Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine

Endpoint summary

Description of key information

NOAEL from a 90-day repeat dose study in rats was 51 mg/kg day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 8 December 1980 to 19 March 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study run to a reliable method but not to GLP and no guideline followed. However it has been inspected and audited by QA

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Five groups of 20 male and 20 female rats of the Fischer 344 strain were dosed at concentrations of the test substance in the diet calculated to give dose levels of 50, 150, 450, 1350 or 4000 mg test substance/kg/day for male rats and 50, 115, 270, 620 or 1500 mg test substance/kg/day for female rats. One group of 20 male and 20 female rats received untreated diet and acted as contemporary controls. Study duration was 13 weeks, at the end of this period animals were killed and subjected to full necropsy.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.S.A. via Charles River (U.K.) Limited, Manston, Kent, England
- Weight at study initiation: 40-60 g
- Housing: Rats were housed in a barrier maintained animal room. Rats were housed one animal per cage in suspended polypropylene cages (overall dimensions ca 480 x 150 x 120 mm) with stainless steel wire grid tops and bottoms. Each cage had a polypropylene water bottle (total capacity 300 mL) with rubber washer and melamine cap.
- Diet (e.g. ad libitum): rodent diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 2°C
- Humidity (%): ca. 50%
- Air changes (per hr): 14 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From: 8 December 1980 To: 19 March 1981

No additional data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diets were prepared once each week.
- Mixing appropriate amounts with (Type of food): The concentration of test compound was adjusted each week to give as constant a mg/kg/day level as possible by prediction of mid-week body weight and weekly food consumption for the week in question.
Diets were prepared by direct admixture of the required amount of the test substance to diet and blending for 20 min in a Winkworth change drum tumble mixer.

No additional data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A 100 g sample of diet from each group/sex was taken and retained immediately after diet preparation at the beginning of each study week. In addition, 4 samples of 100 g were taken from each group/sex at the beginning of Weeks 1, 2, 3, 4, 7, 10 and 13. The latter samples were analysed for test substance levels.

A suitable weight of diet (2.5 g or 5 g) was weighed accurately into clean glass 8 oz jars. To this was added I ml of internal standard solution (dinitrobenzene in acetonitrile at a suitable concentration) and 50 ml of acetonitrile as extracting solvent. The jars were shaken mechanically for 1 h then left to settle, preferably overnight. A suitable aliquot was transferred to a sample vial and analysed by HPLC.

Standard solutions of test substance were prepared by adding a known amount of test substance (equivalent to that of the group being analysed) to a sample of untreated diet. These were treated with internal standard solution and extracting solvent as described for the formulated diet samples.

Three quality control samples were included with each batch of test samples and standards. For this purpose a solution of the test substance in acetonitrile was prepared by an independent analyst and these solutions used by the analyst to spike blank diet samples in exactly the same way as the standards.

HPLC Conditions
Instrument: Hewlett Packard 1084B with variable wavelength detector and automatic sampler.
Column: 100 x 5 mm stainless steel packed with ODS Hypersil (5 μ).
Solvent: Acetonitrile : Water (40:60 v/v).
Flow: 1.5 mL/min

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

at least 91 consecutive days

Remarks:

Doses / Concentrations:
Male: 50, 150, 450, 1350 or 4000 mg test substance/kg/day; Female: 50, 115, 270, 620 or 1500 mg test substance/kg/day
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
Male: 51.0, 153.5, 461.0, 1394.6 or 4101.3 mg test substance/kg/day; Female: 50.3, 115.6, 273.3, 627.7 or 1511.9 mg test substance/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

20 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

None stated

Positive control:

None stated

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded at weekly intervals commencing one week before the start of treatment up until the end of treatment. In addition, animals were also weighed on Day 4 of the first 4 weeks.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was assessed visually for any intergroup differences.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before dosing commenced and during Week 13 of dosing
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during Weeks 5 and 12 of treatment
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: No data
- How many animals: 10 males and 10 females from top and control groups

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during Weeks 5 and 12 of treatment
- Animals fasted: No data
- How many animals: 10 males and 10 females from top and control groups

URINALYSIS: Yes
- Time schedule for collection of urine: Collections of individual urine samples were made over a 4 h period of food and water deprivation during Weeks 5 and 12 of dosing.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: Pharmacokinetic Sampling: Blood samples were obtained at post mortem by the removal of at least 3 mL whole blood via the caudal vena cava into heparinised tubes. Samples were taken from 5 male and 5 female animals selected at random from each group. These samples were centrifuged and the plasma deep frozen and stored at IRI.

