Topical Lotion Formulation PDF

DESIGNING AND DEVELOPMENT OF TOPICAL CHAPTER 4
LOTION FORMULATION: PHARMACEUTICAL

CHARACTERISATION AND EVALUATION FOR
DECONTAMINATION EFFICACY AND DERMAL
TOXICITY
4.1 Background
Topical lotion formulation was developed using central composite design (CCD) with two
factor and 3 levels (23). Polymer and active drug concentration were chosen as
independent variables while spreadability, extrudability and viscosity as dependent
factors. Regression analysis of the selected formula also supports the development and
found statistically significant (p <0.05). The present study aimed at:
 Development of the topical lotion formulation at different concentrations of

polymer and active drugs
 Characterisation of different critical pharmaceutical parameters of the formulation
 Evaluation of the decontamination efficacy over Sprague Dawley rat as well as
human tissue equivalent models
 Dermal toxicity analysis in rat Sprague Dawley rat model using both sexes (male
and female)
4.2 Experimentation
 Formula was designed using central composite designs (CCD) software and
analysed for regression and quadratic response.
 The best suited models were selected and formulation prepared, characterised for
the different pharmaceutical parameters such as pH, spreadability, extrudability,
viscosity, stability and shelf-life.
 In vitro and in vivo decontamination efficacy was determined using
pharmacoscintigraphy technique. Human tissue equivalent and Sprague Dawley
99m 131 201
rat (male and female) were chosen as experimental models. Tc, I and Tl
were employed as radiological contaminants mixed appropriately in normal saline.
After different time intervals of contamination (5 – 60 min), decontamination was
performed using topical lotion soaked in cotton swabs. Residual activity was
measured and scintigraphs recorded with gamma camera. Decontamination factor
(DF) was calculated and efficacy determine in percentage.
 Dermal toxicity studies were performed according to the OECD Test guidelines
404, 410 and Schedule Y of Drugs and Cosmetics Act, 1940, as amended.
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4.3 Results
In order to carry out an overview and a critical analysis of skin decontamination, topical
lotion formulation was developed successfully and analysed for decontamination efficacy
and dermal toxicity applying the different formulas and statistical approaches. Most often,
the decontaminating products available have not been tested for their mechanism of
action, effectiveness, or side effects. This section describes the formulation development
procedure, characteristics as well as all the criteria necessary for a suitable product
development.
4.3.1Formula design
Model was analysed for the data fitting, regression analysis and the effect of independent
factors (over the disodium edetate/DTPA and prepared lotion formulation). Most
repeatedly and closely related formula with significant p-value and positive quadratic
response were selected for further analysis. Formula for the development of topical lotion
formulation, loaded and unloaded (disodium edetate/DTPA) was prepared successfully.
Different compositions of the optimised formulation according to the category are
illustrated in Table 4.1 while formula of different batches (%) given in Table 4.2.
Table 4.1: Optimised formula of disodium edetate topical lotion formulation
Unit formula Qty for batch

S. No Ingredients Category
(per 10 ml) (per 100 ml)
Disodium EDTA /
1 API 50mg/100mg 0.5 g/1g
DTPA
2 Propylene glycol Humectant 0.5ml 5 ml
Viscosity
Sodium Carboxy
3 increasing 100mg 1g
methyl cellulose
agent
Methyl paraben
4 Preservative 2mg 0.02g
sodium
Propyl paraben
5 Preservative 0.2mg 0.002g
sodium
6 Purified Water Vehicle Qs to 10 ml Qs to 100
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Table 4.2: Composition of the topical lotion formulation (TLF) of disodium edetate /
DTPA
Ingredients Composition
Na2EDTA/DTPA 0.25 0.5 1.0 2.0 3.0 4.0 5.0
Propylene glycol (ml) 5 5 5 5 5 5 5
Sodium Carboxy methyl 3 1 1 5 4 2 1
cellulose (%)
Methyl paraben sodium
0.02 0.02 0.02 0.02 0.02 0.02 0.02
(%)
Propyl paraben sodium
0.002 0.002 0.002 0.002 0.002 0.002 0.002
(%)
Purified Water (ml) Qs to Qs to Qs to Qs to Qs to Qs to Qs to
100 100 100 100 100 100 100
4.3.2 Fitting model to the data
A two factor three level statistical central composite experimental design as the response
surface methodology (RSM) requires 13 runs. All the responses observed for 13
formulations prepared were simultaneously fitted to first order, second order and
quadratic models using Design Expert® (Version 8.0.7.1, Stat-Ease Inc., Minneapolis,
MN). It was observed that the best fitted model was quadratic and the comparative values
of R2, SD, and % CV with the regression equation generated for each response are
presented in Table 4.2. All statistically significant (p< 0.05) coefficients are included in
the equations. A positive value represents an effect that favours the optimisation, while a
negative value indicates an inverse relationship between the factor and response. It is
evident that all the two independent factors, namely the disodium edetate (X1) and sodium
CMC concentration (X2) have interactive effects on the three responses, e. g., Y1 -
Viscosity (PaS), Y2 - Spreadability (cm), Y3 - Extrudability (gm). The total 13 runs with
triplicate centre point were generated and the responses so observed are illustrated in
Table 4.3.
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Table 4.3: Actual response for the central composite experimental designs
Independent factors Dependent factors

Std Run Factor 1 Factor 2 Response 1 Response 2 Response 3
A:Disodium B:Sodiu Viscosity Spreadabilit Extrudabilit
edetate / DTPA m CMC (Y1) y (Y2) y (Y3)
5 1 -0.53 2.25 95 12 0.5
1 2 1.00 0.50 99.5 12.4 0.3
11 3 2.13 2.25 85 10 0.1
12 4 2.13 2.25 85.5 10.3 0.1
3 5 0.25 4.00 84 10.5 0.2
6 6 4.78 2.25 86 9.5 0.1
9 7 2.13 2.25 86.5 9.2 0.1
13 8 2.13 2.25 86 9.8 0.1
10 9 2.13 2.25 86.4 9.7 0.2
2 10 4.00 0.50 85 10 0.25
7 11 2.13 -0.22 95 10.5 0.2
8 12 2.13 4.72 88 9.5 0.2
4 13 4.00 4.00 83 7 0.1
4.3.3 Regression analysis of the model
The three dimensional plots were prepared for all the three responses. These plots are
known to study the interaction effects of the factors on the responses as well as are useful
in studying the effects of two factors on the response at one time quantitatively compared
the resultant experimental values of the responses for responses Y1, Y2 and Y3
respectively with that of predicted values as illustrated in table 4.3 – 4.5 and figure. 4.1 –
4.2.
Table 4.4: Summary of results of regression analysis for responses Y1, Y2, and Y3 for
fitting to quadratic model
Quadratic R2 Adjusted R2 Predicted R2 SD % CV

