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Repeated dose toxicity: other routes

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Administrative data

short-term repeated dose toxicity: other route
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report which meets basic scientific principles (test substance purity not known).

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Subacute toxicity (5 consecutive days) upon intravenous injection with 33-day observation period.
GLP compliance:
Limit test:

Test material

Details on test material:
- Analytical purity: No data
- Lot/batch No.: B-1
- Storage condition of test material: room temperature
- Stability: The test article is stable at 60°C for 30 days. Additionally, aqueous solutions of Benzisoquinolinedione are stable at room temperature for 14 days. Results by infrared and ultraviolet spectroscopy indicated that the test article did not decompose under the storage conditions.

Test animals

other: Charles River CD2F1
Details on test animals and environmental conditions:
- Source: Animal Genetics and Production Branch, DTP, DCT, NCI
- Age at study initiation: < 8 weeks
- Weight at study initiation: 18.3 to 22.5 g for males and 16.6 to 22.0 g for females (Weight range of females was out of the protocol range of 17.0 - 23.0 g for this sex. Weight ranges within each sex were 4.2 g for males und 5.4 g for females which were out of the protocol 4 gram range within each sex.)
- Housing: group housed by dose and separated by sex with five mice per cage
- Diet: ad libitum (Purine Certified Rodent Chow)
- Water: ad libitum (tap water)
- Acclimation period: for a minimum of one week

- Temperature: 21°C
- Humidity (%): 32.1% ± 1.3% (range: 13 - 61%)

Administration / exposure

Route of administration:
other: 0.9% sterile saline with hydrochloric acid (0.5 N)
Details on exposure:
Dosing solutions were prepared by diluting an initially prepared stock solution of 50 mg/mL of the test item in vehicle until obtaining the targeted test concentrations. The dosing preparations were formulated on day 1 of treatment and were stored at room temperature. A sample of each dosing preparation was taken for concentration analysis. One dosing preparation was selected, at random, for homogeneity analysis. Homogeneity analysis consisted of concentration analysis of a top, middle, and bottom portion of the solution. Concentration analyses were performed by means of high performance liquid chromatography (HPLC).
The lateral tail vein was used for all injections. To facilitate doing, mice were placed in a restrainer with the tail exposed. A lighted box was placed under the mouse with the light shining through a sIit where the mouse's tail was situated. lnjections were completed in approximately 4 seconds. The volume of injection was 0.015 mL/g body weight for all dose groups. The highest concentration used in the study was 3.93 mg/mL.
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
5 days
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
17, 21, 26, 32, 39, 48, 59 mg/kg bw (all within 7% of the theoretical concentrations; additionally, homogeneity tests show homogeneity of the dosing preparations)
No. of animals per sex per dose:
15 (Due to dosing difficulty, one male of the 48 mg/kg bw group was taken off the study on day 5.)
Control animals:
other: vehicle control and nontreated control group
Details on study design:
- Post-exposure period: 33 days


Observations and examinations performed and frequency:

- Time schedule for examinations: Individual body weights were taken for all surviving mice on days 1 - 5, 12, 19, 26 and 33.

Results and discussion

Results of examinations

Details on results:
100% mortality was observed in both males and females at the doses of 59.0, 48.0 and 39.0 mg/kg bw and in females at the dose of 32.0 mg/kg bw. No deaths occurred in either sex at the doses of 21.0 und 17.0 mg/kg bw. In general, the higher the dose, the earlier the mortality. Probit results for days 7, 14 and 33 are: on day 7, LD10, LD50 und LD90 were 27.5, 35.0 und 44.6 mg/kg bw for males, 29.3, 47.2 und 76.2 mg/kg bw for females, and 27.2, 40.4 und 60.0 mg/kg bw for the combined sexes. Due to slightly later deaths in females, lethality doses in this sex were slightly higher than those in males on day 7. Since all the deaths occurred between days 2 und 10, probit values were the same for days 14 and 33. On these two days, lethality doses in females could not be calculated because there was only one data point which was neither 0% nor 100% mortality. On days 14 and 33, LD10, LD50 and LD90 were 24.3, 28.12 and 34.2 mg/kg bw for males and 24.9, 28.4 und 32.5 mg/kg bw for the combined sexes. Based on the probit results in males and combined sexes, there was no apparent sex difference in the final lethality doses.
Drug-related clinical signs observed, in the order of frequency, were rough hair coat, decreased activity, dehydration, hunched posture, labored breathing, loss of locomotor activity, exophthalmus, necrotic and/or swollen tails, ataxia, swollen face and eyes closed with discharge. No drug-related clinical signs were observed at the two lowest doses of 21.0 and 17.0 mg/kg bw. Clinical signs of labored breathing, loss of locomotor ability and exophthalmus occurred immediately or shortly after dosing. ClinicaI signs of death, rough hair coat, decreased activity and hunched posture occurred either shortly after dosing or as long as a few days after dosing. ClinicaI signs of dehydration, necrotic and/or swollen tails, moribundity, ataxia, swollen face and eyes closed with discharge had late occurrences. Drug-related clinical signs were dose-related and males and females were similarly affected. All other observations were considered to be non-drug-related.

The test substance caused a dose-related slight to severe weight loss (6% to 15%) from days 1 to 5 in both sexes at the four highest doses of 59.0, 48.0, 39.0 and 32.0 mg/kg bw. From days 1 to 5, males and females at the three lowest doses of 26.0, 21.0 and 17.0 mg/kg bw also had a slower weight gain than that of the control groups. A further weight loss, although very slight, was observed from days 5 to 12 in males treated with 32.0 mg/kg bw and in females treated with 26.0 mg/kg bw, while the remaining surviving dose/sex groups had a substantial weight gain from days 5 to 12. A normal to substantial weight gain was observed in all dose/sex groups from days 12 to 19. Mean body weights of the surviving dose/sex groups were comparable to the controls on days 26 and 33.

Effect levels

open allclose all
Dose descriptor:
Remarks on result:
not determinable
no NOAEL identified
Dose descriptor:
other: LD50
Effect level:
28.4 mg/kg bw/day (nominal)
Basis for effect level:
other: Mortality

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion