The Impact of Melamine Spiking on the Gel Strength and Viscosity of Gelatin
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- Lenard Lyons
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1 The Impact of Melamine Spiking on the and of atin Introduction The primary purpose of this research was to assess the impact of melamine spiking on the gel strength and viscosity of gelatin. A secondary purpose of this study was to demonstrate whether or not observable physical characteristics of dry gelatin and gelatin in liquid suspension were impacted by the presence of melamine in the mix. To this end, the study assessed the effects of melamine spiking at 0, 5, 10 and 16% levels. With respect to the primary purpose, the null hypothesis to be tested statistically is H 0 : β 1 0, where β 1 is the change in the visco-elastic properties (gel strength and viscosity) of gelatin per unit increase of melamine. Therefore, the alternative hypothesis to be tested, represented by H 1 : β 1 < 0, is that the addition of melamine diminishes these visco-elastic properties. The secondary purpose of the study will be assessed via several exhibits of photographs of the spiked gelatin in the dry state and the liquid suspension state taken in the laboratories where the experiments were conducted. The protocol for the study design, including the statistical analysis, has been previously reviewed and commented upon by the FDA. A copy of that approved protocol is attached and thus the design and testing procedures of the study are not being represented here. Standardized samples of the three pharmaceutical gelatin types derived from bovine bone, bovine hide, and porcine skin were prepared with each sample being split into three equal portions and distributed to the three testing laboratories involved in the study, i.e., Nitta in Japan, ita in Germany and Rousselot in the United States. All testing was conducted in March/April Collected raw data and photos were sent to GMIA which compiled the data and analyzed these data for statistical significance. Experimental Design Summary Table 1 indicates the experimental design, listing the three types of gelatin, the three participating laboratories, and the melamine spiking levels. For each of three laboratories, three gel types were tested. For all nine combinations of lab and gel type, four melamine levels were tested, for a total of 3 x 3 x 4 = 36 tests. Each of these 36 tests was replicated. At melamine level zero, each test was performed 5 times in order to establish a repeatability measure. The other three melamine levels were tested 3 times. This resulted in a total of 9 x14 = 126 tests. Table 1. Experimental Design Three Laboratories Three Types ita Nitta Rousselot For 9 lab/gel pairs, four melamine levels 0 % 5 % 10 % 16 % Porcine Bovine bone Bovine hide 1
2 Results and Discussion of Data Data Box plots provide an initial view of the data, and are presented below. Tabulation of raw data by gel type can be observed in Appendix Tables 1-3. Figure 1. Box Plots of vs. Melamine, by Laboratory and Type Type=Porcine Box Plot of grouped by Mel-Pct; categorized by Lab Exclude condition: NOT( "-Type" = 101 ) Type=Bov-Bone Box Plot of grouped by Mel-Pct; categorized by Lab Exclude condition: NOT( "-Type" = 102 ) Type=Bov-Hide Box Plot of grouped by Mel-Pct; categorized by Lab Exclude condition: NOT( "-Type" = 103 ) Lab: GELITA Lab: Nitta Lab: Rousselot Mel-Pct Median 25%-75% Non-Outlier Range Outliers Extremes Lab: GELITA Lab: Nitta Lab: Rousselot Mel-Pct Median 25%-75% Non-Outlier Range Outliers Extremes Lab: GELITA Lab: Nitta Lab: Rousselot Mel-Pct Median 25%-75% Non-Outlier Range Outliers Extremes Plots shown in Figure 1 demonstrate a high level of consistency within each laboratory, as well as consistency among laboratories. The plots also show the different gel strength levels among gel types, and the clearly negative impact of melamine level on gel strength. Box plots for viscosity, shown in Figure 2, indicate similar trends in results for viscosity testing, i.e., consistency within and across laboratories, differences among gel types, and the clearly negative impact of melamine on viscosity. Figure 2. Box Plots of vs. Melamine, by Laboratory and Type Type=Porcine Box Plot of grouped by Mel-Pct; categorized by Lab Exclude condition: NOT( "-Type" = 101 ) Type=Bov-Bone Box Plot of grouped by Mel-Pct; categorized by Lab Exclude condition: NOT( "-Type" = 102 ) Type=Bov-Hide Box Plot of grouped by Mel-Pct; categorized by Lab Exclude condition: NOT( "-Type" = 103 ) Lab: GELITA Lab: Nitta Lab: Rousselot Mel-Pct Median 25%-75% Non-Outlier Range Outliers Extremes Lab: GELITA Lab: Nitta Lab: Rousselot Mel-Pct Median 25%-75% Non-Outlier Range Outliers Extremes Lab: GELITA Lab: Nitta Lab: Rousselot Mel-Pct Median 25%-75% Non-Outlier Range Outliers Extremes Repeatability and Reproducibility Repeatability, representing the ability of a single lab to obtain repeatable measurements on the