo.nninrYtN':
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DATA
TWENTY FOUR MONTH CHRONIC TOXICITY/CARC I NOGEN ICITY STUDY OF
TRINITROTOLUENE (INI) IN THE B6C3F1 HYBRID MOUSE
INDIVIDUAL ORGAN WCIGHT VALUES (g) - TEST WFEK 105
CD Z < O IT
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mtninncoiocDcrO'-icOnr-r'Trinr'-f'-in — 0)(n--Oi'-oor , -‘0~-r-<NincD^tnTTncNcn
nc^nrj-TTTc^nfnnofnnnrrnnnrTnnmDncnrrromnoDDDn
i rrnr:Dnnt T :rinDr)nnnnnr)nnf , '' , onnr , )frnDnnn>*5n( v )nr)(*i( y )n( T i
i or. r ru;rC'^or^x / >.^Lnc3r'CCcr)OJnr'C^O T ?u'<X'ccc>0'^<^f^'7u*)vDr'*'^iroofN
: OOOGO---**”tN^^rifNrv>'Nnnr. ^'7n’tn77LnuninL'iininm^iv^(DiiiN
i nmnm'nrnmnmnnr. nr'nnnronmmnfnnonnnnnnnnr, nnnnnr)
Table VII.6c (continued)
TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF
TRINITROTOLUENE (TNT) IN THE BGC3F1 HYBRID MOUSE
INDIVIDUAL ORGAN WEIGHT VALUES (g) - TEST WEEK 105
i A Cl j uj w 2
I UJ < C* K
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moo > i k
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•“ — ■*- c\ rn — rv — <n ^
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TWENTY FOUR MONTH CHRONIC TOXICITY/CARC INOGENICITY STUDY OF
TRINITROTOLUENE (TNT) IN THE BGC3F1 HYBRID MOUSE
INDIVIDUAl ORGAN WEIGHT VALUES (g) - TEST WEEK 105
APPENDIX VI I I
CHIORTETRACYCLINE CONTENT OF 5002
CHRLORTETRACYCLINE CONTENT OF 5002
ANALYTICAL RESULTS (ppm)
SOURCE Of ANALYSIS
SAMPLE JDEN JIF I CAT .I& N
A & £ 0
TEI ANALYTICAL*
9.9
TEI ANALYTICAL* 12
SCIENTIFIC ASSOCIATES** 1.76
WOODSEN-TENENT LABS, INC.** N. D.
HARRIS LABS, INC.** <0.05
9.9
1 .72
N. D.
<0.05
7.7
1 .20
N. D.
<0.05
10.2
1 .64
N. D
<0.05
Sample A = Lot No Sept.18.81
Sample B = Lot No Dec.10.81
Sample C = Lot No March.24.82 (Original lot)
Sample D * Lot No Sept.10.82
♦Method: Snell and Snell, Colorimetric method of analysis.
Vol. IV AAA, pg. 184
♦♦Method: A0AC, XIII, pg.722-723, paragraph 4 2.211-42.214;
N. D.
None Detected
NITRATE, NITRITE, AND MERCURY CONTENT OF 5002
LOT NUMBER
N1TRATE S(ug/g)
NITRITES(ug/g)
MERCURY(ug/g)
/AUG 04-81 IT
28
0.5
0.03
/SEPT 18-81 1 A
<2.0
<0.1
0.05
/OCT 07-81 1 J
6.3
0.2
0.15
/NCV 12-81 1 G
1 6
0.4
<0.02
/DEC 10-81 1 A
1 2
<0.2
0.09
/JAN 22-821K
1 4
<0.2
<0.05
/FEB 09-821C
7.2
0.4
0.05
/MARCH 24-822G
1 9.0
0.24
<0.05
/MAY 12-822F
1 6. 4
0.1
<0.05
/JUNE 04-821K
17.0
0.1
<0.05
/JULY 29-821G
11.8
0.1
0.06
/SEPT 10-822J
5.0
0 . 1
0.2
/OCT 20-822L
4.7
0 . 1
0.2
/NOV 23-821 M
1 5. 4
0.2
0.05
/APRIL 14-83 1 B
1 9.6
0.1
<0.02
/JULY 07-832G
<5.0
<0.1
0.06
314
SUMMARY OF OPHTHALMIC FINDINGS
Ophthalmic examination, consisting of Indirect ophthalmoscopy
and b1om1croscopy, was performed on alI animals before
administration of the test article and at test weeks 25, 51, 76,
and 103. Only animals found to be free of clinically apparent
ophthalmic lesions In the pretest examination were used In the
study. The results of these examinations are summarized by test
week, followed by Individual animal observations.
