Perhydro-1,3,5-trinitro-1,3,5-triazine

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

RDX

IUPAC Name:

RDX

Test material form:

not specified

Details on test material:

- Source: IITRI Kingsbury Ordnance Plant (KOP) Explosive Facility, La Porte, IN.
- Batch n°: HOL 435-37
- Storrage conditions: ambiant room temperature, relative humidity, and in the dark.
- Analytical purity: Analyzed three times during the study: 91.0 +/-2.9% (May 1981); 89.2 +/- 8% (May 1982); 98.7 +/-2% (April 1983)
- Impurities: 3-10% of HMX (other impurities not determined)
- Particule size: < 22 µm (51.7%) or between 22-44 µm (43.2%)

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Madison, WI
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 111 +/-11 g (males) 93 +/- 8 g (females)
- Fasting period before study: no data
- Housing: three per polycarbonate cage with Absorb-dri bedding. Animals were transferred to clean cages twice weekly. Some of the male rats at the 40 mg/kg/day dose level were individually housed at the onset of test week 30. By test week 40 all males at this dose level were housed separately.
- Diet (e.g. ad libitum): available from powdered diet feeders
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 30-70% relative humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark cycle

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

2 years

Frequency of treatment:

Weekly

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.3 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1.5 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
8.0 mg/kg/day
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
40.0 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

75/sex/group dose

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Examinations

Statistics:

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 for repeated measurements model.
Variables that were measured a single time, e.g. organ weights, were analyzed using both unvariate and multivariate analysis of variance procedures.
In the presence of significant ANOVA results, a series of post-hoc analyses were conducted.
Individual between group comparisons at each time-point were performed using Tukey's b test for multiple comparisons.
Frequency data, such as incidence of mortality, gross necropsy observations ans histopathologic lesions were compared using log linear analysis techniques where appropriates.
Time to death data were analyzed using Kaplan-Meler and Cox regression analyses.
Ophtalmic lesions were analyzed by Chi-square test.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
RDX at 40 mg/kg/day was lethal to most of the males and many of the females during the two year treatment period. Mean survival time for these high dose males was 14.6 months compared with 22.3 months for control males. For these high dose females, a 20.6 months mean survival time was seen versus 22 months for control females. Both of these reductions were statistically significant.
Prior to their death, tremors and/or convulsions, first observed around Test week 25, were often seen for 40 mg/kg/day animals. In addition, many of the males and some of the females were on occasion hyperreactive to approach. This was first apparent by Test week 9, and this increased sensitivity to stimull apparently resulted in fighting among many of the co-habited males. As a result, high dose males which were fighting were singly housed during Test week 30. Subsequently in Test week 40, the remaining high dose males were placed in separate cages.
Discolored and/or opaque eyes were seen in an increased frequency for high dose females. This was first observed approximately midway through the study and continued until termination.

BODY WEIGHT AND WEIGHT GAIN
Dose-related reductions in body weight gain were seen throughout the study for males receiving 8 or 40 mg/kg/day. At this latter dose, a 20-30% reduction was apparent for most of the study. For males administered 8 mg/kg/day, an approximate 5% reduction was observed. Body weights of female rats were less affected than males. Although females receiving 40 mg/kg/day often showed 10-15% reductions in body weight gain, there were several points in time when their body weights were actually higher than control females. Occasional slight but statistically significant reductions in body weight gain, about 5%, were also seen for 8 mg/kg/day treated females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food intake was slightly but significantly reduced for male rats receiving 40 mg/kg/day. For females at this dose, slight decreases but more often slight increases were observed. Occasionnal increases or decreases were also seen for other dose levels, however they were sporadic and were not considered to be treatment-related.

OPHTHALMOSCOPIC EXAMINATION
Statistically significant increases in the incidence of cataracts were seen during Test weeks 78 and 104 for females but not males administered 40 mg/kg/day. The incidence of cataracts at this latter evaluation period was significantly higher thant that observed for Test week 78. All other ophtamologic abnormalities seen occurred in random fashion, and were not considered to be treatment-related.

HAEMATOLOGY
Reductions in hematocrit, hemoglobin, and RBC's were seen throughout the study for rats administered 40 mg/kg/day. Males were in general more affected thant females. The observed anemic state was slight and physiologic compensatory responses were not in evidence.
Thrombocytosis was seen throughout the study for rats of both sexes administered 40 mg/kg/day. In addition, males receiving 8 mg/kg/day demonstrated elevated platelet counts during Test weeks 13 and 26. Total white blood cell counts were sporadically increased for the 8 and 40 mg/kg/day-treated rats, however, a dose-related pattern was not evident. No other hematology parameters were apparently altered by RDX treatment.

CLINICAL CHEMISTRY
Hypoglycemia was observed throughout the study for rats administered 40 mg/kg/day. Whereas males were more sensitive than females during the first year of the study, the opposite was, in general, seen during the second year. Hypochloesterolemia was seen for high dose (40 mg/kg/day) animals at all time points tested except for Test week 104. Males were more affected, with females only showing significant reductions in serum cholesterol levels at Test week 52. Reductions in serum cholesterol levels occurred for males and females administered 40 mg/kg/day. This was seen, in general, at all time points tested with both sexes being similarly affected.
Serum GPT (ALT) levels were significantly lowered during Test weeks 26 and 52 for male rats receiving either 8 or 40 mg/kg/day. Females receiving the latter dose demonstrated this effect only at Test week 26. For females administered 8 and/or 40 mg/kg/day, reductions in total serum protein were observed. Slight increases in alkaline phosphatase were seen for females receiving 40 mg/kg/day. No other clinical chemistry parameters appeared to be altered by RDX treatment.