model
Response (Y1) 0.9882 0.9083 0.9311 2.22 2.52
Response (Y2) 0.9019 0.9604 0.9281 0.76 1.61
Response (Y3) 0.9756 0.9153 -0.9859 0.072 3.09
Regression equation of the fitted quadratic model**
Y1 = +107.76-7.06 X1 -8.40 X2 + 1.30 X1 X2 + 0.46 X12 + 0.74 X22
Y2 = + 12.48 - 0.890 X1 – 0.392 X2 – 0.066 X1 X2 + 0.092 X12 + 8.68 – 0.03 X22
Y3 = + 0.48 – 0.168 X1 – 0.088 X2 + 4.00 – 0.03 X1 X2 + 0.024 X12 + 0.012 X22
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**
Only the terms with statistical significance are included
Fig. 4.1: Linear correlation plot (A, C, E) between actual and predicted values and the
corresponding residual plots (B, D, F) for various responses.
Response 1 (Y1): Effect on viscosity of Lotion
The model proposes the following polynomial equation for viscosity Y1 = +107.76-7.06 X1
-8.40 X2 + 1.30 X1 X2 + 0.46 X12 + 0.74 X22 where Y1 is the viscosity of the formulation,
and X1 is the concentration of the disodium edetate, X2 is the concentration of sodium
CMC polymer. A positive value for the coefficient is an indicative of the favourable effect
whereas a negative value for the coefficient indicates an unfavourable effect of that
particular factor on the response. The predicted R-square of 0.9311 is in reasonable
agreement with the adjusted R- square of 0.9083. Adequate precision is within the
desirable limit. Therefore, this model can be used to navigate the design space as
presented in Table 4.5. The three dimensional surface response plot which shows the
effect of independent variables on viscosity, spreadability and extrudability. In our study,
it is revealed the viscosity decreased with the increase in concentration of the polymer as
the drug remains entrapped in the polymer film network (Singh et al., 2005; Xiong et al.,
2005). Factor B (polymer) appeared to have more profound effect on viscosity than factor
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A. As the polymer level increase, the viscosity decreased dramatically Factor A which is
the concentration of the disodium edetate used, affected the viscosity in opposite direction
to that observed with factor B (polymer concentration). The polymer has a more fuzz ball
type of structure containing interstitial micro voids forming channels in the polymer
hydrogels which are influenced by the concentration of the polymer and the degree of
swelling. Increasing the amount of polymer will decrease the size of the channels, as dose
an increase in swelling degree decreases the release of drug. Disodium edetate is widely
used chelating agent in decorporation and decontamination formulations. Besides this, it
has a low systemic toxicity and high local tolerability when they are applied topically. In
topical decontamination formulations the solubility is important as it influences the
partition coefficient of the drug between the formulation and the skin which in turn affects
the chelating agent and radio-isotopes complex formation. In our findings, disodium
edetate has good chelation property, as the proportion used in the formulation (0.5% and
1.0%) favours the maximal radio-contaminants removal (Gannu et al., 2010).
Table 4.5: Viscosity actual and predicted values for all the runs
Standard order Actual value Predicted value Residual

1 99.50 97.81 1.69
2 85.00 85.60 -0.60
3 84.00 85.64 -1.64
4 83.00 86.25 -3.25
5 95.00 94.92 0.077
6 86.00 83.64 2.36
7 95.00 96.17 -1.17
8 88.00 84.94 3.06
9 86.50 85.99 0.51
10 86.40 85.99 0.41
11 85.00 85.99 -0.99
12 85.50 85.99 -0.49
13 86.00 85.99 0.012
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Fig. 4.2: 3D response surface plot sh
Fig. 4.2: 3D response surface plot showing effect of polymer and disodium edetate
concentration over viscosity, spreadability and extrudability parameters.
Response 2 (Y2): Effect on spreadability of Lotion

The model proposes the following polynomial equation for spreadability of lotion
formulation Y2 = + 12.48 - 0.890 X1 – 0.392 X2 – 0.066 X1 X2 + 0.092 X12 + 8.68 – 0.03
X22 where Y2 is the spreadability, X1 is concentration of disodium edetate and X2 is the
concentration of sodium CMC polymer. The predicted R2 of 0.9281 is in reasonable
agreement with the adjusted R-square of 0.9604. Adequate precision is within the limit.
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Therefore, this model can be used to navigate the design space. Fig. 4.3 represents the 3
dimensional contour plot shows the effect of different invariables on spreadability. The
model was found to be significant (F-value = 5.67; p < 0.02 while the lack of fit was
found not significant (F-value = 0.5; p < 0.753). The signal to noise ratio was found to be
satisfactory as the observed 8.287 indicates an adequate signal (more than 4). Thus this
model could be used to navigate the design space. From the polynomial equation for
spreadability, a positive sign represented a synergistic effect, while a negative sign
indicted an antagonistic effect. Factor B (polymer) appeared to have more profound effect
on spreadability than factor A. As the concentration of polymer increased, the
spreadability decreased dramatically. The positive coefficient value of factor A indicated
the increase in spreadability with an increase in factor A (disodium edetate concentration).
Actual vs. predicted value of spreadability for all the 13 runs are shown in Table 4.6.
Table 4.6: Spreadability actual and predicted values for all the runs
Stand Actual value Predicted value Residuals

order
1 12.40 11.46 0.94
2 10.00 10.09 -0.087
3 10.50 10.77 -0.27
4 7.00 7.92 -0.92
5 12.00 12.22 -0.22
6 9.50 8.80 0.70
7 10.50 11.14 -0.64
8 9.50 8.69 0.81
9 9.20 9.86 -0.66
10 9.70 9.86 -0.16
11 10.00 9.86 0.14
12 10.30 9.86 0.44
13 9.80 9.86 -0.060
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Fig. 4.3: Contour plot showing effect of polymer and disodium edetate concentration
over viscosity, spreadability and extrudability parameters.
Response 3 (Y3): Effect on extrudability of Lotion
The following polynomial equation was proposed by the model for extrudability of lotion
formulation Y3 = + 0.48 – 0.168 X1 – 0.088 X2 + 4.00 – 0.03 X1 X2 + 0.024 X12 + 0.012
X22. The positive coefficient values of factor B indicate an increase in the extrudability
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(disodium edetate concentration). It was observed that higher concentration of the

independent variables decreases extrudability. Table shows the actual and predicted
values for the extrudability of lotion. The interaction between the independent factors was
also found to be significant. Overall, the values for predicted R-square (0.9859) and
adjusted (0.9153) R-square values are in reasonable agreement. Extrudability of the
formulation is a crucial feature as it is applied to the skin. Therefore it is preferable to
formulate plastic samples because of their low resistance to flow when applied under high
shear conditions, whereas at rest the flow under the stress caused by gravity is zero. In our
study, the extrudability of the lotion increases with the increase in disodium edetate
concentration while decreases with the increase in polymer concentration.
Mathematically, the formulations are showing good response in terms of decontamination
efficacy and no observable dermal toxicity. Actual vs. predicted value of extrudability for
all the 13 runs are given in Table 4.6.
Table 4.7: Extrudability actual and predicted values for all the 13 runs
Stand Order Actual value Predicted value Residuals

