AD

)IRI

Report No.

1963

SEHMX:

14 Day Toxicity Study in Rats by Dietary Administration

Final Report by: R.J. Greenough P. McDonald July,41985

Supported by: U.S. Army Medical Research and Development Command Fort Detrick Frederick, Maryland, 21701

Contract No. DAMD 17-80-C-0053 IRI Project 415669 SR

Inveresk Research International Limited Musselburgh, ER21 7UB, Scotland

Contracting Officer's Technical Representative: Jesse J. Barkley, Jr. Army Medical Bioengineering Research and Development Laboratory Fort Detrick, Frederick, Maryland 21701-5010 U.S.

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 authorised documents.

Non

Classified
MWhonDore Irnfe'.d)

StCURITY CLASSIFICATION OF THIS PAGE

REPORT DOCUMENTATION PAGE

I.
REPORT NUVRER .GOTACCESSION NO.

READ INSTRUCTIONS BEFORE COMPLETING FORM

3. RECIPIENT'S CATALOG NUMBER

4.

TTLE

and

ubtile)S.

TYPE OF REPORT A PERIOD COVERED

HMX:

14 Day Toxicity Study in

Rats by
Oct

Final
1980-Nov 1980
PERFORMING ORG. REPORT NUMBER

DeayAdministration Dietary6. ________________________________________

7. AUTHtOR(*)

41 5669S R/ 1963
6. CONTRACT OR GRANT NUMBER(s)

R.J. Greenough
P. McDonald
NAME AND ADDRESS

DAMD 17-80-C-0053

S.

PERFORMING ORGANIZATION

10.

PROGRAM ELEMENT, PROJECT, TASK

Inveresk Research International Limited,

Musselburgh,
III.

62 104
12.

0A31

70A

5.

EH21

7UB,

Scotland

CONTROLLING OFFICE NAME AND ADDRESS

REPORT DATE

Jesse J. Barkley, Jr, U.S. Army Medical Research and Development Command, Ft. Detrick, Maryland, U.S.A.
4I. MONITORING AGENCY NAME & ADORESS(if different from Contfrolling Office)

30 July 1985
13. NUMBER OF PAGES
'.f

78
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Non classified
IS&. DECL ASSI FICATION/ DOWNGRADING SCHEDULE

16.

DISTRIBUTION

STATEMENT (of this Report)

IDISTHIDUTION
STATEMENTl A
Apprvedforpublic release; Distribution Unlimited. .

17.

DISTRIBUTION STATEMENT (of the abstract entered in Block 20, it dIffereit hoea Report).

IS.

SUPPLEMENTARY

NOTES

Principal Investigator:

A.B.

Wilson

19. KEY WORDS (Continue an reverse side If necessary and Identify by block nunaber)

HMX, Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, Rat, Subacute, Toxicity

Explosives,

20.

A@GTV ACT (Cauthme -i reers

seb

Nt necessar

I Identify by block nmirbet)

See Overleaf.

%p

SECURITY CLASSIFICATION OF THIS PAGEr(TMbe

Date Anierd)

Abstract , Male and female rats were dosed via the diet for 14 days with HMX Concentrain order to select dose levels for a 13 wee k study. tions were selected to give doses of 0, 100, 1000, 3000 ald Deaths occurred in males at 9000 mg 9000 mg HMX/kg/day. There HMX/kg/day and in females at 1000 mg HMX/kg/day and above. were substantial effects on food intake and upon body weight. Histopathological examination revealed centrilobular toxic There was also degeneration in the lives of male and female rats. hepatocyte hyperplasia, increased cyto plasmic eosinophilia in "the liver and lymphocytic depletion in the spleen and thymus.

,%

4..

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AD IRX Report No. 1963 HMX: 14 Day Toxicity Study in Rats by Dietary Administration

Final Report by: R.J. Greenough P. McDonald 16 July, 1985

Supported by: U.S. Army Medical Research and Development Command Fort Detrick Frederick, Maryland, 21701

Contract No. DAMD 17-80-C-0053 IRI Project 415669 SR

Inveresk Research International Limited Musselburgh, EH21 7UB, Scotland

Contracting Officer's Technical Representative: Jesse J. Barkley, Jr. U.S. Army Medical Bioengineering Research "and Development Laboratory Fort Detrick, Frederick, Maryland 21701-5010

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 authorised documents.

FOREWORD "I, the undersigned, hereby declare that this work was performed under my supervision, according to the procedures herein described and that this report represents a true and accurate record of the results obtained."

A.B. Wilson,

B.V.Sc., D.A.B.T.

M.R.C.V.S.,

7:, /,
NSPF rLD

Accesion For NTIS CRA&M

' .2 ;

0l Justification J stfiaton...................
By ........ ................................. Di:ti ibution I

DTIC TAB Unannounced

......... .... ...... .

"Availability Codes
Dist Avail arnd I or Special

AI ,Project No. 415669SR

I
1963

Report No.

QUALITY ASSURANCE AUTHENTICATION The conduct of this study has been subjected to periodic inspections by the IRI Quality Assurance Unit. The dates of inspection are given below. IRI Project No. 415669SR Date of Q.A. Inspection Report No. 1963

Date of Report to Management 21 October 1980 3 November 1980

20 October 1980 31 October 1980

This report has been audited by the Quality Assurance Personnel according to the appropriate Standard Operating Procedure. The report is considered to describe accurately the methods and procedures used in the study and the original "data generated during the study.
,_Signed:
_ _Date:

DMLA

f4

(Quality Assurance : Manager)

D/

W
f. 21.