ORGAN WEIGHTS
Relative liver and kidney weights were elevated at Test weeks 27, 52, and 104 for male and female rats administered 40 mg/kg/day. During Test week 52, females at the 8 mg/kg/day dose level also showed these relative organ weight increases. Slight increases for relative adrenal weights were seen during Test week 27 for 40 mg/kg/day males and at Test weeks 52 and 104 for 40 mg/kg/day females. Statistically significant reductions in testes weights were osberved at Test week 52 for 40 mg/kg/day males. Due to testicular masses for nearly all of the males (including the controls) at the terminal sacrifice (Test weeks 105 to 106), testes weights were not evaluated at that time.

GROSS PATHOLOGY
Histopathologic lesions observed for rats administered RDX for up to 6 months were confined to the 40 mg/kg/day dose level. Splenic extramedullary hematopoiesis and spermatic granuloma of the prostate were present in these animals although gross morphologic changes were not in evidence.
By twelve months of RDX treatment, pathologic lesions of the urinary bladder, kidneyx, testes and spleen were considered to be treatment-related for 40 mg/kg/day males. Onlys splenic lesions were seen for females receiving this dose. Urinary system lesions consisted of distended urinary bladder containing dark-red fluid, red-brown fluid in the abnominal cavity, and dark-brown kidneys with renal pelvis dilatation. Corresponding histologic observations were luminal distention and cystitis of the urinary bladder, and renal medullary papillary necrosis. Small testes with germinal cell degeneration, enlarged red-brown seminal vesicles, and enlarged prostate distented with red-brown fluid were also seen. Splenic lesions observed for both males and females consisted of enlarged dark-red spleens with histologic evidence of sinusoidal congestion.
Urogenital lesions observed for male rats during the 12-24 month treatment period were similar to those described above. Statistically significant treatment-related lesions were confined to the 40 mg/kg/day dose level, except for suppurative inflammation of the prostate observed at 1.5 and 8 mg/kg/day. Splenic lesions at the 24 month scheduled sacrifice were seen for both males and females. Males receiving 1.5, 8 or 40 mg/kg/day demonstrated increased levels of a hemosiderin-like pigment whereas extramedullary hematopolesis was in evidence for 40 mg/kg/day females. Lenticular cataracts were also seen for females at this dose level.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: convulsions/seizures

2

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: convulsions/seizures

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The administration of RDX to male and female F344 rats resulted in a reduction in the survival rate for both sexes given 40 mg/kg/day.

Those animals that died often demonstrated convulsions prior to death. Surviving animals at this dose were hyperreactive to approach and appeared to fight with their cagemates to a greater extent than that seen for animals at lower doses. Histologic evaluation failed to detect treatment-related lesions of the central nervous system.

Anemia consisting of reduced hematocrit, hemoglobin and RBC's was seen for males and females receiving 40 mg/kg/day. The effect was mild and none of the usual physiologic compensatory responses were in evidence. The anemia appeared to be peripheral in origin as bone marrow appeared within normal limits and secondary splenic lesions including extramedullary hematopoiesis, sinusoidal congestion, and increased quantities of a hemosiderin-like pigment were seen. Although gross necropsy observations suggested enlarged spleens, organ weight analysis failed to substantiate this.

Liver injury, primarily at 40 mg/kg/day, was evidence by several observations. Hepatomegaly was seen at 40 mg/kg/day and to a much lesser extent for females at 8 mg/kg/day although histologic changes were not apparent. Hepatotoxicity was also suggested by hypocholesterolemia, hypotriglyceridemia, reduced serum albumin/total protein levels, and possibly by increased alkaline phosphatase activity.

RDX-induced renal damage occurred primarily at the 40 mg/kg/day dose level. Kidney weights were elevated at this dose and possibly for 8 mg/kg/day females. Lesions of this organ included dark brown kidneys with medullary papillary necrosis for males receiving 40 mg/kg/day for longer than 6 months. Additional toxic effects on the urogenital system, primarily seen at 40 mg/kg/day, included urinary bladder distention with luminal distention and cystitis, testicular atrophy with germinal cell degeneration and enlarged seminal vesicles. In addition, enlarged prostate accompanied by spermatic granuloma and suppurative inflammation occurred for male rats administered 1.5 mg/kg/day or greater following 24 months of treatment.

Additional toxic effects seen primarily at 40 mg/kg/day included cataracts (females only), hypoglycemia, thrombocytosis and enlarged adrenals although microscopic changes were not seen. RDX was not found to be carcinogenic under the conditions of the present study.

Applicant's summary and conclusion

Conclusions:

According to the results obtained in this study, the NOAEL identified was 8 mg/kg/day and the LOAEL was 40 mg/kg/day for tremors and convulsions in rats exposed to RDX in the diet for 2 years.