1 0.30 0.30 0.465
2 0.25 0.17 0.077
3 0.20 0.29 -0.089
4 0.100 0.11 -0.011
5 0.50 0.44 0.061
6 0.100 0.15 -0.050
7 0.20 0.26 -0.056
8 0.20 0.13 0.068
9 0.100 0.12 -0.020
10 0.20 0.12 0.080
11 0.100 0.12 -0.020
12 0.100 0.12 -0.020
13 0.100 0.12 -0.020
4.3.4 Data analysis of the formulation developed
The optimum formulation was selected by applying following constraints (Table 4.3) on
the responses Y1 (83-100 cP), Y2 (7-13 cm), Y3 (0.01-0.5 gm). Point prediction of the
design expert software was used to determine the optimized composition which predicted
the optimised processed parameters to be 100 Cp viscosity, 12.4 cm spreadability and
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0.30 gm extrudability of the lotion formulation at polymer concentration 1.0% and 0.5-
1.0% disodium edetate. Based upon the seven formulations were selected randomly at the
responses of viscosity, spreadability and extrudability were evaluated. Formulation was
studied for in vitro and in vivo decontamination efficacy against a number of radio-
isotopes.
The validation for RSM involving all the three check-point formulations was found to be
within limits. The percentage predition error assures the valdity of generated equations
and thus depicts the domain of applicability of RSM model. Figure 4.4 shows the
desirability of independent variables in the optimized formulation. Sufficient replicates of
topical lotion were prepared based on the optimised formula and then carried out the
evaluation studies.
Fig. 4.4: Desirability of the independent variables (disodium edetate and sodium carboxy
methylcellulose) of the optimised formulation.
4.4 Characteristics of Pharmaceutical Parameters of the Lotion Formulation
(i) pH
pH values of the topical lotion (disodium edetate / DTPA) ranged 7.3±0.2.
(ii) Visual observation
It was performed once in 15 days for 3 months. Visual observation was recorded in the
following manner:
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i. If transparent lotion was obtained, it was recorded as clear transparent lotion

(CTL).
ii. If milky lotion was obtained, it was marked as contaminated lotion (CL).
(iii) Spreadability
Spreadability of the formulation was recorded to be 13.4±0.2 cm.
(iv) Viscosity
Viscosity of 95 to 100 P ensures its application over the skin without runoff or wastage.
(v) Extrudability
The extrudability of formulation was found to be 0.30±0.021 g which implies the ease of
application of the lotion.
(vi) Homogeneity
Appearance of the formulation after 3, 6, and 9 months were clear and transparent without
any aggregates or phase separation.
(vii) Drug content
96-98% of drug content recorded after 3, 6 and 9 months of the study.
4.5 Decontamination Protocol Standarisation
DE of the lotion was investigated in both the experimental models using the commonly
99m
used medical radioisotopes. Protocol was standardised using Tc on human tissue
equivalent model followed by extensive studies with the 131I and 201Tl radio-isotopes.
(i) Animal model vs. human tissue equivalent
Animal group with the hair removal using paired scissors was found to be better than the
tissue equivalent model. Developed formulations were found more effective for the
animal skin contaminants removal as illustrated in Figure 4.5.
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Fig. 4.5: Decontamination efficacy of developed formulation (90±5% for animal model
and 83±6% for human tissue equivalent) against 99mTc after different time intervals of
contamination observed more effective for the rat model.
(ii) Mode of hair clipping: Scissors vs. Chemical depilatory
Animal group in which hairs were removed using chemical depilatory recorded with 40-
50% of efficacy may be due to quick uptake of the radionuclides through skin due to
removal of stratum corneum and the opening of hair follicles may be providing direct
entry into the systemic circulation. Direct uptake of the 99mTc into the systemic circulation
also proved by whole body scintigraphs recorded after 0.5 h of the contaminant
application is presented in Figure 4.6 and 4.7.
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Fig. 4.6: Application of chemical depilatory resulted in direct absorption of 99mTc through
skin. Decontamination efficacy reduces up to 45-60% for this group.
Fig. 4.7: Rat hair shaved applying chemical depilatory and allows contaminating with
99m
Tc. After 30 min of contamination, level of radioactivity and whole body scintigraphs
recorded. Decontamination was performed and residual activity measured. A:
Scintigraph after 30 min of contamination. B-D: Four consecutive scintigraphs with
decontamination attempts representing that radioactivity reaches throughout the body.
(iii) Consecutive decontamination attempts required
First 2-3 attempts were found most efficacious (80±5%) than the successive attempts that
was able to decontaminate 3-5% only. Rest 5-7% of the residual radioactivity left over
could not be removed by any means due the co-ordination binding of the contaminants
with skin constituents becomes impossible.
(iv) Contact time between contaminants and formulation
Before 2 min of the start the decontamination procedure, formulation was applied over the
contaminated areas for 0.5, 1, 2, 3, 4 and 5 min. Decontamination was performed and
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analysed that 2 min contact time was enough for maximum complexation between the
formulation APIs and the applied radio-contaminants.
(v) Decontamination procedure length
Decontamination attempts were performed within 0.5, 1, 2, 3 4 and 5 min and observed
that 1 min time period is enough to performed the decontamination.
(vi) Length of contaminant exposure
Decontamination must be done as soon as possible. Long time of contaminants deposition

over the body surface makes it difficult to remove. During study, decontamination was
started at different time intervals, e. g., 5, 15, 30, 45 and 60 min after contamination to
ensure the effectiveness of the formulation at maximum length of contaminants exposure.
Formulation was found highly efficacious for up to 1 hr even after that it was reduced up
to 5-7% only. 1 h was assuming the time period when contamination would be suspected
in case of accidental release of the radio-isotopes into the environment and
decontamination should be followed. Decontamination protocol is presented in figure 4.8.
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Fig. 4.8: Schematic representation of the decontamination efficacy protocol
4.6 Decontamination Efficacy of Disodium Edetate Lotion Formulation
Decontamination efficacy (DE) of the lotion formulation (with Disodium edetate / DTPA)
was compared with the water and placebo as well as between both the experimental
models (human tissue equivalent and Sprague Dawley). Lotion formulation was studied
for the decontamination efficacy according to the protocol standarised. Fig. 4.9 represents
99m
the efficacy of the lotion applied against Tc. Decontamination done after long time
period of contamination (up to 1 h) could effectively remove most of the contaminants
from both the experimental models.
Fig. 4.8: Decontamination efficacy of the disodium edetate lotion found to be 90±5%.
Efficacy data found statistically significant (p< 0.05).
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Fig. 4.9: Decontamination efficacy of the disodium edetate lotion against 99mTc after
applying chemical depilatory was found to be 45-65%. Efficacy data found statistically
non-significant (p> 0.05).
Decontamination of 99mTc after 0.5 h of contamination
Rat model
100
Tissue equivalent
90
Decontamination efficacy (%)
80
70
60
50
40
30
20
10
0
1 0.75 0.5 0.25 Placebo
Concentration of disodium edetate (%) in lotion formulation
Fig. 4.10: Decontamination efficacy of the lotion formulation found higher for rat than
the human tissue equivalent. Efficacy data found statistically significant (p< 0.05).
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Fig. 4.11: Effective of time over decontamination efficacy of the developed lotion within
1 h could remove most of the contaminants (p< 0.05).
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Fig. 4.12: DE of the disodium edetate lotion studied against 131I radiological contaminant.
A: Effect of time over the %efficacy of the formulation studied using quantitative
scintigraphy over both the experimental models. B: Effect of API concentration over the
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DE using the optimized formulation (0.25-1.0%). C: Number of consecutive