CONTENTS Page SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TABLES 1 2a 2b 3 4
5a

1 3 4 11 17 18 Incidence of Mortality Group Mean Body Weights - Males Group Mean Body Weights - Females Group Mean Food Consumption Achie%id Dosages
Absolute Organ Weights: Group Mean Values

18 19 20 21 22
23

5b 6 FIGURES 1 2

for Animals Killed at Term Group Mean Values Relative Organ Weights: for Animals Killed at Term Incidence of Histopathological Findings

24 25 26

Group Mean Body Weight Changes - Males Group Mean Body Weight Changes - Females

26 27 28 28 29 31
32 38 42 44

APPENDICES la lb 2
3a 3b 4 5a

Analysis of Diet Analysis of Water Pre-experimental Analysis of Diet for Stability and Homogeneity
Body Weights - Individual Values - Males Body Weights - Individual Values - Females Formulated Diet Analysis Individual Values Absolute Organ Weights:

-i

5b 5c 5d

for Animals Killed at Term Individual Values Relative Organ Weights: for Animals Killed at Term Individual Values Absolute Organ Weights: for Premature Decedents. Individual Values Relative Organ Weights: for Premature Decedents.

46 48 49

CONTENTS (continued) Page APPENDICES (Continued) 6 Gross Pathology and Histopathology for Individual Animals 50 77
78

PERSONNEL INVOLVED

"-

DISTRIBUTION LIST

FINAL PAGE OF REPORT

78

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SUMMARY The object of this study was to provide information on the subacute toxicity of the test substance and to give an indication of suitable dose levels for subsequent studies in rats. Groups 0. 66 and 6Y Fischer 344 rats were fed diets containing Octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine (HMX) for 14 days at nominal dose levels of 0, 333, 1000, 3000 and 9000 mg/kg/day. At the end of this period all the surviving rats were killed and subjected to necropsy and gross pathology. Subsequent histopathological examination of selected target organs was carried out on all control and group 5 animals, together with all the premature decedents from groups 3 and 4. The results obtained are summarised below: Mortality There were 13 premature decedents distributed as follows: Males 3 4 Females 3

Dose Group Nominal Dose Level mg HMX/Kg/

day

1 0 0

1 0 0

333 1000 3000 9000 0 0 0 83

333 1000 3000 9000 0 17 17 100

% Mortality

"Clinical Signs
piloerection and frequent incidences of red staining around the eyes and nostrils were observed in group 4 and 5 males and all HMX treated females. One convulsion occurred among the group 5 females. Body Weight *
.

"Emaciation, hunched posture, subdued appearance,

Males receiving HMX showed a dose related reduction/loss in body weight gain. Groups 4 and 5 showed an actual loss in body weight at start of dosing. This weight loss was not regained by the group 5 animals. Females receiving HMX all showed a significant body weight

"loss.
.4

'

Food Consumption Marked reductions in food consumption were observed for the HMX treated animals. The female rats consumed 50% or less compared with control animals during the first week of dosing. Terminal Studies Gross Pathology and Histology Histopathological examination revealed centrilobular toxic degeneration in the livers of the group 5 male rats receiving an average achieved dose of 8504 mg HMX/kg/day. Hepatocyte hyperplasia and increased cytoplasmic eosinophilia in the liver, together with lymphocyte depletion in the thymus and spleen were observed for the group 5 females receiving an average achieved dosage of 3055 mg HMX/kg/day. These findings were also observed for the premature decedents from groups 3 and 4 receiving 1280 and 3474 mg HMX/kg/day respectively. Organ Weights Absolute liver and kidney weights were reduced for all HMX treated groups when compared to control animals. Relative liver weights were reduced for males whilst relative kidney weights were increased for both males and females. The increases observed in relative kidney weights, for HMX treated animals, can probably be attributed to the reductions observed in body weight gain.

1%

5259

_P

7Z

INTRODUCTION (HMX) Octahydro-l,3,5,7-tetranitro-1,3,5,7-tetrazocine explosive and is found in the water effluent from the manufacturing processes for RDX and HMX. is an

*-',

This report describes a 14 day dietary toxicity study in rats conducted to set dose levels for a proposed 90 day study. The study was undertaken at the Elphinstone Research Centre of Inveresk Research International Limited over the period 9 October - 31 October 1980.

o -= . . . . . . . . . . . . . . . . . . . . * .* *

-----------------------------------------------------------------------------------.-

"4

MATERIALS AND METHODS The HMX (Lot No. HMX-IRI-001) was manufactured by the Royal Ordnance Factory, Somerset. The bulk of the material (5 kg: wetted with 15.25% water and packaged in individual 50 g lots) was stored at Nobel's Explosive Company, Muirside, Dunfermline. Small amounts of HMX were transported, in an approved container, to the IRI laboratories as required. The samples used in this study were obtained on 4 August, 16 September and 7 October 1980. The test compound was stored in a Miristry of Defence approved container under ambient conditions in the company dispensary. Prior to use the required amount of HMX wag removed from this container and dried at approximately 90 C overnight. The dried HMX was then stored in a glass dessicator until used. Animals Thirty five male and 35 female Fischer 344 rats (40-60 g body weight) were obtained from Charles River (U.S.A.) Limited, Sixty rats (306 and 39) Wilmington, Mass. on 1 October 1980. to their new environment for 8 were allowed to acclimatise days before treatment commenced. Pre-experiment Acceptance Testing All animals were examined on arrival for signs of disease. Five animals of each sex were selected, firstly on the basis of "a clinical examination and secondly in a random choice manner, "and subjected to a microbial examination and histopathological evaluation of main organs. The results of these tests showed "that the delivery of animals was of an acceptable standard for use on this study. Animal Management

"The rats were housed in an animal room dedicated to this

experiment with a light intensity of approximately 200 lux, a 12 h light-dark cycle, temperatures 8utomatic 8 lly maintained at 20C + 2 C with extreme limits of 21 C and 24 C, and humidity ca 50% with extreme limits of 34% and 59%. Caging The rats were housed one per cage in polypropylene cages (overall dimensions 48 cm x 15 cm x 22 cm) with stainless steel wire grid tops and bottoms. The cages were suspended over trays lined with absorbent paper.