Orbital bleeding was performed on randomly selected animals
from each test group at test weeks 14, 26, 52, 78, and 104.
Animals used for orbital bleeding were denoted on the ophthalmic
Incidence data sheets by using "L" or "R" when It was known which
orbit was used for bleeding. "U" was used to denote animals bled
when both orbits were used or when It was not recorded which
orbit was used. At test week 26, 0/100 animals bled showed
ophthalmic lesions. At test week 51, 13/100 animals bled were
observed to have ophthalmic abnormalities. At test week 76,
21/98 animals bled and examined were found to have eye lesions.
At the final examination In test week 103, 42/77 animals bled and
examined had ophthalmic lesions. Lesions observed In eyes which
had been orbitally bled Included: ocular discharge,
enophthaImI a, phthisis, cataracts, uveitis, buphthelmla, hyphema,
retinal vascular attentuatlon, and retinal degeneration. The
aforementioned abnormalities could have been caused by ocular
trauma or penetration at the time of orbital bleeding.
Ocular conjunctivitis, discharge, keratitis, and corneal
scarring were observed In all test groups throughout the study
and probably were a result of chronic external Irritation.
Increasing Incidences of cataracts and retinal lesions were noted
as the study progressed with test week 103 having the highest
Incidence. Retinal lesions Included: retinal folds, retinal and
choroidal vascular attenuation, retinal hyperrefIectIv1ty,
retinal hemorrhage, retinal degeneration, choroidal hemorrhage,
and subretlnal exudate. The frequencies of occurrence of these
changes were similar among control and treatment groups.
Scleral Icterus was noted In 1 animal (dose group 2.0
mg/kg/day) at test week 76 and In 10 animals at test week 103 (3
animals In dose group 0.4 mg/kg/day and 7 animals In dose group
50.0 mg/kg/day). These changes were probably related to
disorders elsewhere In the body.
In summary, ophthalmologic abnormalities observed In this study
occurred In random fashion, and were not considered to be related
to TNT treatment. , N ^
... v t i . ! ,
C. Sue West, D.V.M.
Dlplomate, American
College of Veterinary
Ophthalmologlsts
Date
Study Number L611&-*? OPHTHALMIC INCIDENCE TABLE
Test Article TNT SUMMARY - TEST WEEK 51
a a a a
n a a a
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32
116-9 OPHTHALMIC INCIDENCE TABLE
TNT SUMMARY - TEST MEEK 103
FINAL PATHOLOGY REPORT
OF TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCI NOGEN ICTY
STUDY OF TRINITROTOLUENE (TNT) IN THE
B6C3F1 HYBRID MOUSE
Decemb er 8, 1984
IITRI Project Number L6 1 1 6
Study No. 11
QUALITY ASSURANCE STATEMENT
L6116 SN11
Necropsy and histology procedures were Inspected on April 15
and October 7, 1982, October 12 and 13, 1983 and February 21 and
April 3, 1984 by J. Reed. Draft pathology reports were audited on
October 29 to November 29, 1982, January 19/ February 2 to 7, June
21 to 23, 1983, March 21 to 23 and November 12 to 14, 1984 by J.M.
Reed and J. McPhlllps. The study was found to meet Life Sciences
Quality Assurance criteria. Specimens and raw data generated during
the study will be retained In the IITRI Life Sciences Archives as
specified
IABLE. QL £Q!iI£tiIS.
