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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
publication
Title:
Convulsant properties of Cyclotrimethylenetrinitramine (RDX): spontaneous, audiogenic and amygdaloid kindled seizure activity
Author:
BURDETTE LJ, COOK LL and DYER RS
Year:
1988
Bibliographic source:
Toxicology and applied pharmacology, 92, pp 436-444

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male Long Evans rats treated with 0-60 mg/kg RDX po were monitored for spontaneous seizures during an 8 hr interval between dosing and audiogenic (AG) seizure testing. The objective of the present series of experiments was to document plasma RDX concentrations associated with the presence of spontaneous seizures. The authors also explored the involvement of different brain regions in RDX-induced seizures by evaluating the effects of this convulsant on sensitivity to audiogenic (AG) seizures and the rate of development of amygdaloid kindled (AK) seizures.
GLP compliance:
no
Test type:
other:
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Source: Class 5 particle size, obtained from Chemtronix, Swananoa, NC)
- Storage: Due to its sensitivity to electrostatic charge, dry RDX was stored and handled in a static free environment. Once in solution, RDX does not require special handling.

Test animals

Species:
rat
Strain:
Long-Evans
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles river Breeding Co (Willmington, MA)
- Age at study initiation: 60 days
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: Subjects used for spontaneous and AG (audiogenic) seizure experiments were housed in pairs; rats with chronic electrode implants for the AK (Amygdaloid kindled) experiment were housed individually
- Diet and water (e.g. ad libitum): All animals had continuous access to food and water. No details available.
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hr light-dark cycle, beginning at 6 AM and 6 PM, respectively.

IN-LIFE DATES:
No data available.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Carboxymethylcellulose and distilled, deionized water
Details on oral exposure:
A stock solution was prepared for each experiment by adding RDX to a 2% mixture of carboxymethylcellulose and distilled, deionized water.
To ensure uniform dispersion, large RDX particles were break up with an ultrasonic probe.
Doses:
The concentration of the stock RDX solution was based on the highest dosage administered in each experiment (6.0, 37.5, 50 and 60 mg/mL); lower concentrations were prepared by dilution with the vehicle.
All dosages were delivered by gavage in a 1 mL/kg vol.
No. of animals per sex per dose:
No data available
Control animals:
not specified
Details on study design:
No data available.
Statistics:
The proportion of animals in each RDX treatment group exhibiting AG or spontaneous seizures was analyzed using Fisher's exact probability test; group comparisons were made for spontaneous seizure incidence during the first 2 hr (0-2) and the last 2 hr (6-8) after treatment.
The total number of spontaneous seizures observed for each rat during the 8 hr observation period and plasma RDX levels were analyzed by analysis of variance (ANOVA), followed by orthogonal contrasts using the F statistic.
The number of stimulation trials and the cumulative AD duration required to achieve the kindled state were submitted to general linear model multivariate analysis of variance (ANOVA) procedures.
In the event of significance, each variable was analyzed by ANOVA, followed when appriopriate, by a repeated-measures ANOVA to indentify at what stage during the kindling process group differences occurred.
Significant interactions were analyzed further with simple effects test.
The alpha level was adjusted for all analyses by the number of comparisons made.
Probability values are expressed according to the adjusted alpha level for the analysis, unless otherwise noted.

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
other: LOAEL
Effect level:
25 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Neurological effets
Sex:
male
Dose descriptor:
LDLo
Effect level:
50 mg/kg bw
Based on:
test mat.
Mortality:
In the group treated with 50 mg/kg, 3/12 of the rats died during spontaneous seizure before AG seizure testing.
No deaths were observed in any of the other groups.
An additional 6 of these animals died during AG seizure testing in the first dose-effect study. The absence of deaths resulting from AG seizures in the second dose-effect experiment is explained by the exsanguination procedures that immediately followed testing; 4 of 9 animals in the 50 mg/kg group were still in seizure when placed in the halothane chamber.There were no deaths observed before or during testing for any of the other groups in either study.
Clinical signs:
Spontaneous seizures were observed even at the lowest RDX dosage (12.5 mg/kg) administered, with the number of spontaneous seizures elicited during the 8 hr interval (relative to controls) reaching statistical significance at dosages of 25.0 mg/kg or higher. Spontaneous seizures began with slow forelimb clonus that inceased in frequency and generally was accompanied by rearing.

Audiogenic seizures were elicited in all groups treated with RDX except for the 12.5 mg/kg group. The proportion of animals exhibiting AG seizures, relative to the absence of AG seizures in the control group, was significant for animals treated with 50-60 mg/kg. The seizures were initiated with uncontrolled running that terminated with the animals falling onto their sides in forelimb and hindlimb clonus.
Body weight:
Not measured
Gross pathology:
No data
Other findings:
Spontaneous seizures:
The time course of spontaneous seizures differed according to the RDX dosage administered. The percentage of animals exhibiting spontaneous seizures was greatest for all groups within the first 2 hr after treatment. A significant and similar proportion of the animals in the 25 and 50 mg/kg treatment groups exhibited spontaneous seizures during the first 2 hr relative to the absence of seizures in the control group. The proportion of animals in the 12.5 mg/kg group that experienced seizures did not differ from controls. In contrast, the percentage of animals in the 50 mg/kg group experiencing seizures remained significantly elevated for the duration of the 8 hr observation period.