No additional data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, All animals which died or were sacrificed were necropsied. The gross dissection and necropsy was performed under the supervision of a pathologist. The necropsy is defined as external examination including body orifices, weighing of the following tissues: Brain, Heart, Kidney, Ovaries, Liver, Lungs, Testes, Adrenals, Spleen
HISTOPATHOLOGY: Yes, examination and fixation of the following tissues: Brain, Spinal cord, Peripheral nerve (sciatic), Eyes, Pituitary, Thyroid, Parathyroid, Salivary glands (submaxillary), Heart, Lungs, Spleen, Liver, Pancreas, Adrenals, Lymph nodes (mesenteric cervical submaxillary bronchial), Kidneys , Bladder, Testes (plus epididymides), Prostate, Ovaries (minus fallopian tubes), Uterus, Fallopian tubes, Stomach, Small intestine (duodenum jejunum ileum), Large intestine (caecum colon rectum), Skeletal mucle (thigh), Skin (abdominal), Mammary gland, Any gross lesions e.g. tissue masses, suspected tumours (including surrounding normal tissue), Sternum, Adipose tissue (perirenal), Nasal tubinate, Trachea, Thymus (where possible)

Other examinations:

None stated

Statistics:

Statistical evaluation of quantitative data was performed where it seem appropriate. Males were treated independently of females. The level of probability chosen as significant was P<0.05, but in any case the actual level is reported. For evaluation of mean differences a "two tail" distribution was used.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical Signs and Mortality:
None was observed which could be attributed to dosing with the test substance.
Only 3 premature deaths occurred during the study, all from different treatment groups.

Body Weight:
Body weight gains were reduced in male and female animals receiving test substance in dose related fashion.

Food Consumption:
Food consumption trends were variable throughout the study but treated groups consumed less food than untreated groups during dosing.

Water Consumption:
Visual assessment of water consumption revealed no intergroup differences.

Ophthalmic Examination:
No abnormalities were detected which could be attributed to dosing with the test substance.

Haematology:
(i) Significant reductions in haemoglobin (Hb) levels and packed cell volume (PCV) (P<0.001) in males and females receiving 4000 and 1500 mg test substance/kg/day respectively were seen. Other parameters fell within normal ranges except for isolated cases.
(ii) Significant reductions were seen in Hb, PCV and red blood cell (RBC) levels (P<0.01) in females receiving the high dose level of the test substance, with corresponding reductions, though not significant, in the same parameters in males. Slight rises in methaemoglobin in both males and females receiving high dose of the test substance were seen, significant (P<0.05) in males only. Female white blood cell (WBC) count was significantly raised (P<0.05) probably reflecting significant increases (P<0.05) in neutrophils.

Clinical Chemistry:
(i) There were increased alkaline phosphatase levels in males and a marginal increase in females (P<0.01 in males only) receiving 4000 or 1500 mg test substance/kg/day. There was also a reduced ALT level in males receiving 4000 mg test substance/kg/day (P<0.01). A significant increase was also seen in BUN levels of females receiving 1500 mg test substance/kg/day. Albumin levels were also increased in males receiving 4000 mg test substance/kg/day (P<0.001). Except for isolated cases other parameters were considered to fall within normal reference ranges.
(ii) Significant increases were seen in AP levels in males (P<0.001) and females (P<0.05) receiving the top dose level of the test substance and also in albumin levels in females (P<0.001). The male control value for AP was rather lower than normally anticipated for animals of this age and thus the results should be interpreted with caution. Total protein levels of females receiving 1500 mg test substance/kg/day also showed a slight increase (P<0.05) in line with the raised albumin levels. BUN levels were also raised significantly (P<0.001) in females receiving 1500 mg test substance/kg/day. Other parameters were considered to be within normal ranges except for isolated cases.