I. INTRODUCTION. 516
II. MATERIALS AND METHODS. 516
1. Gross Pathology..... 516
2. H I stopatho I ogy. 517
3. Statistical Evaluations..... 518
III. PATHOLOGY RESULTS. 518
SIX MONTH INTERIM SACRIFICE. 518
A. Gross Observations. 518
B. Microscopic Observations. 518
TWELVE MONTH INTERIM SACRIFICE. 518
A. Gross Observations. 518
B. Microscopic Observations. 518
TWENTY-FOUR MONTH TERMINAL SACRIFICE. 519
A. Gross Observations. 519
B. Microscopic Observations. 519
Table I Statistical Evaluation of
H IstopathoIogIc Lesions for the 12-24
Month MS/SD and Terminal Sacrificed
Males. 520
Table II Statistical Evaluation of
Histopathologic Lesions for the 12-24
Month MS/SD and Terminal Sacrifice
F ema I es. 521
IV. SUMMARY ANO DISCUSSION. 522
V. REFERENCES. 523
32P»
1L&LL GE. GGtLlfctili
PATHOLOGY APPENDIX I. 524
SIX MONTH INTERIM SACRIFICE
A. Necropsy Observations - Table I. 525
B. H I stopatho I ogy Incidence - Table.. 536
C. Incidence Comparison Tables. 573
PATHOLOGY APPENDIX II. 576
TWELVE MONTH INTERIM SACRIFICE
A. Necropsy Observations Table II.. 577
B. H I s top at ho I ogy Incidence Table. 593
C. Incidence Comparison Tables. 642
PATHOLOGY APPENOIX III. 647
TWENTY-FOUR MONTH TERMINAL SACRIFICE
A. Necropsy Observations - Table I I I — A
Scheduled Sacrifice.. 648
Spontaneous Death/Moribund Sacrifice
Tab I e- I I I -B. 663
B. Overall Incidence for Males... 681
C. Combined Neoplastic and Non-NeopIast1c
Incidence for Males. 698
D. Non-Neop I ast 1 c Incidence for Males. 714
E. Neoplastic Incidence for Males. 7 26
F. Overall Incidence for Females. 731
G. Combined Neoplastic and Non-NeopIast I c
Incidence Table for Females. 745
H. Non-NeopIastIc Incidence for Females. 759
I. Neoplastic Incidence for Females. 769
J. Table of animals with Neoplasia. 774
L. Single Tabulated Animal Report
Individual Microscopic Observations. 823
FINAL PATHOLOGY REPORT OF TWENTY-FOUR MONTH CHRONIC
TOXICITY/CARCINOGENICITY STUDY OF TRINITROTOLUENE (TNT)
IN THE B6C3F1 HYBRID MOUSE
I. INTRODUCTION
In accordance with the experimental protocol necropsy and
histopathologic examination were performed on organs and tissues
of 600 (300 males and 300 females) B6C3F1 hybrid mice for IITRI
Project L6116, Study Number 11.
The mice were divided Into four groups, each containing 75
males and 75 females. Each group was fed either tr I nItroto1uene
(TNT) as a dietary admixture or an untreated rodent chow diet
until death or sacrifice. The treatment group number, treatment,
number of mice per group, sex, and corresponding dose levels are
shown below.
Treatment
Group
T reatment
Number of
Males
Number of
Females
Dose leve
mg/k g/day
1
_
75
75
0.0
1 1
TNT
75
75
1 .5
1 1 1
TNT
75
75
10.0
1 V
TNT
75
75
70.0
Scheduled sacrifices were conducted at 6, 12, and 24 months.
Ten mice per dose level oer sex were sacrificed at 6 and 12
months. All surviving mice were sacrificed at 24 months. A
pathology report was made for each of these Intervals. These
pathology reports constitute Pathology Appendices I, II, and III.
Each report lists the number of mice examined that died
spontaneously or were sacrificed as moribund along with those
scheduled to be sacrificed at 6, 12, and 24 months of the study.
II. MATERIALS AND METHODS
1. Gross Pathology
The mice were anesthetized with carbon dioxide,
exsangIunated from the orbital sinus or abdominal aorta, and
necropsled. Mice dying spontaneously during the study or
sacrificed JLa extremis were also necropsled. At necropsy,
abdominal, thoracic and cranial cavities of each mouse were
opened and organs were examined and collected In 10$ neutral
buffered formalin. The eyes were fixed in 3$ gIutaraIdehyde
solution and the testes in Bouln's solution. The lungs were
fixed by Intratracheal perfusion of formalin. The brain, heart,
liver, spleen, and testes were weighed before fixation. Organs
were not weighed for mice which either spontaneously died or were
sacrificed moribund.
2 . HI stop athoiogy
The following tissues were collected at necropsy, processed
using standard histologic technique, paraffIn-embedded, sectioned
at 5u , stained with hematoxylin and eosln, and examined
microscopical ly. Those tissues marked with an asterisk In the
list below were processed for microscopic examination only at the
control and high dose level (70.0 mg/kg/day). The brain, spinal
cord, spleen, kidneys, heart, liver, gonads, as well as all gross
lesions were processed at .all dose levels.