Audiogenic seizures:
Pilot observations suggested that rats treated with high dosages of RDX were resistant to AG seizures within the first 2 hr following treatment. This contrasted with the highest proportion of animals experiencing spontaneous seizures during the first 2 hr after dosing for all RDX groups. The time course of audiogenic seizures was evaluated using a dosage of 37.5 mg/kg to allow a margin for lower or higher AG seizure incidence at test intervals other than the 8-hr time point used in the dose-effect studies. The results of testing 2, 4, 8 and 16 hr following treatment with 37.5 mg/kg confirmed earlier observations. At 8 hr post treatment, 4/13 exhibited AG seizures. In contrast, 1/13 rats demonstrated AG seizures at 16 hr, and no AG seizures were elicited at 2- or 4-hr time points.

Plasma RDX concentration:
Circulating levels of RDX were elevated 2 hr after treatment and remained elevated with no significant change for 16 hr. At 8 hr after dosing, the mean plasma RDX concentration of 7.7 µg/mL following treatment with 37.5 mg/kg falls between the plasma RDX concentrations for the 25 mg/kg (5.52 µg/mL) and 50 mg/kg (8.4 µg/mL) groups obtained at this interval for the dose-effect determinations. According to the plasma RDX concentrations associated with significant incidence of AG seizures in the dose-effect study, it was anticipated that 60-70% of the animals would exhibit AG seizures in the time course study.The relatively low percentage (38%) of animals experiencing AG seizure 8 hr after treatment cannot be explained at this time.

Amygdaloid kindled seizures:
Rats treated daily with 6 mg/kg RDX until kindled exhibited no signs of systemic toxicity or spontaneous seizures. Prestimulation EEG records also appeared unremarkable. Rats treated with RDX required fewer stimulations to reach the kindling criterion. This observation is supported by a significant treatment effect identified in the MANOVA for kindling variables. No group differences were identified in either threshold determinations or in wet dog shake frequency. All animal except one (a control) exhibited a stage 5 seizure when stimulated 1 week after dosing ad been discontinued, suggesting that the accelerated kindling was not an acute effect of RDX treatment.

Any other information on results incl. tables

The present results provide the first characterization of dose-effect relationships for the convulsant properties of RDX using two seizure models.

Treatment with 50 -60 mg/kg was effective in sensitizing resistant rats to audiogenic seizures.

At a lower (25 mg/kg) dosage, a significant incidence of spontaneous seizures was observed, while a still lower dosage (6 mg/kg) accelerated the rate of amygdaloid kindling in the absence of any obvious signs of toxicity.

Although the present results suggest increased risk of seizures at dosages above 6 -12.5 mg/kg, these results must be qualifed by the size (class 5) of RDX particles. The authors suggest that RDX toxicity is inversely related to the size of the particle, according to the results obtained in a other study.

Significant risk of spontaneous and audiogenic seizures is incurred with plasma RDX concentrations in excess of 5 µg/mL and 8 µg/mL, respectively.

In the present series of experiments, the first spontaneous seizures were observed within 25 -30 min after treatment, with an average time to first seizure of 47 min for rats treated with 12.5 mg/kg and 48 min and 62 min for rats treated with 25 and 50 mg/kg, respectively.

Evaluation of the time course of plasma RDX concentrations following administration of 37.5 mg/kg RDX demonstrated that plasma levels were elevated at the first post-treatment time examined (2 hr) and remained elevated for the duration of the 16 -hr test interval.

The relatively long plasma RDX half-life observed at dosages greater than 37.5 mg/kg may explain the sustained incidence of spontaneous seizures exhibited by animals treated with 50 mg/kg.

The time course of plasma RDX concentrations obtained with dosages below 37.5 mg/kg is not known.

A shorter plasma half-life may be associated with lower RDX dosages, since the authors found that RDX could be detected in plasma only at 7 hr.

Although the mechanism by which RDX alters seizure susceptibility has yet to be studied, the results obtained in the present series of experiments implicate limbic system involvement.

This possibility was suggested first by the characteristics of the spontaneous seizures.

At lower dosages, seizures were initiated by slow forelimb clonus that increased in frequency during rearing.

In addition to forelimb clonus, consecutive rearing and falling movements were observed often in the higher dosage groups.

Applicant's summary and conclusion

Interpretation of results:
moderately toxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the results obtained in this study, the ingestion of high doses of RDX induce mortality during spontaneous seizures in male rats. The lowest letal dose was 50 mg/kg and the low observed adverse effect level was 25 mg/kg where the number of spontaneous seizures was significant.