4011

Cages, Diet

trays and absorbent paper were changed as necessary.

Food and tap water were freely available to the rats at all times. The diet was BP Nutrition Rat and Mouse No. 1, a ground diet adequate for all stages of growth in rats. Typical analyses of both water and diet are presented in Appendix 1. Allocation of Rats to Cages and Treatment Groups Empty cages were placed on racks, then upon receipt, starting first with male rats, a transporting box was opened and a rat placed in the first cage at the top left hand corner of the rack. A second rat was removed from the same transporting box and placed in the next cage and so on until 35 cages each
contained one male rat.

This process was repeated using new cages and female rats. * Following a 2 day transportation recovery period the animals were allocated to specific treatment groups using a stratified body weight sequenced randomisation procedure. Four body weight ranges were selected for each sex. Starting first with the males, the animals were removed from their cages, weighed and then segregated into 1 of 4 large stainless steel holding cages according to their body weight. Six rows of cages, each row containing 5 cages, were then arranged on racks. Two sets of computer generated random number permutations were obtained, the first set gave 5 random sets of numbers from 1-6 corresponding to the number of sequences of cages and the second gave 6 random sets of numbers from 1-5 corresponding to the number of treatment groups. Starting with the lowest weight range of male rats one animal was placed in a cage according to the sequence position indicated by the first number in the first set of random numbers. The cage was then assigned to a treatment group using the second random number set. This process was repeated until all the male rats from the 4 different weight ranges had been assigned to cages and treatment groups. This complete process was repeated for the female rats. Animal Identification, Treatment Groups and Dose Levels

Each animal received a unique ear punch which identified it individually within the study and wnlch corresponded to that 5301
S.

.-.

'

-6

animal's number. Each rat was ascribed a cage card which identified that animal by project number, cage number, animal number, sex and treatment group. Each cage card was colourcoded according to the treatment group. The treatment groups were as follows:

Dose Group 1 2 ""

3

Dose Level HMX (mg/kg/day) 0 (Control) 333 1000 3000 9000

Colour Code Green Blue

Yellow

Animal Numbers d3Y 501-506 507-512

513-518

531-536 537-542
543-548

4 5

Buff Red

519-524 525-530

549-554 555-560

Animal Room Sanitation

Floors were mopped each morning, before other work in the room had begun. In the afternoon floors were swept and then mopped with a disinfectant solution after work in the room was finished. Diet Preparation Diets containing HMX were prepared freshly at the beginning of The concentration of test compound in the diet each study week. was calculated weekly after predicting the mid-week body weight The following and food consumption for the forthcoming week. equations were used in this calculation: Dose level (mg/kg/day) x predicted midweek body weight (g)

4'

Concentration

(PPM)

= predicted daily food consumption (g/day)

"'[ Amount of test substance required (g) ;'

=

PPM x required of diet weight 1000

After drying to a constant weight HMX was sieved through a The requisite plastic 100 4m mesh sieve immediately before use. amounts of HMX and powdered diet were measured out, transferred 0500
*-' 1 . .. .. -. ". . . " . ". " . " " " . ' ' " " " ." " ."" ', '

to plastic containers, sealed and mixed for 20 min using a Winkworth change drum tumble mixer. Dietary Sampling Ten gram samples were taken from the top, middle and bottom of the container holding the freshly mixed diet to confirm stabThese samples, along and homogeneity of mixing of HMX. ility with a 100 g sample taken for archives, were taken from all diet mixes including controls. Analysis of HMX in Analytical Method HMX was analysed by high performance liquid chromatography (HPLC). Three samples (of an appropriate size depending on the nominal concentration of HMX in the diet) were weighed into 3 x 8 oz glass jars. To this was added an appropriate volume of internal standard solution (1,3-dinitrobenzene in acetonitrile) The glass and sufficient acetonitrile for complete extraction. jars were then capped and shaken mechanically for 1 h after which the jars were removed and the contents allowed to settle. An aliquot of the liquid fraction was injected into the HPLC and analysed either directly or after suitable dilution using acetonitrile:water (20:30 v/v). Standard solutions of HMX were prepared by adding known These were amounts of HMX to samples of untreated diets. treated with internal standard solution and extracting solvent Further as described above for the formulated diet samples. details are reported under project DAMD 17-80-C-0053. Stability and Homogeneity of HMX in Diets Diets

Before commencement of the study experiments were performed to of HMX treated diets. determine homogeneity and stability Accordingly diets containing 1,250, 10,000 and 25,000 PPM Ten representative samples from (w/w) of HMX were formulated. each diet mixture were analysed immediately after preparation. Ten samples from each diet were subjected to accelerated ageing by storage at 40"C in an environmental cabinet and The remainder of each diet was stored analysed after 14 days. at room temperature and analysed after 21 days. Results of these analyses showed that mixing procedures and stability were satisfactory (see Appendix 2).

0578
Jr 1111 11* 1 111,

x,

K : --:.>

.1.