same sample, is the smallest measure of variability while reproducibility, representing the ability of separate labs to obtain repeatable measurements when those measurements are taken on the same sample by different labs, is the larger measure of variability. The repeatability and reproducibility of this study are expressed in standard deviations which measure the average distance that a measurement will fall from its respective mean value. Reproducibility was assessed using the melamine-free samples, of which five measurements were taken by each of three labs on each of three gelatin types, giving a set of 45 tests. Table 2 gives lab means and standard deviations for each of the three gels. These results indicate some systematic differences among the labs where, for every gel type, ita measurements are a bit greater than Rousselot s, and 2
3 Nitta reported smaller averages for each gel type. However, these observed differences lacked statistical significance. Table 2. Intra-laboratory means and Repeatability Standard Deviations for Porcine Bovine Bone Bovine Hide Lab Mean Std Dev Mean Std Dev Mean Std Dev ita g g g 1.76 Rousselot g g g 0.45 Nitta g g g 1.10 The table of means and standard deviations for viscosity are provided in Table 3. Here again, while there was some variation among laboratories with Nitta again reporting lower averages, the differences were not statistically significant. Table 3. Intra-laboratory means and Repeatability Standard Deviations for Porcine Bovine Bone Bovine Hide Lab Mean Std Dev Mean Std Dev Mean Std Dev ita 31.24mps mps mps Rousselot 31.12mps mps mps Nitta 30.48mps mps mps The inter-laboratory reproducibility results, obtained by pooling the three labs together, are provided in Table 4. This table gives the means and standard deviations of gel strength and viscosity for all labs. Table 4. Inter-laboratory means and Reproducibility Standard Deviations for and Porcine Bovine Bone Bovine Hide Lab Mean Std Dev Mean Std Dev Mean Std Dev g g g mps mps mps The inter-laboratory reproducibility standard deviations are somewhat larger than the repeatability standard deviations, reflecting the fact that they have absorbed the small differences among the labs. These reproducibility standard deviations are very small compared to the observed differences in gel strength and viscosity among the melamine spiking levels depicted in Figures 1 and 2. This comparison clearly shows the significant negative effect of melamine. Regression Analysis Statistical modeling employs independent observations. Since the set of 126 individual tests were not independent, as these were repeat tests on the same object, the data analysis was performed on the smaller set of individual treatment means, 3 x 3 x 4 = 36 total results. The issue of the potential differences among laboratories was resolved using analysis of covariance (ANCOVA).This analysis modeled the quantitative responses, i.e., gel strength and viscosity, as a function of both the quantitative variables, i.e., melamine levels, and qualitative variables, i.e., laboratory and gel type. Thus, melamine levels were treated as a measurement variable, while the other two 3
4 predictors, gel type and laboratory, are categorical. Additionally, the potential interaction between laboratory and gel type was examined. Table 5 contains the results of this initial analysis. Significant effects are indicated by red text with the statistical importance of each effect shown by t values or by the p values. The insignificant effects are laboratory and laboratory by gel type interactions, while statistically significant effects for both gel strength and viscosity are clearly gel type and melamine content. This unquestionably shows that the variations of both gel strength and viscosity are determined by the gel type and melamine content alone. Table 5. Analysis of Covariance Results for and 0Parameter Estimates Level of Effect Effect Param. Std.Err t p Param. Std.Err t p Intercept Mel-Pct 0GELITA Lab*-Type Lab Lab Rousselot Type Porcine Type 0Bov-Bone Lab*-Type Lab*-Type Lab*-Type The data were further analyzed using linear regression, fitting a measurement response (gel strength and viscosity) versus measurement variables. Initial regressions allowed the possibility of separate equations for gel properties versus melamine. These regressions established that there are no differences between the equations for lab type. The coefficient tables for these tests are given in Table 6. Table 6. Linear Regression Result for and N=36 2Intercept GELITA Nitta BBone BHide Mel-Pct N_Mel G_Mel Regression Summary for R²=.979 Std.Error of estimate: 8.76 b Std.Err. t(28) p-value of b N=36 3Intercept GELITA Nitta BBone BHide -Mel-Pct N_Mel G_Mel Regression Summary for R²=.9932 Std.Error of estimate:.596 b Std.Err. t(28) p-value of b In these tables, the variables GELITA and Nitta represent shifts in the intercepts (averages for zero melamine samples), and N_Mel and G_Mel represent offsets to the slopes of the responses with respect to melamine. Any differences among the laboratories in the equations relating gel properties to melamine would be revealed by the t tests. In fact, there are no significant differences among the laboratories for either response. However, as indicated in Table 6, there are significant differences among the three gel types. After all of these considerations, there is no evidence at all of any significant inter-laboratory differences for either response. Final analysis therefore only uses gel type and percent melamine as predictors. 4