decontamination attempts required for study.
131 201
Lotion formulation was further investigated for the I and Tl on human tissue
equivalent and rat experimental models successfully. Figure 4.13 representing the DE of
131
the lotion against I at different time point (DE vs. length of exposure), efficacy for
different dosages (DE vs. concentration of Disodium edetate) and efficacy up to fifth
round of decontamination attempts (DE vs. number of decontamination attempts). Most of
131
the I removed from the skin (≥95%), decontaminated within 1 h of the contamination
occurred while over DE was reduced by ~5-7%.
99m 131 201
Likewise two other two radio-contaminants, e. g., Tc and I, Tl was also
decontaminated effectively from the surfaces of the rat as well as human tissue equivalent
models. Figure- 1(a-c) shows the decontamination of the 131I radio-iodine, 201Tl and 99mTc
with lotion (0.5% EDTA) at 0.5 h length of exposure was highly effective. Results clearly
indicate that the decontamination attempt within 0.5 h of contamination could easily
remove ~90% of the applied activity while over that it was reduced by 5-7% only.
Decontamination lotion was found more efficacious (85-90%) for the rat skin than the
human tissue equivalent model (80-82%). Radio-isotopes possess tendency to tightly
bind to the skin protein or may move towards the hair follicles reduces its efficacy up to
85-88% at 1 h study. Figure 4.7 demonstrates the quick reduction in percentage activity
removed with the first and second decontamination attempt.
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C
Fig. 4.13: Decontamination efficacy (DE) of the disodium edetate lotion against 201Tl. A:
Effect of length of contaminant exposure over DE. B: Effect of API concentration over the
DE. C: Number of consecutive decontamination attempts required
4.7 Decontamination efficacy of DTPA lotion
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99m
Tc DE of the lotion found to be 85 ±5% as F3 batch of the formulation with 1.0% of
the DTPA was the most effective when compared with water, placebo and F2 formulation
(0.5% DTPA) . It was also interesting to note that the less percentage difference of DE
between F2 and F3 batches formulations found. As shown in figure 2, the DE of the
201
lotion formulation against Tl was recorded to be 80-90% for both the experimental
99m
models. Figure 4 shows scintigraph of the whole body of rat recorded after 0.5 h of Tc
contamination (group IV). DE was recorded to be 65-75% may be due to fast uptake of
99m
the contaminant through skin. It should be noticed in Figure 4 (A-E) that Tc directly
absorbs into the systemic circulation through the skin and reaches to the nostril and tail.
This may be due to the fact that the chemical depilatory removes the stratum corneum
which is a limiting barrier and opening of the hair follicles results direct deposition of the
contaminants into themselves.
Fig. 4.14: DE of the DTPA lotion studied against 99mTc after different length of
contaminant exposure concluded efficacious (85±5%). Efficacy data found statistically
significant (p< 0.05).
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Fig. 4.15: DE of the DTPA lotion studied against 99mTc. A: Effect of AP concentration over
DE. B: Number of consecutive decontamination attempts. Efficacy data found
statistically significant (p< 0.05).
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Decontamination of Thallium after 0.5 h of contamination
100 Rat model

Tissue equivalent
Decontamination efficacy (%)
90
80
70
60
50
40
30
20
10
0
1 0.75 0.5 0.25 Placebo
Concentration of DTPA (%)
Fig. 4.16: DE of DTPA lotion studied against 201Tl. A: Number of consecutive

decontamination attempts required for maximum extent of contaminants removal. B:
Effect of the length of contaminant exposure over the DE. C: Effect of API concentration
over DE of the lotion applied. Efficacy data found statistically significant (p< 0.05).
I II
Fig. 4.17: Scintigraphs of the rat model after 30 min of contamination. I: Rat hair shaved
using scissor presents no uptake of the contaminant through percutaneous route. II:
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After application of chemical depilatory, direct absorption of radio-contaminant through

skin and systemic circulation in whole body.
4.8 Dermal Toxicity Studies
4.8.1 Primary skin irritation
All animals survived, gained weight and appeared active and healthy. There were no signs
of gross toxicity, adverse pharmacologic effects or abdominal behaviour. Skin patch
where formulation was applied was found to be normal, e. g., no erythema or edema. No
adverse health effects or deaths recorded during the study.
Table 4.8: Erythema and edema scoring method for skin reaction
Skin Reaction Score

(A) Erythema and Eschar formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema beet redness to eschar formation 4
(B) Edema formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well raised) 2
Moderate edema(raised approx. 1 mm) 3
Severe edema (raised more than 1 mm and extending 4
beyond area of exposure)
Table 4.9: Evaluation of reactions (Draize’s method) for Disodium edetate and
DTPA lotion Formulations
Rabbits number Average Combined Index
1M 2M 3M
24 Hrs.
Erythema Score 0 0 0 0.00 0.00
Edema Score 0 0 0 0.00 0.00
48 Hrs.
Edema Score 0 0 0 0.00 0.00
72 Hrs.
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Edema Score 0 0 0 0.00 0.00
Primary Irritation Index (PII)
All animals appeared clinically normal throughout the study. No irritation was observed
on the skin of the rabbits. The Maximum Irritation Response was not applicable. The
Primary Irritation Index of the test formulations was calculated to be 0.0. The irritation
calculations are shown above. Under the conditions of this study, no erythema and no
oedema were observed on the skin of the rabbits. The Primary Irritation Index for the test
article was calculated to be 0.0. The response of the test article was categorised as
negligible.
Table 4.10: Evaluation of Primary Skin Irritation Index (PII)
Evaluations Score
Non irritant 0.0
Negligible irritant 0.1-0.4
Slight irritant 0.41-1.9
Moderate irritant 2.0-4.9
Severe irritant 5.0-8.0
4.8.2 Sub-chronic toxicity study
1. Body weight: Body weights of all the experimental animals were increased in all
groups over the course of the study observed in sighting at both the dose levels as
well as in the control. In the later study, with administration of 5 mg/kg body
weight to 4 animals, weight increase post administration of was 15.41% between
1- 7 days, 13.39% between 7- 14 days and 12.32% between 14-21 days as
illustrated in Table 4.12.
2. Food consumption: During dosing and the post-dosing recovery period, the
quantity of food consumed by animals from different dose groups was found to be
comparable with that by control animals.
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3. Mortality: During 21 days period of study, no mortality was recorded. All animals
survived until scheduled euthanasia
4. Clinical Signs observations:
5. Clinical Signs observations including:
A careful cage side examination was made daily
Daily observations include changes in Skin, fur, eyes and mucous membrane
Convulsions, lethargy, sleep, coma, salivation, diarrhea and death of animals
6. Functional Observations: These tests conducted on the experimental animals at

termination and recorded and did not reveal any abnormalities.
7. Macroscopic examination of animals sacrificed at termination revealed no

abnormalities.
8. Urine analysis: Urine analysis data Table 8 of control group and treated group of
animals determined in week 1st, 2nd and 3rd week, did not reveal any significant
changes in the metabolite constituents.
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Table 4.11: Group means body weight
Sex- Male
Group Dose Day
No. 0 7 14 21 (Day of
termination)
I Control Mean 190 205 215 225
± SD 5.24 3.15 4.321 4.13
II OD Mean 191 206 217 223
± SD 5.44 4.15 4.321 2.23
III ODX 5x Mean 191.5 208 216 221
± SD 4.13 4.15 6.321 3.33
IV ODX10x Mean 191 207 215 220
± SD 3.13 4.15 5.321 4.05
Sex- Female
I Control Mean 135 140 145 151
± SD 3.673 4.321 5.877 4.23
II OD Mean 137 142 146 153
± SD 4.23 4.321 5.21 4.6
III ODX 5x Mean 136 141 147 155
± SD 4.23 4.321 5.21 5.3
IV ODX Mean 137 142 147 154
10x ± SD 3.23 3.321 4.21 5.76
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Table 4.12: Individual cage side observation
Groups No. of Finding Days