HPLC Conditions HPLC: Altex pump, Pye Unicam LC3 UV Spectrophotometer

Column: Solvent: Flow: Wavelength:

Recorder:

Chart Speed:

Hypersil ODS (10 cm x 0.5 cm) Acetonitrile:water (20:30 v/v) 1 ml/min 228 nm Servoscribe Is - 10 mV 300 mm/h

Observations on the Animals Mortality All animals were inspected for any deaths at the start of each day and again during the afternoon clinical signs check. Clinical Signs The rats were observed at intervals throughout the day for any signs of ill health or reaction to treatment. Physical Examination Each animal was given a weekly detailed physical examination for external lesions or palpable masses. Body Weight The weight of each rat was recorded on the day dosing commenced Animals were also weighed and twice each week thereafter. twice prior to the start of treatment. Food Consurintion The quantity Food consumption was recorded on a weekly basis. of food eaten by each rat was calculated by measurement of the amount of food given at the beginning of each week and deducting that remaining in the food hopper at the end of each week and any that may have been scattered on the cage floor during that week. Water Consumption The quantity of water consumed by each rat was assessed each week by visual assessment of the calibrated water bottle.

7756
p.O

Terminal Studies * -, On day 15 of dosing all the surviving rats were sacrificed by nitrogen asphyxiation. Blood samples of ca 2 ml were taken into heparin via the posterior vena cava from all animals. Plasma was separated by centrifugation and stored deep frozen at -20 C for analysis at a later date. Gross Necroosv A full necropsy was performed as detailed below. Each rat was examined externally including the body orifices along with examination of internal organs and tissues. The following organs were taken at necropsy: Brain Heart *Kidneys *Liver Spleen Thymus The fresh weights of the organs marked * were recorded before preservation. All organs were examined in situ, then dissected from the carcass, re-examined, including cut surfaces, and then preserved in 10% neutral buffered formalin. Tissues were fixed after slicing to a thickness not exceeding

0.5 cm.
Liver lobes were sliced, the kidneys longitudinally bisected and the cut surfaces examined before fixation. All gross lesions were recorded in narrative, descriptive terms, including size (in mm), number, shape, colour and texture. Carcasses of animals were discarded immediately following autopsy and the placing of all tissues listed above in fixative. Processing of Fixed Tissues The fixation time was 14 days. Tissues were trimmed to a maximum thickness of 0.3 cm for processing. Parenchymal organs, e.g. liver, were trimmed to allow the largest surface area possible for examination. 6269

NO%

10

Multi-longitudinal sections through the entire cortex and medulla of each kidney were submitted. Three cross-sections of brain including (a) frontal cortex and basal ganglia, (b) parietal cortex and thalamus and (c) cerebellum and pons were submitted. Histological Technique Tissues were cut to 4-6 4m thickness and stained with haematoxylin and eosin (H & E). All staining methods used are described in "Histological Laboratory Methods" by Disbrey and Rack (E.S. Livingstone Ltd., Edinburgh, 1970). Histopathological Examination * Histopathological examination of the tissues listed above was carried out on all control and group 5 (top dose) animals, "together with all the premature decedents. Statistical Analysis

Whenever considered necessary, numerical data were subjected to statistical analysis using Student's 't' test. The levels of significance
* **

as indicated in

the report are: P<0.05 P<0.01 P<0.001

* Archivina

Significantly different from controls, Significantly different from controls, Signficiantly different from controls,

On completion of all practical work the biological material and data generated during the study was stored, together with samples of the diet formulations and a sample of the test compound used, in the Scientific Archives of Inveresk Research International Limited. All materials relating to this study will be retained for a minimum period of 5 years.

5302

"RESULTS
Dosing period: Necropsy: Observations Mortality There were 13 premature decedents during the course of this study. The time of death and mortality distribution is presented in Table 1. All the premature decedents showed a pronounced body weight loss during the period they were dosed. Clinical signs observed prior to death or sacrifice in extremis were emaciation, unkempt appearance, hunched posture and piloerection. Clinical Signs Males Group 1 (Control) - No abnormalities were observed. Group 2 - No abnormalities were observed. (333 mg/ Group (1000 mg/ kg/day) Group 4 (0mslightly 518d slight loss of body tone at the start of week 2, no abnormalities were observed for remaining animals in group. all animals observed to have a emaciated appearance. 16-30 October 1980 31 October 1980

- Day 4:

Day 11: mild piloerection in 3/6 animals. Piloerection observed for remainder of dosing period.

Group 5 (9000 mg/

animals observed to have a Day 4: all slightly emaciated appearance, No. 530d had red staining around nostrils and a hunched appearance. animals emaciated and disall Day 7: playing a hunched posture with piloerection - these signs were observed for Nos. the remainder of the dosing period. 528d and 5296 were observed to have some bald patches. Day 9: 528d found dead in cage, hyperkinesia observed in surviving animals. Day 11: emaciated, unkempt appearance.

5234

12

Day 12: 527d found dead in cage. Dal 13: 525d, cage. Females
Group 1 - No abnormalities detected.

526d and 5296 found dead in

(Control) Group 2 (333 mg/ kg/day)

- Day 4: all animals observed to have a slightly emaciated appearance. Day 7: 5399 had red staining around nostrils, mild piloerection observed in all animals. Day 11: mild piloerection in 2/6 animals.

Group 3 (1000 ng/ "ka/day)

Day 4: all animals observed to have an emaciated appearance with a hunched posture and piloerection - these signs persisted for the remainder of the dosing period. Day 11: 5489 ataxic, extremely emaciated with a hunched posture and marked piloerection - killed in extremis.

Group 4 (3000 mg/ kg/day)

Day 4: all animals observed to have an emaciated appearance and piloerection these signs persisted for the remainder of the dosing period. Blood stains were observed on the tray papers for Nos. 5499, 5539 and 5549. Day 7: of Nos. Day 8: red staining observed around nose 5509 and 5549. 5539 observed to have eaten the

"4

tip of its own tail, later observed to be ataxic and vocalising. Day 9: 5509 found dead in cage. viving animals hyperkinetic. Sur-

Day 11: all surviving animals hyperkinetic when aroused. The tail of 5539 was slightly swollen. Day 12: 5539 was observed gnawing at remainder of tail.