The following tissues were collected:
AdrenaI *
Brain (frontal, parietal, cerebellar)*
Cecum*
Colon*
Costochondral juncture
Duodenum*
Epididymides*
Esophagus*
Eyes with optic nerves*
Gall bladder
Gross lesions*
Heart*
I I e u m *
Jejunum*
KIdneys*
Larynx
Liver*
Lungs and malnstem bronchi*
Lymph nodes:
Mandibular
Mesenter lc*
Mammary gland*
Muscle, skeletal
Nasal turbI nates
The grading system for lesions and the abbreviations used In
the pathology tables are as follows.
Grade 1 = minimal severity
Grade 2 = mild severity
Grade 3 = moderate severity
Grade 4 = marked severity
N = Within Normal Limits
M = Tissue Not Present
- = Tissue Not Applicable
P = Lesion Present, No Grade
Ovaries*
Pancreas *
Pituitary
Prostate*
Rectum*
Salivary gland
SemInal vesicles
Sciatic nerve
Skin/Abdomlna!
Spinal core (cervical,
thoracic, lumbar)*
SpIeen*
Sternum with marrow*
Stomac h
Testes *
Thymus
Thyrolds*/parathyrolds*
Tissue masses*
Tr ac hea *
Urinary bI adder
Uterus*
Bone marrow smear
3. Statistical Evaluations
Statistical evaluation of pathological lesions were
performed using models for qualitative data. For the comparison
of treated vs control mice in terms of the presence or absence of
a specific lesion, Fishers exact test was used for cases when the
expected value of any cel I was less than or equal to five.
Otherwise, a chi-square analysis was performed.
III. PATHOLOGY RESULTS
The necropsy summary tables and the h I stop at ho IogIst' s
report. Including the h1stop atho Iogy Incidence and summary tables
for the 6 and 12 months Interim sacrifices and the 24 month
terminal sacrifice, are presented In Pathology Appendices I, II
and III, respectively. A summary of the pathology results
fo I lows.
1. SIX MONTH INTERIM SACRIFICE
A. Gross Observations
All lesions observed at necropsy during this study period
were regarded as Incidental findings ascribed to natural ly
occurring disease or method of sacrifice. These lesions were
present In both control and treated mice.
B. Microscopic Observations
Lesions observed In kidneys as cytoplasmic vacuolization of
tubular epithelial cells In male mice and urethral calculi were
considered to be spontaneous In nature In females. A pulmonary
hemorrhage and increased splenic extramedul lary hematopoiesis
were also considered spontaneous and unrelated to the compound
administration. All other lesions, neoplastic and non-neopI ast|c
observed microscopically In mice of both sexes, treated with TNT
for 6 months were considered unrelated to the TNT administration.
2. TWELVE MONTH INTERIM SACRIFICE
A. Gross Observations
Lesions observed at necropsy in mice treated with TNT for 12
months were considered to be spontaneous In nature and unrelated
to TNT administration. These lesions were regarded as Incidental
findings of naturally occurring disease and were present In both
control and treated mice.
B. Microscopic Observations
332
epithelial cell degeneration. Increased splenic extramedullary
hematopoiesis, cytoplasmic vacuolization of renal tubular
epithelial cells, urethral calculi and a variety of Inflammatory
dermal lesions (dermatitis and ulceration) were observed In
control and treated mice. These lesions occurred spontaneously
and were not considered to be related to the compound
adm I nIstrat I on. In the brain of females, corpora amylacea were
observed In treated mice, but these bodies are frequently seen In
untreated adult mice and they were not considered
treatment-re1ated (1,2). Increased extramedullary hematopoiesis
In spleen, pulmonary congestion, renal lymphocytosis and chronic
active dermatitis were observed In control and treated females
and were sporadic and not treatment-reIated .
The cause of death and morbidity among mice during 6-12
months of the study was ascribed to natural ly occurring disease.
Administration of TNT for twelve months did not Induce
non-neopIast I c toxic or neoplastic lesions In male or female
mice.
3. TWENTY-FOUR MONTH TERMINAL SACRIFICE
A. Gross Observations
Lesions observed at necropsy which appeared to have an
Increased Incidence In female mice at the 70.0 mg/kg/day dose
level were: enlarged spleen (splenomegaly) and enlarged lymph
nodes ( Iymphadenopathy) , which may have been treatment-related.