Animals
Group I Vehicle control (VC) 12 (6M + 6F) Active and healthy 0-21
Group II Optimised dose (OD) 12 (6M + 6F) Active and healthy 0-21
Average dose (OD x 5) 12 (6M + 6F) Active and healthy 0-21
Highest dose (OD x 10) 12 (6M + 6F) Active and healthy 0-21
Table 4.13: Individual necropsy observation
Groups No. of Animals Tissues Findings

Group I Vehicle control 12 (6M + 6F) Skin No gross bnormalities
(VC)
Group II Optimised 12 (6M + 6F) Skin No gross abnormalities
dose (OD)
Medium dose (OD 5X) 12 (6M + 6F) Skin No gross abnormalities
Highest dose (OD 10X) 12 (6M + 6F) Skin No gross abnormalities
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Table 4.14: Functional observation
Time
Stimulation (CNS) 30 min 1h 2h 6h 24h 48h
Hyperactivity - - - - - -
Piloerection - - - - - -
Twitching - - - - - -
A
Rigidity - - - - - -
Irritability - - - - - -
Jumping Colonic - - - - - -
Convulsions - - - - - -
Tonic convulsion - - - - - -
Ptosis - - - - - -
Depression (CNS)
Sedation - - - - - -
Loss of Pinna - - - - - -
B
Reflux Catatonia - - - - - -
Ataxia Loss of - - - - - -
Muscle Tone - - - - - -
Analgesics - - - - - -
Time
Stimulation (CNS)
30 min 1h 2h 6h 24h 48h
Straub Trail - - - - - -
Laboured Resp - - - - - -
C
Cyanosis - - - - - -
Blanching - - - - - -
Reddening - - - - - -
Remarks: - = Normal, + Mild or moderate effect, ++= Marked effects
Interpretation: None of the rats showed any side effect and mortality as shown above.
All were normal.
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Table 4.15: Post-mortem gross examination of six each of the vital organs
Group Dose Sex Organs

Peritonea Plural Liver Kidney Heart Lungs Spleen
l Cavity Cavity
I Contro M Normal Normal Normal Normal Normal Normal Normal

l F Normal Normal Normal Normal Normal Normal Normal
II Test M Normal Normal Normal Normal Normal Normal Normal
F Normal Normal Normal Normal Normal Normal Normal
Haematology and Blood Chemistry
No signs of toxicity and no deaths were observed. The study includes control and
treatment group, each consisting of male and female rats. The multiple dermal dose of the
lotion/gel did not produce mortality or significant changes in the body weight, food and
water consumption. The relative weights of the internal organs were normal. However,
hemoglobin (HGB), hematocrit (HCT), red blood cell (RBC) and total protein were
within the normal range. Enzymes SGOT and SGPT biochemistry parameters
demonstrated no significant changes as compared to the control. Total protein, creatinine
and glucose level were also within the normal range recorded.
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Table 4.16: Haematology±SD (Male)
S. No. Test Saline Placebo Optimised

dose
1 WBC (m/mm3) 10.5±1.3 11.3±0.2 10.5±0.26
2 Lymphocytes 76±2.5 77±3.1 78±2.3
3 Monocytes 11±0.5 10±0.58 11±2.0
4 Neutrophil 12±0.4 12.6±0.8 13 ±0.9
5 Eosinophils 5±1.5 5±0.9 5±1
6 Basophils 0.2±0.1 0.2 ±0.1 0.2 ±0.1
7 RBC ( m/mm3) 9±1 9.5 ±1 9 ±1
8 MCV 50±2 51 ±2 49±2
9 HCT 31.6 32.6 30.6
10 MCH 23±0.2 21±3 23±1.5
11 MCHC (g/dl) 40±2 40±2 40±2
12 RDW 10±1 10±1 10±1
13 Hb (g/dl) 15±1.2 15±0.5 15±1.0
14 THR ( m/mm3) 700 ±100 700 ±100 700 ±100
15 MPV 8 ±1 8 ±1 8 ±1
16 Pct 0.28 ±0.1 0.28 ±0.1 0.28 ±0.1
17 PDW 13 ±2 13 ±2 13 ±2
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Table 4. 17: Haematology±SD (female group)
S. No. Test Optimised dose OD X5X OD 10X

(OD)
1 WBC 9±3 9±4 8.8 ±3
2 Lymphocytes 68±2 65±3 69±4
3 Monocytes 4±1 4±1.5 4±2
4 Neutrophil 18±5 18±4 18±5
5 Eosinophils 5±1.2 6±2 5.5±1.5
6 Basophils 8±0.5 7.5±0.5 9±1.5
7 RBC 7.20 ±0.6 7.30 ±0.8 7.50 ±0.9
8 MCV 53±0.4 54 ±0.4 55 ±0.4
9 HCT 38 ±0.2 40±0.2 39 ±0.2
10 MCH 22 ±0.4 20±0.4 22 ±0.4
11 MCHC 35 ±0.2 30 ±0.2 33 ±0.2
12 RDW 10 ±0.6 10 ±0.6 10 ±0.6
13 Hb 14 ±0.8 13 ±0.8 14 ±0.6
14 THR 700 ±100 700 ±100 700 ±100
15 MPV 8±0.5 8±0.41 8±1.0
16 Pct 0.12 0.12 0.12
17 PDW 13±0.2 15±3 14±2
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Table 4.18: Urine qualitative test (Ames multiple sticks)
S. No. Test Male Female