Urinalysis:
(i) Females rats receiving 1500 mg test substance/kg/day showed a reduced pH and specific gravity (SG) with a corresponding increase in urinary volume. Males did not show this trend. Other parameters were considered to fall within normal ranges.
(ii) Female rats receiving 1500 mg test substance/kg/day showed a reduced pH and SG with a corresponding increase in volume. In addition to this effect fern-like crystals were noted in urine of males and females receiving 4000 or 1500 mg test substance/kg/day but not in controls.

Organ weights:
The following changes were seen: Significantly reduced absolute and relative adrenal weights in all treated male groups with an increase in female groups. Relative brain weight increased in males and females receiving 4000 or 1500 mg Htest substance/kg/day. Absolute brain weights increased in top dose females. Absolute heart weights increased in male and female top dose rats.
Female treated rats showed a dose related increase in relative kidney weights.
Males showed reduction in absolute and relative spleen weights. Relative liver and lung weights increased in treated females. Females showed reductions in absolute spleen and ovary weights.

Gross and Histopathological:
No macroscopic lesions were observed which could be attributed to dosing with the test substance. There were a number of lesions found on histopathological examination of the 2 highest dose and control groups but these were consistent with the age and strain of rats.
There were, however 2 lesions found which exhibited a dose related trend. These were found in liver and kidneys which were recognised as target organs and histopathologically examined in all other animals (see below).
Toxic changes in the liver were characterised by enlarged cells, mainly in centrilobular areas, with large, pale nuclei and dark, granular, eosinophilic cytoplasm. In some areas there was dilation of the sinusoids and small foci of necrosis.
This liver effect was most marked in males, all males receiving 450 or 4000 and 90% of males receiving 1350 mg test substance/kg/day exhibiting the condition.
Only one female receiving 270 mg test substance/kg/day showed the effect.
Tubular changes in kidneys were seen mostly in females receiving the higher dose levels of the test substance and were characterised by focal atrophy and dilation of the tubules. Males also exhibited this condition but to a considerably lesser extent and one female control rat also showed this change.

Dose descriptor:

NOAEL

Effect level:

ca. 51 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Significant toxic liver changes in males receiving 150 or more mg HMX/kg/day

Dose descriptor:

NOAEL

Effect level:

ca. 115.6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Tubular kidney changes in females receiving 270 or more mg HMX/kg/day.

Critical effects observed:

not specified

From Henderson, 1985:

No significant increase in plasma levels of the test substance was observed with increasing dose.

The data may well indicate that most of the administered the test material was not systemically absorbed but was excreted unchanged.

The presence of the test substance in the plasma of the control group animals was also indicated. The levels were low but significant. They may approximate to those anticipated following a single dose of 13 -15 mg/kg. The reason why the plasma from the control group animals should have contained the test substance are unknown.

Henderson 1985. HMX: Analysis in Plasma Obtained After 90 Day Toxicity Studies with Rats and Mice

Insufficient plasma was available for successful analysis of the test substance in mouse plasma at the termination of the 90 day mouse toxicity study. Inveresk Research International Limited Musselburgh, EH2l 7UB, Scotland. IRI Projects 415669 AD, 415669 AR, 415669 AM

Conclusions:

Dosing of test substance to male and female F344 rats for 90 days via the diet results in a slight reduction in red cell parameters and possible methaemoglobinaemia. The most significant changes were toxic liver damage in male rats at doses of 150 mg test substance/kg/day and above and renal tubular damage in female rats at 270 mg test substance/kg/day and above. Other effects were of doubtful significance or most likely to be secondary to the hepatic and renal damage.
The NOAEL of the test substance to female Fischer 344 rats is 115.6 mg/kg bw/day (actual dose). The NOAEL of the test substance to male Fischer 344 rats is 51.0 mg/kg bw/day (actual dosel).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

51 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Another subchronic study performed in mice is available which supports the rat study. This study is also performed to a method equivalent to internationally accepted guidelines. It is not a GLP study however it is published and has been inspected and audited by QA