5 Model for An initial model for gel strength indicates that the relationship to melamine is not linear. While the true behavior is probably exponential, a simple quadratic term in melamine was added. The final model for gel strength is given below. Table 7. Regression Summary for with Inter-Laboratory Variable Removed Regression Summary for R²=.9862, Std.Error of estimate: b Std.Err. t(32) p-value N= of 2.2b Intercept BHide Mel-Pct MPsq This regression equation for gel strength depends on the gel type. For porcine and bovine bone gels, the relationship is: = Mel Mel 2 For bovine hide gel, the equation is: = ( ) 7.32 Mel Mel 2 = Mel Mel 2 The difference in the intercepts indicates that the gel strength measurements for bovine hide average about 96 less than for the other two gel types. Extrapolating from this, one can estimate that the curvature of gel strength with respect to melamine would not bottom out until melamine spiking reached levels of over 20%. Actual experience could possibly reveal that strength never bottoms out. Model for There were significant differences among all three gel types for viscosity. However, there was no indication of curvature in this model, thus indicating a constant decrease in viscosity per unit increase in melamine. The final results for viscosity are given in Table 8. Table 8. Regression Summary for with Inter-Laboratory Variable Removed Regression Summary for R²=.9918 Std.Error of estimate: b Std.Err. t(32) p-value N= of 0.b Intercept BBone BHide Mel-Pct The viscosity regression equations for different gel types are: Porcine gel: Bovine bone gel: Bovine hide gel: = Mel = ( ) 0.44 Mel = Mel = ( ) 0.44 Mel = Mel 5
6 Residuals To fully complete the model estimations, an examination of the residuals, consisting of several plots of residuals versus deleted residuals, was conducted. These plots are available upon request. In summary, these various diagnostic plots indicated no departures from the regression model assumptions of normally distributed errors and constant variability for either response variable. Nor did the plots indicate any serious deficiencies in the assumption of linear fit to melamine for viscosity, nor the quadratic model for gel strength. Dilution versus Supra-Dilution Impacts of Melamine One final question deals with the type of deleterious effect that melamine has on gelatin properties. If the negative impact results from a pure dilution effect, we would expect the gelatin properties to decline in proportion to the decline in gelatin percentage. That is, if the impact is from dilution only, then a mixture that it 95% gelatin and 5% melamine would exhibit 95% of the gel strength or viscosity of a pure gelatin. The plot in Figure 3 shows that simple dilution is a reasonable model for viscosity. Median viscosity for the 95% gelatin mixture is about 95% of the viscosity of pure gelatin, and is only slightly lower than pure dilution for the 90% and 84% gelatin mixtures. As indicated by Figure 3, losses of gel strength are far greater than mere dilution proportionality suggests. Median gel strength is less than 90% for the 95% gelatin mixture, about 76% for the 90% mixture, and 72% for the 84% mixture. Figure 3. Box Plots of (%) versus atin (%) and (%) versus atin (%) 105 Box Plot of (%) versus atin (%) 102 Box Plot of (%) versus atin (%) (%) (%) atin (%) Median 25%-75% Non-Outlier Range Outliers Extremes atin (%) Median 25%-75% Non-Outlier Range Outliers Extremes Plots in Figure 3 also demonstrate that the addition of melamine increases the variability of gel strength and viscosity, evidenced by the wider box plots for spiked samples. Therefore, not only does melamine have a deleterious impact on the mean levels of these properties, but the presence of melamine also increases production risk by providing for inconsistencies in product produced. Results and Discussion of Physical Observations While less scientific in approach, useful information about the impact of melamine spiking on gelatin can be obtained from observation of test samples in the laboratory. Several exhibits of photographs from the various laboratories conducting the research follow. 6