0613

Day 13: 5539 very pale. , Group 5 (9000 mg/ kg/day)

-

eyes were observed to be

SubDay 4: all animals were emaciated. dued appearance, hunched posture, reduced body tone, piloerection and unkempt appearance were also observed - these signs persisted until death. Blood staining observed Animal 5569 on tray papers of all animals. had red/brown staining around nostrils. Day 5: animal 5569 had red staining around nostrils. Animal 5579 standing on the tip of its digits - not on the whole foot, ataxia, sneezing, red/brown staining on nostrils and fore paws also observed. Day 6: 5559 very subdued - resists handling. Red staining around nose, eyes, lower jaw and fore paws - killed in extremis. 5569 had red staining on eyes, nose and chin, yellow staining observed around perigenital No. 5579 area. Aggressive when handled. resists handling. No. 5589 observed having a convulsion during morning observations eyes dull with red staining, also red stainNos. 5599 and 5609 had ing on nose and jaw. red staining on eyes, nose and jaw. No. 5599 Day 7: 5569 found dead in cage. hyperkinetic. 8: 5579, 5589 and 5599 found dead in their cages.

SDay

4Day

'

9:

5609 found dead in

cage.

Body Weight Group mean body weights are presented numerically in Tables 2a Individual values and 2b and graphically in Figures l and 2. are given in Appendix 3.
Males

*
'

Significant dose related reductions in body weight gain were Groups 4 and 5 receivinag300n or 9000 rnci/Ka/ resDectobserved. GrouD ively showed an actual body weight loss by Day 4 of dosing. 4 animals' body weights were slightly above initial body weights by Day 7.

0549
V.N.xa*-

14

Females Animals fed HMX treated diet showed a significant loss in body weight during the first 4 days of dosing. Group 5 animals continued losing weight and eventually died, or were killed in extremis. Groups 2, 3 and 4 showed a marked deoression in body weight gain with only group 2 animals exceeding their ore dose body weight values. Food Consumption Group mean food consumption values are presented numerically in Table 3. During the first week of dosing HMX treated animals showed a marked reduction in food intake when compared to controls. Food consumption values were increased for all treated groups during week 2 of dosing although groups 4 and 5 male and 2, 3, and 5 female were still significantly less than controls.

Water Consumption No differences were detected between control and HMX treated animals. Achieved Dosage The actual amounts of HMX consumed by each dose group are presented in Table 4. The achieved dosage for group 59 during the second week of dosing has not been calculated due to the premature decedency of the test animals. Results obtained from analysis of the freshly prepared diets are presented in Appendix 4. Terminal Studies Organ Weights Group mean values for organ weights expressed in absolute terms and as a percentage of body weight are presented in Tables 5a and b. Values for group 59 are not presented due to premature decedency of all animals. Individual values for all animals are given in Appendix 5.

V%

Liver weights in all the HMX treated groups showed statistically significant reductions compared with control animals. These reductions were significant in absolute and relative terms for the males but only in absolute terms in the females.

5928

....

15

Kidney weights showed a significant dose related reduction in absolute terms, this being reflected in the dose related effects on body weight resulting in elevation of relative kidney weights compared with controls. Organ weight profiles for most of the premature decedents showed reductions in absolute terms. Relative weights were considered to be slightly elevated, due primarily to the observed body weight loss following dosing with HMX.

"Pathological Examination
Gross pathology and histopathological findings for individual animals are presented in Appendix 6. Histopathological findings are summarised in Table 6.

"N'

Gross pathological examination revealed smaller than normal spleens and enlarged adrenals in 4/6 group 5 females. Enlarged adrenals were also observed in the group 4 female premature decedent. It was also noted that the group 3 premature decedent had a smaller than normal liver and small kidneys. The brains from one male and 2 females in group 5 were described as friable. Blood was observed under the skull of 2 group 5 males. Dark red lungs were also described in 2 control females and 4 group 5 males, this high incidence possibly being the result of autolysis. Histopathological examination revealed centrilobular toxic degeneration in the livers of the group 5 male rats treated with a target dose level of 9000 mg HMX/kg/day (actual achieved dose 8504.3 mg HMX/kg/day). In the case of female rats hepatocyte hyperplasia and increased cytoplasmic eosinophilia was found in the liver together with lymphocyte depletion in the thymus and spleen. These lesions were found in female rats dosed at a target level of 9000 mg HMX/kg/day (actual achieved dose 3055 mg HMX/kg/day for a maximum of 9 days), and the premature decedents from groups 3 and 4 (target 1000 and 3000 mg HMX/ kg/day dose levels). Two male rats from group 5 also showed haemorrhaging into the cerebral ventricles and meninges. The 2 control females and 4 males from group 5 which were described as having dark red lungs showed areas of congestion on histological examination, autolytic changes being confirmed. On the basis of these findings it is recommended that the histopathological evaluation is extended to include

0617
S -V

16

.-'

examination of the liver, thymus, spleen and brain of all Such an extension will provide the HMX treated rats. information regarding whether or not there is a 'no effect' "level, together with clarification of the sex difference in lesions diagnosed in the animals so far examined.

"o.4

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"o.'...

