designing and development of topical lotion...

DESIGNING AND DEVELOPMENT OF TOPICAL

LOTION FORMULATION: PHARMACEUTICAL

CHARACTERISATION AND EVALUATION FOR

DECONTAMINATION EFFICACY AND DERMAL

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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

rat (male and female) were chosen as experimental models. 99m

Tc, 131

I and 201

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

S. No Ingredients Category Unit formula

(per 10 ml)

Qty for batch

(per 100 ml)

1 Disodium EDTA /

DTPA API 50mg/100mg 0.5 g/1g

2 Propylene glycol Humectant 0.5ml 5 ml

3 Sodium Carboxy

methyl cellulose

Viscosity

increasing

agent

100mg 1g

4 Methyl paraben

sodium Preservative 2mg 0.02g

5 Propyl paraben

sodium Preservative 0.2mg 0.002g

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

cellulose (%)

3 1 1 5 4 2 1

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

100

Qs to

100

Qs to

100

Qs to

100

Qs to

100

Qs to

100

Qs to

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

Std

Run

Independent factors Dependent factors

Factor 1

A:Disodium

edetate / DTPA

Factor 2

B:Sodiu

m CMC

Response 1

Viscosity

(Y1)

Response 2

Spreadabilit

y (Y2)

Response 3

Extrudabilit

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

model

R2

Adjusted R2

Predicted R2

SD % CV

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 X2

2

Y3 = + 0.48 – 0.168 X1 – 0.088 X2 + 4.00 – 0.03 X1 X2 + 0.024 X12 + 0.012 X2

2





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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

order

Actual value Predicted value Residuals

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

used medical radioisotopes. Protocol was standardised using 99m

Tc on human tissue

equivalent model followed by extensive studies with the 131

I and 201

Tl 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 99m

Tc 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 99mTc. 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

the efficacy of the lotion applied against 99m

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).

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).

0

10

20

30

40

50

60

70

80

90

100

1 0.75 0.5 0.25 Placebo

Deco

nta

min

ati

on

eff

ica

cy (%

)

Concentration of disodium edetate (%) in lotion formulation

Decontamination of 99mTc after 0.5 h of contamination

Rat model

Tissue equivalent





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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).

A





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B

C 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.

Lotion formulation was further investigated for the 131

I and 201

Tl on human tissue

equivalent and rat experimental models successfully. Figure 4.13 representing the DE of

the lotion against 131

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

the 131

I removed from the skin (≥95%), decontaminated within 1 h of the contamination

occurred while over DE was reduced by ~5-7%.

Likewise two other two radio-contaminants, e. g., 99m

Tc and 131

I, 201

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 131

I radio-iodine, 201

Tl and 99m

Tc

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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A

B

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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99mTc 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

lotion formulation against 201

Tl was recorded to be 80-90% for both the experimental

models. Figure 4 shows scintigraph of the whole body of rat recorded after 0.5 h of 99m

Tc

contamination (group IV). DE was recorded to be 65-75% may be due to fast uptake of

the contaminant through skin. It should be noticed in Figure 4 (A-E) that 99m

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).

A





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B

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:

0

10

20

30

40

50

60

70

80

90

100

1 0.75 0.5 0.25 Placebo

Deco

nta

min

ati

on

eff

ica

cy (%

)

Concentration of DTPA (%)

Decontamination of Thallium after 0.5 h of contamination

Rat model

Tissue equivalent





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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

beyond area of exposure)

4

Table 4.9: Evaluation of reactions (Draize’s method) for Disodium edetate and

DTPA lotion Formulations

Rabbits number

Average Combined Index

1 M 2 M 3 M

24 Hrs.

Erythema Score

Edema Score

0

0

0

0

0

0

0.00

0.00

0.00

0.00

48 Hrs.

Erythema Score

Edema Score

0

0

0

0

0

0

0.00

0.00

0.00

0.00

72 Hrs.





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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.

Erythema Score

Edema Score

0

0

0

0

0

0

0.00

0.00

0.00

0.00





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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, 2

nd and 3

rd 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

No.

Dose Day

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

10x

Mean 137 142 147 154

± SD 3.23 3.321 4.21 5.76





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Table 4.12: Individual cage side observation

Groups No. of

Animals

Finding Days

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

(VC)

12 (6M + 6F) Skin No gross bnormalities

Group II Optimised

dose (OD)

12 (6M + 6F) Skin No gross abnormalities

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 1 h

2h 6h

24h

48h

A

Hyperactivity - - - - - -

Piloerection - - - - - -

Twitching - - - - - -

Rigidity - - - - - -

Irritability - - - - - -

Jumping Colonic - - - - - -

Convulsions - - - - - -

Tonic convulsion - - - - - -

Ptosis - - - - - -

Depression (CNS)

B

Sedation - - - - - -

Loss of Pinna - - - - - -

Reflux Catatonia - - - - - -

Ataxia Loss of - - - - - -

Muscle Tone - - - - - -

Analgesics - - - - - -

Time

Stimulation (CNS)

30 min

1 h

2h

6h

24h

48h

C

Straub Trail - - - - - -

Laboured Resp - - - - - -

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

l Cavity

Plural

Cavity

Liver Kidney Heart Lungs Spleen

I Contro

l

M Normal Normal Normal Normal Normal Normal Normal

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)

OD X5X OD 10X

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

gravity (SG)

1.020 1.020

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)

Qualitative

Absent = 0

Trace = +

Small amount of analyte = ++

Moderate amount of analyte = +++

Large amount of analyte = ++++

Group

No

Dose

(mg/ml)

Volume BLD BIL URO

(mg/dl)

Ketones

(mg/dl)

Protein Nitrites GLU pH SG LEU

I

Control

Mean 6 0.0 0.0 0.1 5.0 0.0 0.0 0.0 7.0 1.020 0.0

± 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





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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,

making it difficult to remove. Decontamination of the 99m

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

decontamination efficiency of the 99m

Tc radiopharmaceuticals, e. g., 131

I (as iodide and





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orthoiodohippurate), 67

Ga (as gallium citrate) and 111

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

201Tl

+ ion and

99mTc

4+ 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).

designing and development of topical lotion...

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