1 BLD --- ---
2 BIL --- ---
3 URO 0.1 mg/dl 1 mg/dl
4 Ketones 5 mg/dl 5 mg/dl
5 Protein --- ---
6 Nitrites --- ---
7 Glucose --- ---
8 pH 7 7.0
9 Specific 1.020 1.020
gravity (SG)
10 LEU --- ---
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Table 4.19: Group mean urine analysis of male and female (Sex-Male and Female at the Day- 15th and 21st)
Group Dose Volume BLD BIL URO Ketones Protein Nitrites GLU pH SG LEU
No (mg/ml)
(mg/dl) (mg/dl)
Mean 6 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0
I Control ± SD 0.32 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0
II OD Mean 6.5 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0
± SD 0.68 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0
III OD 5X Mean 6.9 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0
± SD 0.97 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0
IV OD 10X Mean 7.0 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0
± SD 1.034 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0
Qualitative
Absent =0
Trace = +
Small amount of analyte = ++
Moderate amount of analyte = +++
Large amount of analyte = ++++
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ECG intervals
RR intervals
No changes of RR intervals were observed and heart rates were normal in treated group at
baseline as compared to the control group. Therefore, normal of PR waves on indicates the
no possibilities of tachycardia caused by formulation.
Decreased Heart rate variability (HRV)
Heart rate variability (HRV) was not changed in treated group as compared to control.
Further, no changes of HRV were observed throughout the experiments.
QRS intervals
No changing of QRS intervals of treatment group was observed therefore no abnormal intra-
ventricular conduction is predicted.
QT and QTc intervals
QT intervals were normal as compared to control rats which indicate that no ventricular
repolarization caused by treated formulation. Treated animals showed no significant
widening of QTc intervals as compared to control rats.
Histopathological studies
Histopathological studies were carried out for elucidation of the effect of the formulation on
cellular structure, hair growth, epidermal keratinocytes, granulocytes, and melancytes of the
skin. Thickness of the epidermis was found to be comparable with the treated animals and
intact with the dermis. The sweat duct is clearly ending at its coiled secretory portion.
Cornified (keratinized) stratified squamous epithelium makes up the epidermis. The stratum
granulosum is very dark; the stratum lucidum is bright red. The stratum corneum is thick, and
very pale. Stratum spinosum showing ‘prickle’ appearance of cell contacts and the cross-lines
were once thought to be intercellular bridges. Melanin pigment is produced by stellate shaped
melanocytes of the dermal layer and then deposited in the basal cells of the epidermis. The
darker circles in the lower part are ducts; the lighter cross-cuts above are the secretory
portions.
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Fig. 4.18: Histology of skin was found comparable with the control group
4.8.3 Dermal toxicity biomarkers:
Dermal toxicity biomarkers studied as a model of the radiation burn and their responses over
level of expression when applied. Levels of expression of both the proteins were found non-
significant and comparable to the control group. On the basis of the results formulations were
concluded safe to use during radiation emergency scenarions.
Fig. 4.19: Level of expression of the Col 3A1 and VEGF were found non-significant (p>0.05)
when compared with the controlled untreated group animals. C- Control, E- Exposed with
heat shock, E + E- Exposed and Formulation.
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4.9 Discussion
Accidental cutaneous contamination by radio-nuclides may occur due to accidental

confinement disruption, mechanical damage or gloves defect. Like materials, humans may
also be contaminated with radiological contamination. This may result from a radiation
dispersal device or a nuclear reactor accident. Alpha and beta particles have only a very
limited range of penetration while beta particles are capable of causing burns if in direct
contact with skin (Schulte 1966; Moduler 1971; Moore and Mettler, 1980; Czeizel et al.,
1991). The current use of radio-nuclides decontaminating products is based on empiricism.
There are few scientifically valid data certifying the effectiveness of radio-nuclides
decontaminating products. The current procedure neglect decontamination of the hair and
eyes; for example, protein shampoos should be avoided because they promote binding of the
contaminants to hair (TMT 2009). In addition, most of the documents mention the efficiency
of undressing following contamination but rarely the efficiency of the wash itself. Multiple
contamination cases are very complex. The priority is to target and decontaminate the
element that is most radiotoxic (β emitter) with specific products. Fixed contaminants are
also difficult to remove; for example, cesium is fixed quickly and is very difficult to remove
by decontamination treatments. In such a case with no decontamination solution, the person
will remain contaminated until natural desquamation and skin renewal, which takes around
21 d. In addition, the effect of decontaminating products is sometimes insufficient for radio-
nuclides that spread easily and quickly through the skin, such as tritium, iodine, cesium
(WHO-IAEA 2002) and technetium (Bolzinger et al., 2010).
The delay of treatment constitutes an overriding issue. The faster the decontamination is
done, the more effective it will be, because the contaminant still remains on the skin surface.
Yet, in many cases, due to lack of organisation and capacity, it is not performed thoroughly.
The problem of the toxicity of some treatments is also of concern, especially in case of a
mass contamination. It should be noted that many of these decontaminants have irritant
properties or are toxic internally. In this last part, prospects and ways of improving
decontamination approaches will be detailed to include recent techniques and products in this
field. Disodium etidrnate (EHDP), marketed under the name of Didronel®, is a drug used in
the treatment of bone resorption in Paget’s disease. It turns out that EHDP may have an
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application in radio-toxicology as a potential decontamination of uranium. These effects have

been demonstrated y ex vivo tests using human skin explants. It would therefore be necessary
to perform additional studies of safety to allow an extension of the marketing authorisation.
A new molecule, patented by French Institute for Radiological Protection and Nuclear Safety
(IRSN) in 2009, has recently been developed: a calixarene nanoemulsion used in oil/water as
a chelator of uranium. The use of ex vivo pig ears, intact or injured, showed a decrease of
98% in the incorporation of uranium (Spagnul et al., 2010a) and an extraction of 80% from a
contaminated aqueous solution (Spagnul et al., 2010b). Nevertheless, calixarenes present a
potential hepatic and blood toxicity (Spagnul 2009), limiting their indications to healthy skin
and careful application without abrasion.
The products that do not require rinsing constitute an important research approach in case of
emergency situations with limited access to water. The use of flour or Fuller’s earth is
common and recommended by U.S. and European organizations for Chemical, Radiological
and Nuclear (CRN) skin decontamination. On the same principle, it would be interesting to
extend the range of products such as Ca-DTPA, in powder form, which could have both soft
abrasive and binding effect. It would also be interesting to develop gel forms, currently used
for the decontamination of surfaces in Fukushima. Peel-off formulation would also be a way
to develop research as iit consists of applying the product on the skin and after a period of
drying, the gel is removed by peeling. However, it would be essential to assess the risk-
benefit formula for this kind of treatment involving removal of a part of the stratum corneum,
which is protective; as such a procedure can promote the percutaneous passage and the
accumulation of toxic compounds.
Radiological decontamination is performed in an identical manner to doctrinal chemical

decontamination. Water has been used as universal decontamination agent that is followed by
use of liquid soap and water bath. Chemical decontamination agents are based on the
oxidation and reduction reactions that make contaminants soluble and easily removable from
the body. Chelating agents form complexes with radioisotopes to facilitate removal. Various
protocols and kits for skin decontamination have been proposed for the removal of the
radioactive contamination from skin. A number of chemical decontaminants have been
advocated (EPR- Medical 2005, AERB Manual No. SM/MED-2). The ‘SHUDHIKA’ skin
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decontamination kit contains a number of recommended chemical decontamination agents