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A key subchronic dietary study in rats was undertaken (Inveresk Research Ltd on behalf of the U.S Army Medical Research and Development Command, 1985). The study was performed using a method similar to international guidelines and the study was QA audited. Five groups of 20 male and female Fischer 344 rats were dosed at concentrations of 60, 150, 450, 1350 & 4000 mg HMX/kg/day in diet (males) and 50, 115, 270, 620 & 1500 mg HMX/kg/day in diet (females). The study duration was 13 weeks. There were no clinical signs attributable to dosing with the test substance. Body weight gains were reduced in male and female animals in dose related fashion. Two dose related lesions were seen: Toxic liver change, characterised by enlarged centrilobular cells with pale nuclei and dark cytoplasm, in significant numbers of males receiving 450, 1350 or 4000 HMX/kg/day. Tubular kidney change characterised by focal atrophy and dilatation in significant numbers of females receiving 270, 650 or 1500 mg HMX/kg/day. Significant toxic liver changes were observed in males receiving 150 or more mg HMX/kg/day and tubular kidney chagnes in females receiving 270 or more mg HMX/kg/day. HMX administration tends to reduce red blood cell parameters and possibly cause methaemoglobinaemia. The NOAEL of the test substance to female Fischer 344 rats is 115.6 mg/kg bw/day achieved dose in diet.

The NOAEL of the test substance to male Fischer 344 rats is 51.0 mg/kg bw/day achieved dose in diet.

A supporting study in mice was undertaken (Inveresk Research Ltd on behalf of the U.S. Army Medical Research and Development Command, 1985). Mice were fed diets containing 0, 5, 12, 30, 75 or 200 mg HMX/kg/day (males) or 0, 10, 30 , 90, 250 or 750 mg HMX/kg/day (females). There were mortalities attributable to HMX at 200 mg HMX/kg/day amongst males and at 250 and 750 mg/kg/day amongst females. Neither gross autopsy nor histopathology revealed any findings likely to be associated with the administration of HMX. The NOAEL of the test substance to female B6C3F1 mice is 93.1 mg/kg/day achieved dose in diet. The NOAEL of the test substance to male B6C3F1 mice is 75.1 mg/kg/day achieved dose in diet.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Study performed to a method equivalent to internationally accepted guidelines. It is not a GLP study however it is published and has been inspected and audited by QA

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Based on the CLP guidance values to assist in category 2 classification and the data available for HMX which includes adaptative responses and small changes in clinical biochemistry, haematology or urinalysis parameters of minimal toxicological importance occuring at high doses, HMX is therefore not classifed in accordance with Regulation (EC) No1272/2008.

CNS: in the two subchronic studies, no significant CNS effects were seen in rats or in mice administered up to 1500 and 200 mg/kg/day in the diet, respectively (Everett et al, 1985; Everett and Maddock, 1985). Although mortality in mice was marked at 200mg/kg/day in the males and 250mg/kg/day in the females, little evidence of toxic changes were found by histopathology.

Liver: the centrilobular cells of male rats exposed to 153.5 mg/kg/day HMX for 13 weeks were enlarged with pale nuclei and dark cytoplasm (Everett et al., 1985). Higher doses of 1511.9 mg/kg/day (females) and 4101.3 mg/kg/day (males) produced significant elevations in serum alkaline phosphatase levels in rats.

Kidneys: focal tubular atrophy, dilatation, and increased kidney weights were observed in female rats exposed to 273.3 mg HMX/kg/day and above, while blood urea nitrogen was elevated in female rats exposed to 1511.9 mg/kg/day HMX in the diet for 13 weeks (Everett et al., 1985). In the same study, decreased pH, increased volume, and crystal formation was observed in the urine of male and female rats exposed to 4101.3 and 1511.9 mg HMX/kg/day, respectively. However no gross or histopathological effects were observed in male rats exposed to 4101.3 mg HMX/kg/day for 13 weeks. Haematological system: A statistically significant decrease in hemoglobin and packed cell volume, and a non-significant increase in methemoglobin were noted in female rats exposed to 1511.9 mg HMX/kg/day in the diet for 13 weeks (Everett et al., 1985). A significant elevation in methemoglobin concentration was observed in male rats exposed to 4101.3 mg/kg/day (Everett et al., 1985). Non-significant haematological effects were noted in female mice exposed to 784.5 mg HMX/kg/day for 13 weeks.