7 Exhibit 1. Melamine Powder in Original Container Exhibit 1 shows the physical appearance of pure melamine while Exhibit 2 shows a typical dry mixture of gelatin and melamine. As evidenced in Exhibit 2, the presence of melamine in gelatin would be easily observed in the dry state because melamine is a bright white, fine grained substance, while gelatin is a more yellowish color and has, in the vast majority of instances, a coarser granulation than melamine. Exhibit 2. Dry Mixture of Melamine and atin 7
8 The first step in preparing gelatin for use in manufacturing either hard or soft capsules is to mix the gelatin with water, thus forming a liquid suspension. Exhibit 3 shows a photograph of liquid suspensions in which there is 0% melamine, 5%, 10% and 16% melamine spiking. As is evident in these photographs, the presence of melamine whitens the appearance of the suspension due to un-dissolved crystals. Exhibit 3. atin and Melamine/atin Mixture at 5, 10 and 16% Spiking Levels In Liquid Suspension Prior to Heating After melamine spiked gelatin is heated, the samples are chilled to produce solid gels (as is done when capsules are formed). The melamine appears in the cooled solid gel as white dots or crystals suspended in the gel, which actually are re-crystallized melamine that was un-dissolved in the solution. These white dots or crystals are best seen by viewing the bottom of bloom jars (see Exhibits 4 through 6). The evidence of the presence of melamine begins to appear at the 5% level of contamination, and it is more clearly seen at the 10% and higher levels of contamination. Further, this evidence is apparent whether the gel is of bovine bone, porcine skin, or bovine hide origin. 8
9 Exhibit 4. Bovine Bone atin and Melamine/Bovine Bone atin Mixtures at 5, 10, and 16% Spiking Levels After Heating then Chilling (bottom of the bloom jars). Exhibit 5. Melamine/Pig Skin atin Mixtures at 10 and 16% Spiking Levels After Heating then Chilling (bottom of the bloom jars). 9
10 Exhibit 6. Bovine Hide atin and Melamine/Bovine Hide atin Mixtures at 5, 10, and 16% Spiking levels After Heating then Chilling (bottom of the bloom jars). 10
11 Finally, as demonstrated by the photograph in Exhibit 7, the crystallization/re-crystallization of melamine increases over the time span of one week storage in gel form. Exhibit 7. atin after Storage in Refrigerator for One Day (left) and One Week (right) - 10% Melamine Spiking Conclusions This research study evaluating the effect of melamine spiking on the visco-elastic properties of gelatin clearly shows that both gel strength and viscosity are negatively impacted by melamine additions. Therefore, the null hypothesis that the addition of melamine to gelatin has no effect on the visco-elastic properties of gelatin is rejected while the alternative hypothesis is accepted. More specifically, statistical analysis of the experimental data showed that there was a highly significant negative relationship between gel strength and the percentage of melamine spiking of the gelatin. This highly statistically significant negative relationship was also revealed between gel viscosity and percentage of melamine spiking. Testing methods were validated through the evaluation of the results for repeatability and reproducibility. These test results showed no statistically significant differences in testing within each of the three laboratories and across the three laboratories. As expected, there were significant differences in gel strength for the three gel types tested, with porcine skin having the greatest gel strength and bovine hide the lowest. was greatest for bovine bone and lowest for porcine skin. These rank orders for gel strength and viscosity according to type of gel were maintained as spiking levels were made and as these levels were increased. Tests evaluating whether or not there existed deleterious effects of melamine spiking beyond pure dilution proportionality revealed that gel strength was less than 90% for the 95% gel mixture and about 76% and 72% for the respective 90% and 84% mixtures. On the other hand, changes in viscosity with increasing percentage melamine were only slightly less than simple dilution. 11
12 This study has also clearly shown through photographs of laboratory test samples that the presence of melamine in gelatin can be seen by the naked eye in the dry form, the room temperature liquid suspension state, and the chilled solid gel. The presence of melamine in the gelatin mix becomes more and more evident with increased percentage spiking levels. It can be soundly concluded that the spiking of gelatin with melamine, even at very low levels, has significant deleterious effects on the valued properties of gel strength and gel viscosity. 12
13 Appendix Table 1. and of Bovine Bone atin With 0%, 5%, 10% and 16% Melamine Laboratory ita Nitta Rousselot Melamine (%)
14 Appendix Table 2. and of Porcine atin With 0%, 5%, 10% and 16% Melamine Laboratory ita Nitta Rousselot Melamine (%)
15 Appendix Table 3. and of Bovine Hide atin With 0%, 5%, 10% and 16% Melamine Laboratory ita Nitta Rousselot Melamine (%)
16 16
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