Estimation of the dose range and percentage of
mortality
An approximate LD50 can be initially determined as a
pilot study by a so called ‘staircase method’ using a
small number of animals (2 each dose) and increasing
the doses of the drug. Five doses can be chosen for
determination of LD50 starting from no death to 100%
mortality. In our study for estimation of LD50 of
thymoquinone, 5 doses were given intraperitoneally to 5
groups of rats, 10 in each group (Table-1).
The animals were observed for first 2 hours
and then at 6th and 24th hour for any toxic symptoms.
After 24 hours, the number of deceased rats was
counted in each group and percentage of mortality
calculated.
Table-1: Results of the lethal doses of
thymoquinone for the determination of LD50 after
intraperitoneal injection in rats (n=10)
Group
Dose
(mg/kg)
Log
dose
%
Dead
*Corrected
% Probits
1 25 1.4 0 2.5 3.04
2 50 1.7 40 40 4.75
3 75 1.88 70 70 5.52
4 100 2 90 90 6.28
5 150 2.18 100 97.5 6.96
*Corrected % Formula for 0 and 100 is given in the text.
Conversion of percentage mortalities to probits
and calculation of LD50
The percentage of animals that had died at each dose
level is then transformed to probit (Table-2).
Table-2: Transformation of percentage mortalities to probits
% 0 1 2 3 4 5 6 7 8 9
0 - 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.59 3.66
10 3.72 3.77 3.82 3.87 3.92 3.96 4.01 4.05 4.08 4.12
20 4.16 4.19 4.23 4.26 4.29 4.33 4.36 4.39 4.42 4.45
30 4.48 4.50 4.53 4.56 4.59 4.61 4.64 4.67 4.69 4.72
40 4.75 4.77 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.97
50 5.00 5.03 5.05 5.08 5.10 5.13 5.15 5.18 5.20 5.23
60 5.25 5.28 5.31 5.33 5.36 5.39 5.41 5.44 5.47 5.50
70 5.52 5.55 5.58 5.61 5.64 5.67 5.71 5.74 5.77 5.81
80 5.84 5.88 5.92 5.95 5.99 6.04 6.08 6.13 6.18 6.23
90 6.28 6.34 6.41 6.48 6.55 6.64 6.75 6.88 7.05 7.33
The percentage dead for 0 and 100 are corrected before
the determination of probits as under:
Corrected % Formula for 0 and 100% mortality3:
For 0% dead: 100(0.25/n)
For 100% dead: 100(n-0.25/n)
The probit values are plotted against log-doses and then
the dose corresponding to probit 5, i.e., 50%, is found
out (Figure-1). In the present case the Log LD50 is 1.76
and LD50= 57.54 mg/kg.
Calculation of Standard Error (SE) of LD50
The SE of LD50 can be calculated from the following
formula:3
Approx. SE of LD50= (Log LD84-Log LD16) … (a)
where N is number of animals in each group.
The probits of 84 and 16 from Table-1 are 5.99
and 4.01
Estimation of the dose range and percentage of
mortality
An approximate LD50 can be initially determined as a
pilot study by a so called ‘staircase method’ using a
small number of animals (2 each dose) and increasing
the doses of the drug. Five doses can be chosen for
determination of LD50 starting from no death to 100%
mortality. In our study for estimation of LD50 of
thymoquinone, 5 doses were given intraperitoneally to 5
groups of rats, 10 in each group (Table-1).
The animals were observed for first 2 hours
and then at 6th and 24th hour for any toxic symptoms.
After 24 hours, the number of deceased rats was
counted in each group and percentage of mortality
calculated.
Table-1: Results of the lethal doses of
thymoquinone for the determination of LD50 after
intraperitoneal injection in rats (n=10)
Group
Dose
(mg/kg)
Log
dose
%
Dead
*Corrected
% Probits
1 25 1.4 0 2.5 3.04
2 50 1.7 40 40 4.75
3 75 1.88 70 70 5.52
4 100 2 90 90 6.28
5 150 2.18 100 97.5 6.96
*Corrected % Formula for 0 and 100 is given in the text.
Conversion of percentage mortalities to probits
and calculation of LD50
The percentage of animals that had died at each dose
level is then transformed to probit (Table-2).
Table-2: Transformation of percentage mortalities to probits
% 0 1 2 3 4 5 6 7 8 9
0 - 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.59 3.66
10 3.72 3.77 3.82 3.87 3.92 3.96 4.01 4.05 4.08 4.12
20 4.16 4.19 4.23 4.26 4.29 4.33 4.36 4.39 4.42 4.45
30 4.48 4.50 4.53 4.56 4.59 4.61 4.64 4.67 4.69 4.72
40 4.75 4.77 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.97
50 5.00 5.03 5.05 5.08 5.10 5.13 5.15 5.18 5.20 5.23
60 5.25 5.28 5.31 5.33 5.36 5.39 5.41 5.44 5.47 5.50
70 5.52 5.55 5.58 5.61 5.64 5.67 5.71 5.74 5.77 5.81
80 5.84 5.88 5.92 5.95 5.99 6.04 6.08 6.13 6.18 6.23
90 6.28 6.34 6.41 6.48 6.55 6.64 6.75 6.88 7.05 7.33
The percentage dead for 0 and 100 are corrected before
the determination of probits as under:
Corrected % Formula for 0 and 100% mortality3:
For 0% dead: 100(0.25/n)
For 100% dead: 100(n-0.25/n)
The probit values are plotted against log-doses and then
the dose corresponding to probit 5, i.e., 50%, is found
out (Figure-1). In the present case the Log LD50 is 1.76
and LD50= 57.54 mg/kg.
Calculation of Standard Error (SE) of LD50
The SE of LD50 can be calculated from the following
formula:3
Approx. SE of LD50= (Log LD84-Log LD16) … (a)
where N is number of animals in each group.
The probits of 84 and 16 from Table-1 are 5.99
and 4.01
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