All other lesions observed at necropsy during this study period
were considered to be spontaneous naturally occurring diseases
for this strain of mice.
B. Microscopic Observations
Treatment-related lesions were observed In the
reticuloendothelial system (spleen) of females at the 70.0
mg/kg/day dose level. These lesions Included; leukemia of
granulocytic or lymphocytic type and/or malignant lymphoma of
histiocytic, lymphocytic or mixed type. Leukemia and malignant
lymphoma were systemic reticuloendothelial neoplasias and did
Involve other organs and tissues (adrenals, bone marrow, brain,
gastrointestinal tract, eyes, kidneys, liver, lung, and the
following lymph nodes; axillary, cervical, hepatic. Inguinal
lumbar, mandibular, mesenteric, popliteal, renal and respiratory.
They were also observed In mammary gland, optic nerves, ovaries,
pancreas, peritoneum, salivary gland, skeletal muscle, spinal
cord, thymus, thyroids, urinary bladder and uterus.
The Incidence of combined IeukemIa/ma I Ignant lymphoma In the
spleen of females appeared to be dose-related. The Increase was
statistically significant at the 70.0 mg/kg/day dose level, and
these lesions were considered to be treatment-related (Tables I
and II).
TABLE I
Twenty-Four Month Chronic ToxIcIty/Care I nogen IcIty Study of
Trinitrotoluene (TNT) In the B6C3F1 Hybrid Mouse.
Statistical Evaluation of HIstopathologIcaI Lesions
for the 12-24 Month MS/SD and Terminal Sacrificed Males
DOSE (mg/kg/day)
flxfl. lxl lflxfl 2^0.
LIVER - INCREASED ti£MAlQEQ-LE21S.
PRESENT 37 13* 7
ABSENT 50 46 39 47
£EL££ti
LYMPHO ID
Tab Ie I I
Twenty-Four Month Chronic ToxI c 1 ty/Care 1 nogen I c I ty Study of
Trinitrotoluene (TNT) In the B6C3F1 Hybrid Mouse.
Statistical Evaluation of HIstopathologIcaI Lesions for the
12-24 Month MS/SD and Terminal Sacrificed Females
DOSE (mg/kg/day)
0.0 1*1 liUl 2£L^1
KIDNEYS r. LEUKEM1A /.MAL1GNMI LXU£M£14A
PRESENT 5 11 8 10
ABSENT 49 43 46 44
S PLEE N - LEUKE MIA/MAL I GNAN.I LIHEHUMA
PRESENT 9 15 17 21*
ABSENT 45 39 37 33
L IVER r
PRESENT 5 11 8 10
ABSENT 49 43 46 44
« P < .05
335
In males. Increased splenic extramedullary hematopoiesis and
lymphoid hyperplasia were observed In control and treated mice.
There was no dose-response relationship and these lesions were
considered spontaneous with no biological significance. The
hepatocellular adenomas and carcinomas were observed In control
and treated mice and the occurrance of these neoplasms were
considered to be spontaneous In nature.
IV. SUMMARY AND DISCUSSION
Lesions observed at necropsy at 6 and 12 month Interim
sacrifice and microscopic examination of tissues during this
study period did not reveal lesions which were considered to be
Induced by the administration of TNT.
Enlarged spleen and lymph nodes In females observed at
necropsy at the terminal sacrifice and In mice that died or were
sacrificed moribund between 12 and 24 months were considered to
be treatment-related. Microscopic examination revealed leukemia
and malignant lymphoma In the spleen of these mice for the 70.0
mg/kg/day dose level. These statistically significant Increases
were considered to be a carcinogenic effect of TNT In the B6C3FI
hybrid females when treated for twenty-four months.
On the basis of compound-Induced histopathologic neoplastic
lesions observed for this study, no effect levels for TNT In
B6C3F1 hybrid mice were 70.0 mg/kg/day for male mice and 10.0
mg/kg/day dose level for female mice.
All other lesions observed at necropsy and microscopically
In tissues from the twnety-four month chronic
toxlclty/carclnogenlclty study of trinitrotoluene (TNT) were
considered sponteneous, naturally occurring degenerative.
Inflammatory and/or neoplastic diseases which commonly occur In
an aging mouse population of the B6C3F1 strain (1,2).
Vladislava S. Rac, M.S., D.V.M.
Sc Ientlf1c Adv1sor
Veterinary Pathologist
Life Sciences Research
336
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