6315

17
4

DISCUSSION Dose levels selected for the 90 day study were 0, 50, 1350 and 4000 mg HMX/kg/day for males and 0, 50, 115, and 1500 mg HMX/kg/day for females. 150, 270, 450, 620

Initial reductions in food consumption and body weight loss/reduced body weight gain were observed for all HMXtreated groups. Female rats were shown to be more sensitive than male rats, 1/6 females dying in each of the groups fed 1000 or 3000 mg/HMX/kg, and all the females dying at 9000 "mg/kg; 5/6 males treated at 9000 mg/kg died during the study. The emaciated appearance of all premature decendents was consistent with a reduced food intake possibly due to unpalatability of the test diet. Surviving animals tolerated the test diet and by the end of the dosing period, showed a slightly increased level of food consumption and a trend towards body weight gain. Unpalatability and extreme body weight effects meant that the achieved dosages were unavoidably erratic. Differences observed in liver weights for HMX-treated animals correlate with the histopathological diagnosis of toxic degeneration seen for the males and hepatocyte hyperplasia seen in the females. The organ weight data is difficult to interpret because of the observed body weight changes. The histopathological findings of toxic degeneration in the livers of male rats and hepatocyte hyperplasia with increased cytoplasmic eosinophilia in the females indicate the liver to The be a target organ for HMX treatment related effects. liver and kidney together with thymus and spleen in which lymphocyte depletion was observed, will need detailed scrutiny in the histopathological evaluation undertaken after the scheduled 3 month dietary study with HMX in rats.

1549

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FIGURE 1

HMX

: 14 Day Toxicity Study in Rats with Dietary Administration

(g)
-

Group Mean Body Weight

Males

200

(n=6)

"(n=6) ""
175 (n=6)

S150

-
Q- 125
*J.

(n=l)

3(n=5)

100
Group I SGroup 2 75

*---a Group 3 : 1000 mg/kg/day HMX "o-a--o Group 4 : 3000 mg/kg/day HMX
Group 5 : 9000 mg/kg/day HMX

: Control : 333 mg/kg/day HMX

Pre Trial 9 .October 16

Week 1 23 1980

Week 2 30

27

"FIGURE 2
4HMX

14 Day Toxicity Study in Rats with Dietary Administration Group Mean Body Weight (g) - Females

&

140

130

Group Group *--a Group 0-0 Group o--* Group

2 3 4 5

1 : Control
: : : :

333 mg/kg/day 1000 mgA/kg/day HMX 3000 mgA/g/day HMX 9000 mg/kg/day HrMX

(nMX

"120

110

(n=5)

SM
p.

100

(n=5)

.i.
-4

80

"".

70

"Pre Trial
9 16

Week 1 23 October 1980

Week 2 30

1)

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4

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. '.'

. . '' .-

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'.4%-.j

APPENDIX la HMX: ,-* 14 Day Toxicity Study in

Rats

With

Diete- y Administration Analysis of Diet

B.P. NUTRITION (U.K.) LTD. SPECIAL QUALITY CONTROL OF LABORATORY ANIMAL DIETS CERTIFICATE OF ANALYSIS PRODUCT: BATCH NO: RAT & MOUSE M.1 (IIOzruED) E2GWANDED FINE GROUND 919 PREMIX BATCH NO: 15TH AUGUST I1910 Contaminant % % % % % % % % % % % mgnmg mg/kg mgflmg mgg5g Total Allaloxins NONE DETECTEDugnrg I ug/kg each oI 1,82,GI..G2 Fluoripe Nitrate as NaNO3 Nitrite as NaNO2 Lead Arsenic Cadmium Mercury Selenium Found Analysis 7.6 12.0 ( 1.0 41.0 0.23 0.13 4 0.01 0.12 mgg mgrkg mglkg mOvg mg/kg mg/kg mgig mg&g Limit of Detection 10.0 1.0 1.0 1.0 0.2 0.2 mglkg mglkg mglkg mg/kg mgrg mg.Okg P1`10

DATE OF MANUFACTURE: Nutrient Moisture Crude Fat Crude Protein Crude Fibre ASh Calcium Phosphorus adium Chlorine Sotassiumn 'Magnesium Iron Copper Manganese Manc Z Found Analysis 7.1 3.50 14.9 2.2 4.8 0.69 0.53 0.22 0.34 1.10 0.1-3 231. 7 5.5 40

0.01 mgfkg 0.02 mg/kg

Total PC.B. Total D.D.T. Dieldrin LUndane Htevtchtor Malathion

NONE DTECTED 0.003 0.001 0.005 NONE DETECTED N1NE DETECTED 3

mg/kg mg/kg mg/kg mg/hg mgflg mgrig

0.001 mgl.g 0.0o1 mgfkg 0.001 mgfkg 0.001 mglkg 0.001 mgnkg 0.02 mg/kg

Vitamin A
-

7000
60

Iunkg
mglkg

'%0gOrganisms O tainE Vitamin C

Total Viable

1.13 x
Mesophilic Spores

0 2

per gi rm

long

mg/kg
3.4

1.5

X 10

per grrrm pNogym

100og Absent in

"'repeat

rpwTJEe

SalonelaD Is.
Presumptive
Coll XDNE DETECTED

17 SEP 1980
JL3E.

per gym per grin per grin

Absent in Absent in

E. CoIl Type I NONE DETECTED Fungat Unliit Antibiotic

Activity

NONE DETECTED

10 rim Absent in 10 arm

Signed .....
Do Dated . e ....... /B .... ...

.......

. I.... . .
... .......

P. Nutrition (UK.) Limited Stephield, Witham. Essex, CMI 3AB

C R POPPLESTONE M Sc. PhD. C Chem I.R I C

- o-.-%-%...