and needed medical supplies (surgical mask, eye pads, water proof bandages, cotton swabs,
nasal catheters, potassium iodide tablets, and nail cutter). Water decontamination along with
liquid soap leaves trace residues of radiation contaminants that are difficult and sometimes
impossible to remove. This by itself is not fully effective in removing traces of leftover
contaminants (Breitenstein 1976, Farina et al., 1991, IAEA TECDOC-1009; Rana et al.,
2012).
Various experimental studies on mice, rats or pigs have examined skin permeability (Hibbard
and Watters, 1985; Khodyreva et al., 1976, Suzuki- Yasumoto and Inaba, 1976), absorption
kinetics (Osanov et al., 1971; Kusama et al., 1986; Peng et al., 1988; Lefaix et al., 1996) or,
more rarely, treatment evaluation (Gerasimo et al., 1997, Stojanovic and Milivojevic 1966) to
investigate the minimal time period required for the decontamination on the basis of
permeability profiles of different radio-nuclides (Bartek et al., 1972). For obvious ethical
reasons, studies on human beings have been few (Ilyin et al., 1975) and have been concluded
for the as earliest possible decontamination.
Chemical decontamination agents are based on the oxidation and reduction reactions that
make contaminants soluble and easily removable from the body. Various protocols and
formulations for skin decontamination have been proposed for the removal of the radioactive
contamination from skin. Intact skin layer is a barrier to for radio-nuclides penetration.
Therefore scrubbing is best avoided during decontamination process to avoid damage to skin
(Moore and Mettler 1980; Felton 1960; Harrison 1963, 1972). Decontamination studies have
shown that skin moistening agents could reduce skin abrasion that to some extent may result
in increased radioactive permeability. Other skin decontamination agents such as kaolin
paste, titanium dioxide paste, potassium permanganate and hydrochloric acid have studied for
decontamination efficacy but none of these to be highly effective or ideal. These agents also
were reported to cause skin roughening, which results in more absorption of the contaminant,
99m
making it difficult to remove. Decontamination of the Tc with saturated solution of
potassium permanganate was concluded that it is effective for long lived alpha emitters rather
than gamma emitting radio-isotopes (Schofield 1971; Merrick et al., 1982). The relative
99m 131
decontamination efficiency of the Tc radiopharmaceuticals, e. g., I (as iodide and
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67 111
orthoiodohippurate), Ga (as gallium citrate) and In (as indium DTPA) with water,
Radicawash and isoclean found that removal of technetium is most difficult with residual of
7% for pertechnetate, 5% for the iodide and 1% or less for others left after 90 second
decontamination attempts (Harrison 1963; Moore and Mettlar, 1980).
The main difference is in timing. In case of humans, if both types of contaminations are
found, then chemical decontamination is the priority, as it is an emergency. Several types of
physical (dry) and chemical (wet) methods are using for decontaminating personnel. The
technique involves washing from the outside toward centre, and it may have to be repeated
many times to decrease the count after every wash. If this simple procedure is unsuccessful,
light abrasives such as cosmetic puff pads or a mild abrasive paste (ground corn) should be
employed. Another method used is known as the ‘sweat technique’. Place a gauze pad over
the contaminated area, cover the site with tape is down and left in position for 4 to 6 hours
and remove this dressing and wash the area again. The final method is using a gauze dressing
or a cotton glove and changing the dressing daily (Waselenko et al., 2004). The basis of this
procedure is that 13% of epithelial skin is shed daily. A significant advantage of most
physical methods is their nonspecificity. Since they work nearly equally well on chemical
agents regardless of chemical structure, knowledge of the specific contaminating agent or
agents are not required. Potassium permanganate was used as a stripping agent for the outer
layer of skin, but this is no longer utilised because of the strong oxidising character of this
agent. Decontamination of causality is an enormous task. The process requires dedication of
both large numbers of personnel and large amounts of time. Even with appropriate planning
and training, the requirement demands a significant contribution of resources. Removal of
outer clothing and rapid washing of exposed skin and hair removes 95% of contamination.
The 0.5% hypochlorite solution used for chemicals will also remove radiological
contaminants. Care must be taken to not irritate the skin. If the skin becomes erythematous,
some radionuclides can be absorbed directly through the skin. Surgical irrigation solutions
should be used in liberal amounts in wounds, the abdomen, and the chest. All such solutions
should be removed by suction instead of sponging and wiping. Only copious amounts of
water, normal saline, or eye solution are recommended for the eye (Reeves 1999; Goans and
Waselenko, 2005; Koenig and Goans 2005; Dainiak and Ricks, 2005).
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Use of dressings or hydrogels could also effectively remove the external contaminants. In
addition to this, chelating agents are prominent category of the decontamination agent
because they could effectively chelate, form stable complexes and remove them from body.
Diluted bleach has also been used, but it is not suitable for use on the face. Decontamination
procedure should be easy and fast to remove the deposited contaminants easily without
eliciting dermal abrasion or irritancy (Mettler 2005; De Lorenzo 2005; Ciottone et al., 2006).
To limit cutaneous systemic exposure with present contaminants, it is prudent that skin
decontamination must be conducted without eliciting abrasion or dermatitis that may enhance
absorption. Potential formulation ingredients sometimes have effects on skin barrier integrity
such as delipidation, membrane fluidisation, or skin irritation ultimately resulting in increase
of uptake. Radionuclides present over skin could penetrate barrier in the form of ion and
preferentially bind to micelles, proteins and membrane to limit their removal from body
(Harper et al., 2007; Goans and Waselenko, 2005; Wolbarst et al., 2010). Cation such as
201
Tl+ ion and 99m
Tc4+ are hydrophilic in nature and do not penetrate skin immediately after
contamination but long time of their deposition may facilitate. Conversely I- anion is less
hydrated and could interact with apolar skin protein constituents making these molecules
more soluble in aqueous systems, destabilise the lipid bilayer structure of cell membranes and
facilitate more absorption through skin. Decontamination of these radio-nuclides must be
done as early as possible to reduce higher internal exposure to the targeted as well as
surrounding tissues (Bolzinger 2010).
This research and analysis of generic protocols have highlighted the need for specific studies
on decontaminating formulations, as well as the need for data in the field of cutaneous
transfer of radionuclides in the case of an intact skin. Current experimental work presents
advancement over the existing decontamination systems. Topical lotion formulation
containing disodium edetate/DTPA proposes chelation reaction mechanism for the removal
of the radio-metal ions presents over the body surfaces. The rates of formation and
dissociation of displacement reactions are important in the practical applications of chelation.
The stability constants of the multidentate complexes usually are from one to several orders
of magnitude greater than those of monodentate complexes. The greater stability of chelates
is largely the result of an increase in entropy resulting from an increase in number of free
molecules, usually solvent or other monodentate ligand, liberated as the chelate is formed.
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This extra stabilization produced by the ring formation is called the chelate effect. Many
parameters influence the stability of chelates. Several of the stability factors common to all
chelate systems are the size and number of rings, substituents on the rings, and the nature of
the metal and donor atoms (Meineke et al., 2003; Musolino and Harper, Flynn and Goans,
2006).
Purified water in the formulation was used as vehicle, in which all pharmaceutical ingredients
of the formulation dispersed. This is the liquid base that carry drugs (disodium edetate and
DTPA) and other excipients in dissolved or dispersed state. Propylene glycol is an
outstanding solvent for many organic compounds. It is colourless and odourless and has a
very slight characteristic taste which is not objectionable. Propylene glycol is a general
solvent and antimicrobial preservative used in a wide range of pharmaceutical preparations
including oral liquid, topical and parenteral preparations.
The toxicity of propylene glycol is quite less in comparison to many other co-solvents
generally used. It is using as solvent and coupling agent in the formulations of lotion,
shampoos, creams and other similar products. At lower concentrations, it acts as an emulsifier
in cosmetics and pharmaceutical creams, very effective humectants, preservative and
stabiliser. Microbiological contamination presents a significant health hazard in formulations.
Therefore, the use of preservatives become inevitable to prevent the growth of
microorganisms during the formulation preparation and shelf life, although it may be most
desirable to develop a ‘preservative-free’ formulation to address the increasing concerns
about the biological activity of these compounds. Most formulations require some kind of
preservative to ensure no microbial growth. The majority of preservatives are bacteriostatic
rather than bacteriocidal, and consists of both acid and non-acid types. Preservatives often
contain reactive functional groups, which are responsible for their antimicrobial activity but
lead to unwanted reactions. Therefore, in addition to the excipient’s antimicrobial activity,
other parameters evaluated during the formulation development for its compatibility with the
API, other excipients, and the container system. Parabens (methyl and propyl) generally used
in combination of methyl and propyl paraben at 0.015% to 0.2% concentrations of (w/v). Its
pH range is 2 to 6 (unstable at 8 or above in of solution). Incompatible with sorbitol, show
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some discoloration in the presence of iron. Parabens have some antimicrobial activity but are
most effective against yeasts and molds. Methyl paraben has the least antimicrobial activity,
used with other long-chain parabens. As the chain length of the paraben’s alkyl moiety is
increased, their antimicrobial activity increases.
Sodium pertechnetate metal oxide (TcO4-) bonds are disrupted by attacking either the metal
ion or the surface oxide group. Redox-based acid decontamination agents (oxalic acid,
sodium bisulphate, hydrochloric acid) break the oxide lattice by proton (H+) attack to form
surface hydroxyl (OH-) entities, allowing a hydrated metal ion to solubilise metals. These
chemical reactions allow more effective removal of contaminates from the body.
Hydrochloric acid solution acts as a strippable coating agent that makes a film, which is then
stripped off, carrying with it contamination. Use of concentrated potassium permanganate
and sodium bicarbonate solution oxidizes pertechnetates to a soluble form which loosen
adhered contamination, resulting in leaching and simple removal.
Decontamination efficacy of the optimised decontamination lotion was compared with water
and placebo (without decontaminating agent) for all the radio-nuclides studied. They were
decontaminated in order to calculate the efficacy with respect to the length of exposure. The
results obtained were summated for each combination of contaminant and lotion, and the
mean count remaining after each decontamination attempt was calculated. The striking result
is that the first two decontamination attempts were able to decontaminate 85±5% of the
applied activity over 0.5h while <5% of the applied activity was removed in the successive
five attempts. Chelating agents forms complexes with the radio-isotopes and facilitate to
remove the contaminant. It shows that lotion could effectively chelate the radio-iodine and
also could prevent entry into systemic circulation through cutaneous absorption.
Decontamination delayed long after radio-nuclide exposure was potentially effective to
remove the radio-iodine from skin surface. Decontamination performed within 0.5 h after
contamination could easily remove ~90% of the applied activity while over that it was
reduced by 5-7% only. Decontamination lotion was found more efficacious (85-90%) for the
rat skin than the human tissue equivalent model (80-82%) may be due to the strong binding
of the contaminant with the polymer surface of the human tissue equivalent. Radio-isotopes
possess tendency to tightly bind to the skin protein or may move towards the hair follicles
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reduces its efficacy up to 85-88% at 1 h study. Quick reduction in percentage activity

removed in the first and second decontamination attempts were observed. This is presumably
due mainly to the chelation reaction between loosely bound contaminant and the active
ingredient of the lotion. Chelating agents form complexes with radio-isotopes to facilitate
removal. These common factors in the entire first and second attempt were apparent to a
much smaller degree in successive attempts. Thus it is in the third, fourth and fifth attempts
that the truly comparative values of the decontamination attempts were assessed (Nishiyama
et al., 1980; Rana et al., 2012, 2012).
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DESIGNING AND DEVELOPMENT OF TOPICAL CHAPTER 4

LOTION FORMULATION: PHARMACEUTICAL

 Development of the topical lotion formulation at different concentrations of

Table 4.1: Optimised formula of disodium edetate topical lotion formulation

Unit formula Qty for batch

4.3.2 Fitting model to the data

Independent factors Dependent factors

4.3.3 Regression analysis of the model

Quadratic R2 Adjusted R2 Predicted R2 SD % CV

Response 1 (Y1): Effect on viscosity of Lotion

Standard order Actual value Predicted value Residual

Fig. 4.2: 3D response surface plot sh

Response 2 (Y2): Effect on spreadability of Lotion

Stand Actual value Predicted value Residuals

Response 3 (Y3): Effect on extrudability of Lotion

(disodium edetate concentration). It was observed that higher concentration of the

Stand Order Actual value Predicted value Residuals

4.3.4 Data analysis of the formulation developed

4.4 Characteristics of Pharmaceutical Parameters of the Lotion Formulation

pH values of the topical lotion (disodium edetate / DTPA) ranged 7.3±0.2.

(ii) Visual observation

i. If transparent lotion was obtained, it was recorded as clear transparent lotion

Spreadability of the formulation was recorded to be 13.4±0.2 cm.

(vii) Drug content

96-98% of drug content recorded after 3, 6 and 9 months of the study.

4.5 Decontamination Protocol Standarisation

(i) Animal model vs. human tissue equivalent

(ii) Mode of hair clipping: Scissors vs. Chemical depilatory

(iii) Consecutive decontamination attempts required

(iv) Contact time between contaminants and formulation

(v) Decontamination procedure length

(vi) Length of contaminant exposure

Decontamination must be done as soon as possible. Long time of contaminants deposition

Fig. 4.8: Schematic representation of the decontamination efficacy protocol

4.6 Decontamination Efficacy of Disodium Edetate Lotion Formulation

Decontamination of 99mTc after 0.5 h of contamination

DE using the optimized formulation (0.25-1.0%). C: Number of consecutive

Decontamination of Thallium after 0.5 h of contamination

100 Rat model

Fig. 4.16: DE of DTPA lotion studied against 201Tl. A: Number of consecutive

After application of chemical depilatory, direct absorption of radio-contaminant through

4.8 Dermal Toxicity Studies

4.8.1 Primary skin irritation

Skin Reaction Score

Rabbits number Average Combined Index

Erythema Score 0 0 0 0.00 0.00

Primary Irritation Index (PII)

Table 4.10: Evaluation of Primary Skin Irritation Index (PII)

4.8.2 Sub-chronic toxicity study

4. Clinical Signs observations:

5. Clinical Signs observations including:

A careful cage side examination was made daily

Convulsions, lethargy, sleep, coma, salivation, diarrhea and death of animals

6. Functional Observations: These tests conducted on the experimental animals at

7. Macroscopic examination of animals sacrificed at termination revealed no

Table 4.11: Group means body weight

Table 4.12: Individual cage side observation

Groups No. of Finding Days

Table 4.13: Individual necropsy observation

Groups No. of Animals Tissues Findings

Table 4.14: Functional observation

Group Dose Sex Organs

I Contro M Normal Normal Normal Normal Normal Normal Normal