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Toxicological information

Acute Toxicity: oral

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acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: based on the requirements of Commission Directive 91/507/FEC, Pharmaceutical Affairs Bureau (Ministry of Health and Welfare) Notifications No.24 and No.88 and US FDA guidance on acute toxicity testing for pharmaceuticals.
not specified
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Pharmaceutical grade Dapsone

Test animals

New Zealand White
Details on test animals or test system and environmental conditions:
Nulliparous, non-pregnant female New Zealand White rabbits of the Crl: NZW/Kbl.BR strain were obtained from Charles River UK Ltd, Margate, Kent on 6 July 1999. The animals were examined and weighed on the day of arrival. The rabbits were in a body weight range of 2.048 to 2.893 kg on Day --L Based on information from the supplier the rabbits were approximately twelve to fourteen weeks old on Day 1.
Rabbits were housed individually in floor-pens throughout the acclimatization and experimental phases of the study. Each pen had a minimum floor area of 0.6 m2. The partitions between pens were constructed of slotted laminate board 1.0 m high. The floor pens had solid floors. Wood chips were provided as floor litter. Each batch of wood chips was analysed for specific contaminants and the data are kept on file in the Covance Archive.
SQC TRB Rabbit Diet 9603 (pelleted) from Harlan Teklad, Bicester was freely available to the animals at all times. Each batch of diet is analysed for specific constituents and contaminants by the manufacturer.
Mains water was provided, ad libitum, from water bottles attached to the pen walls. The potable water supply is periodically analysed for specific contaminants. No contaminants were present in diet, bedding or water at levels that might have interfered with achieving the objective of the study.
The holding room was designed to permit at least 10 air changes per hour and to maintain temperatures of 16 to 22°C. Humidity was not actively controlled but was expected to remain within the range 40 to 80% RH. Recordings of maximum and minimum temperature and humidity were generally made twice daily except on some Saturdays, Sundays or public holidays during the study when only a single record was made without affecting the study integrity. On no occasion during the study did the temperature or humidity values fall outside the expected ranges. The room was illuminated by fluorescent strip-lights for twelve hours daily.

Administration / exposure

Route of administration:
oral: gavage
1% m/v aqueous methyl cellulose
Details on oral exposure:
Individual dose volumes (mL) were calculated from the body weights of the rabbits on the morning of dosing and the selected dose volume, 5 mL/kg. Each rabbit was dosed by passing a catheter along the oesophagus and instilling the test article into the gastric lumen. Plastic syringes and catheters were used.
Dose levels were expressed gravimetrically in terms of test article received (without regard to purity or active content). The test articles were dispersed, separately or in combination, in 1% m/v aqueous methyl cellulose. The formulated concentrations were calculated from the selected dose level and dose volume. All formulations were used on the day of preparation.

No procedures to limit particle size of test article in suspension, to sterilise the test formulations or to measure pH, osmolarity or other physico-chemical characteristic of the test formulations were undertaken. No analyses of the homogeneity, stability or achieved concentration of the test article in administered formulations were undertaken.
Chlorproguanil-200 mg/kg
Dapsone-125 mg/kg plus Chlorpruguanil- 100 mg/kg
Dapsone-250 mg/kg plus Chlorpruguanil - 200 mg/kg
No. of animals per sex per dose:
6 females/dose
Control animals:

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 250 mg/kg bw
Based on:
test mat.
No deaths were seen during the study.
Clinical signs:
other: No effects were seen
Gross pathology:
The mean liver weight showed a slight increase but a comparison of individual weights and organ/body weight ratios indicate that the mean increase is largely· attributable to one rabbit (no 12) which was the heaviest animal on study and this increase can not be directly attributed to a toxic effect of the test article. For the remaining organ weights, values among the treated rabbits were similar to those of the controls. Necropsy revealed a few red or dark foci on the lungs of three rabbits. These were considered likely to be agonal changes rather than treatment related effects.
Other findings:
A single elevated Heinz body count (animal no 7) resulted in a higher group mean percentage in Group 2. 'Methaemoglobin levels were slightly increased for four animals (0.1 to 0.6%) compared with zero values for all control samples. No other haematology parameters measured on Day 2 showed any notable changes.
Two animals (nos 8 & 10) had elevated Heinz body counts on Day 13 resulting in an increased group mean value. Methaemoglobin levels were slightly increased for two animals (0.2 to 0.4%) compared with a single value of 0.1% in the control samples. None of the other haematology parameters showed any marked differences from control values.
Comparison of the results obtained for animals treated with Dapsone with those from the control group revealed no notable differences in any of the measured clinical chemistry parameters

Applicant's summary and conclusion

Interpretation of results:
Category 3 based on GHS criteria
An acute oral median lethal dose (LD50) of Dapsone is > 250 mg/kg bw.
Executive summary:

The study was designed to investigate possible interactions following single oral administration of the two test articles, Chlorproguanil and Dapsone, in combination. The doses of the combined formulation included the MTD of Chlorproguanil (determined in CLE study number 1552/25) and a clinically relevant dose of Dapsone [Chlorproguanil:Dapsone in ratio 1:1.25]. A second, lower dose combination, group was included to provide data on a possible dose related trend. The study also included a single administration of each test article individually to investigate possible synergistic effects and to provide data for comparison with the dose combination groups.

The oral route of administration was used in this study because it is the likely human therapeutic route. Guidance on requirements for new drug registration issued by EU, MOHW and US FDA (1,2,3) requires single dose toxicity studies to be completed (including one non-rodent species for Japan and US). In addition, the expected metabolism of chlorproguanil in the rabbit was an important selection criterion. In humans, chlorproguanil is metabolised to the active metabolite, chlorcycloguaniL There is evidence that this pathway is absent or limited in rodents but that it does occur in rabbits. Hence the rabbit was the chosen test model for this study. Groups of six female rabbits were given a single oral dose of the vehicle control, one of the test articles or one of two doses of the combined formulation.

Administration of the low dose combined treatment resulted in no clearly adverse reactions although some changes suggesting a limited hepatic response were seen.

Administration of the high dose combination treatment resulted in a marked initial loss of appetite and weight loss that was similar to that observed following dosing with the equivalent dose of Chlorproguanil alone. Anogenital soiling, also apparent following dosing with Chlorproguanil, was noted shortly after dosing. However the combination treatment did not result in the death of any animals.

Increases in methaemoglobin concentration and erythrocytes with Heinz bodies, following administration of Dapsone, were more marked in the high dose combination group than in the group dosed with the equivalent dose of Dapsone alone. No such changes were seen in the low dose combination group.

Generally there were no responses seen in either of the two combination groups that indicated any enhancement of effects due to interaction between the two test articles. However there was some indication that the joint administration of the high dose may have slightly increased the normal effects of Dapsone alone on methaemoglobin levels and Heinz body production. No evidence for a clear enhancement of changes due to a synergistic reaction was apparent. There were also no findings that were unique to the combined treatment groups compared with the reactions seen following administration of either Dapsone or Chlorproguanil alone.