290

APPENDIX lb HMX: 14 Day Toxicity Study in Dietary Administration Analysis of Water Rats With

0 0

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30
APPENDIX lb (continued)

G/913882

Polymucle.er Aromatic Hydrocarbons Fluorenthene BeOIzo (ghi) Oerylene Benzo Wk fluoranthene 2, 3.0 - phenylene pyrene Benzo (b) fluoranthene Benzo (a) pyrene Total P.A.H. NDLT 40 og/litre OrganOchiorine Pesticides NULT 20 og/litre NULT 4 ng/litre NULT 4 og/litre NDLT 4 0g./itre RULT 4 ng./litre NULT 4 og/litre

gamaB.H.C.
Reptachior

Aidrin Dieldrin p.p. D.D.T. Polychlorinated biphenyls NDLT 400 n /litre (expressed

NDLT 10 ng/litr: KDLT 20 og/litre MDLT 20 nogitre NULT 40 og/itre NULT 20 og/litre

as AJOCHLOR 1248)

NULT

Not Detected, Less Then

4.............................................

- *~.

.~r

.V *-*.

31
p%.

"APPENDIX

HMX 14 Day Dietary Toxicity Study in Rats Summary Data for Pre-experimental Homogeneity and Stability Testing of HMX Treated Diet

Nominal Concentration Time Analysed Mean 1,250 ppm *Coeff Var%

(ppm) 25,000 ppm Mean *Coeff Var%

10,000 ppm
Mean *Coeff Var%

Immediately after Preparation After 14 d at 40 0 C After 21 d at Ambient

1177 1234 1291 (%)

5.3 3.8 3.9

10198 10130 10311

5.4 3.6 3.0

24817 25004 25598

2.5 4.4 3.5

*Coefficient of Variation

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APPENDIX 5a HMX 14 Day Toxicity Study in Rats with Dietary Administration Absolute Organ Weights (q) Individual Values - Males Surviving 14 Days Dosing

Kidneys Dose Level (mg/kg/day) Animal No. L 0 501 502 503 504 505 506 Mean 0.83 0.77 0.74 0.80 0.79 0.78 0.79
0.03

Liver R 0.80 0.75 0.76 0.79 0.79 0.75 0.77

0.02

9.92 9.19 8.61 10.78 9.18 8.84 9.42

0.80

"+ S.D.
333 507 508 509 510 511 512 Mean

0.74 0.81 0.71 0.87 0.77 0.71 0.77 0.06 0.68 0.72 0.74 0.68 0.68 0.68 0.70 0.03 0.69 0.64 0.70 0.61 0.76 0.59 0.67 0.06 0.60

0.72 0.82 0.65 0.85 0.73 0.72 0.75 0.07 0.65 0.70 0.74 0.69 0.67 0.71 0.69 0.03 0.65 0.59 0.67 0.58 0.73 0.61 0.64 0.06 0.57

8.19 9.07 7.92 9.42 8.19 7.92 8.45 0.64 7.13 7.88 8.44 6.97 7.08 7.70 7.53 0.58 6.15 5.77 6.86 5.94 7.62 5.70 6.34 0.75 5.54

"
1000

S.D. 513 514 515 516 517 518 Mean

"+ S.D.
3000 519 520 521 522 523 524 Mean
+ S.D.

9000

530

7
14.

4%'

"L5
APPENDIX 5a (continued)

"Individual Values

- Females Surviving 14 Days Dosing

Kidneys ,* Dose Level (mg/kg/day) Animal No.

L R

Liver

531 532 533 534

0.61 0.54 0.58 0.56

0.58

0.60 0.56 0.54 0.58

0.56

6.10 5.11 5.24 5.94

5.44

'

535

536 * Mean
+ S.D.

0.59 0.58 0.02 0.52 0.50 0.53 0.49 0.53 0.50 0 51 0.02 0.43 0.54 0.50 0.54 0.51 0.50 0.05 0.45 0.52 0.49
0.45

0.62 0.58 0.03 0.49 0.49 0.52 0.47 0.52 0.49 0.50 0.02 0.39 0.54 0.47 0.55 0.49 0.49 0.06 0.46 0.50 0.48
0.45

5.38 5.54 0.40 4.80 4.19 4.13 4.16 4.21 4.40 4.32 0.26 4.22 4.28 4.22 4.59 4.27 4.32 0.16 3.84 4.49 4.27
3.73

"333

537 538

"539
540 541 542
=

Mean + S.D.

"1000

543 544 545 546 547

"Mean
+ S.D. 3000 549 551 552
.4

553

554 Mean + S.D.

0.49 0.48 0.03

0.51 0.48 0.03

4.28 4.12 0.32

%..

",

APPENDIX 5b {MS 14 Day Toxicity Study in Rats with Dietary Administration Organ Weights as a % of Body Weight Individual Values - Males Surviving 14 Days Dosing

. ..

Dose Level (mg/kg/day)

Animal Number

Body Weight (g) 216 202 200 223 200 194

Kidneys Liver L 0.384 0.381 0.370 0.359 0.395 0.402 0.332 0.016 R 0.370 0.371 0.380 0.354 0.395 0.387 0.376 0.014 0.375 0.390 0.363 0.395 0.374 0.387 0.381
0.012

"0

501 502 503 504 505 506

4.593 4.550 4.305 4.834 4.590 4.557 4.572 0.168 4.266 4.319 4.425 4.381 4.200 4.258 4.308
0.084

Mean
+ S.D.

"333

507 508 509 510 511 512 Mean

+ S.D.

192 210 179 215 195 186

0.385 0.386 0.397 0.405 0.395 0.382 0.392

0.009

1000 ,

513 514 515 516 517 518 Mean + S.D.

160* 180 198 175 170 175

0.425 0.400 0.374 0.389 0.400 0.389 0.396 0.017

0.406 0.389 0.374 0.394 0.394 0.406 0.394 0.012 0.401 0.401 0.396 0.382 0.401 0.430 0.402 0.016 0.445

4.456 4.378 4.263 3.983 4.165 4.400 4.274 0.177 3.796 3.925 4.059 3.908 4.187 4.014 3.986 0.134 4.328

3000

519 520 521 522 523 524 Mean

+ S.D.

162 147 169 152 182 142

0.426 0.435 0.414 0.401 0.418 0.415 0.418 0.012

9000

530

128

0.469

"" Body

weight at day 14 of dosing - not weighed at necropsy.

-v
"'
" . . . . . . . . , . -. - . ' .. . . , ' . "."."-' . .... , "''' ''

47

APPENDIX 5b (continued) Individual Values - Females Surviving 14 Days Dosing

Dose Level (mg/kq/day) 0

Animal Number

Body Weight (g) 143 131 130 137 142 141

Kidneys Liver L 0.427 0.412 0.446 0.409 0.408 0.418 0.420 0.015 R 0.420 0.427 0.415 0.423 0.394 0.440 0.420 0.015 0.438 0.458 0.468 0.420 0.456 0.454 0.449 0.017 4.266 3.901 4.031 4.336 3.831 3.816 4.030 0.224 4.286 3.916 3.721 3.714 3.693 4.074 3.901 0.240

531 532 533 534 535 536 Mean

"+ S.D. "333

537

"538 "539 "540

541 542

112 107 ill 112 114 108

0.464 0.467 0.477 0.438 0.465 0.463 0.462 0.013

"Mean "" S.D.

1000 543 544 545 546 547 Mean + S.D. 3000 op 549 551 552 553
.

104 103 110 112 104

0.413 0.524 0.435 9.482 0.490 0.469 0.045

0.375 0.524 0.427 0.491 0.471 0.458 0.058 0.474 0.476 0.429 0.495
0.500

4.058 4.155 3.836 4.098 4.106 4.051 0.125 3.959 4.276 3.813 4.099
4.196

97 105 112 91
102

0.464 0.495 0.438 0.495

0.480

554

Mean
S.D.

0.474
10.024

0.475
10.028

4.069
0.185

%
-.

~~~~~~
..-.

%...
...

%.,.-

%~\.j 4N'. %a
V.. L.a.

APPENDIX 5c

HMX

14 Day Toxicity Study in with Dietary Administration Premature Decedents

Rats

S.Absolute

Organ Weights

(g)

- Individual Values

Dose Level (mg/kq/day) 9000

Animal Number/ Sex 525 J 526

527*

Kidneys Liver L 0.58 0.55

NDA

R 0.54 0.60
NDA

6.15 7.04
NDA

528 529

0.65 0.59 0.46 0.44 0.41 0.47 0.42 0.56 0.52 0.42

0.63 0.64 0.44 0.44 0.42 0.49 0.44 0.53 0.50 0.44

5.79 6.33 2.46 4.63 2.42 4.99 2.73 3.15 3.53 2.41

"1000
3000 9000

548 $ 550 i 555 i 556 557 558 559 560

5.
.9. 9.l

"*No

organ weights recorded

5W4

-9.

"49

APPENDIX 5d HMX : 14 Day Toxicity Study in Rats with Dietary Administration Premature Decedents Organ Weights as a % of Body Weight Individual Values

Dose Level (mg/kq/day)

is

Animal Number

Body Weight (g) 113 140 113 121 130 79 85 68 82 63 72 73 66

Kidneys Liver L 0.513 0.393 NDA 0.537 0.454 0.582 0.518 0.603 0.573 0.667 0.778 0.712 0.636 R 0.478 0.429 NDA 0.521 0.492 0.557 0.518 0.618 0.598 0.698 0.736 0.685 0.667 5.442 5.029 NDA 4.785 4.869 3.114 5.447 3.559 6.085 4.333 4.375 4.836 3.652

9000

525 d 526 527* 528 529

"1000
""
3000 9000

548 550 $ 555 4 556 557 558 559 560

* No organ weights recorded

P,

I'.

--

q- 1

I-

. WrT T

W U--.-W-T

,-r

50

APPENDIX 6

"HMX:

14 Day Toxicity Study in Rats with Dietary Administration in Individual Animals

Gross Pathology and Histopathological Findings

Abbreviations used: TK KIE FD NAD HE SS Terminal kill Killed in Found dead No abnormality detected Haematoxylin and Eosin Special stain extremis

76

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77

PERSONNEL INVOLVED IN PROJECT NO. Principal Investigator: Project Leader: Technicians: A.B. R.J. Wilson,

415669SR B.V.Sc., B.Sc., M.R.C.V.S. M.I.Biol.

Greenough,

P. McDonald, H.N.C., P.C. Robinson, B.Sc. A. Everett, A.I.A.T. A.T. Soden B.Sc.,

L.I.Biol.

Diet Formulation: Diet Analysis: Pathologists:

M. Henderson, B. Rushton,

Ph.D.

M. Jones, Autopsy Room Supervisor: Histology Supervisor: Pathology/Histology Assistants: E.P. I. C. G. V. A. D. R. D. F. Hall,

Ph.D., B.V.M.S., M.R.C.V.S., R.C.V.S. B.V.M.S., M.R.C.V.S. F.I.M.L.T. A.I.M.L.S.

Nicol,

Petrie, B.Sc. Ash, B.Sc. Derbyshire Kirkwood Frazer Johnston, H.N.C. McBride MacLean Waddell, B.Sc., Ph.D. Baxendine, B.Sc. McLachlan, B.Sc.

Quality Assurance:

A.W. E.M. N.C.

4.

"I
A

1889

78

DISTRIBUTION LIST 5 copies to: Commander U.S. Army Medical Research and Development Command SGRD-RMS Attention: Fort Detrick, Frederick, Maryland 21701-5012

"copies to
Inveresk Research International Limited Musselburgh, EH21 7UB, Scotland

-p'

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