Dose- and time-dependent systemic adverse reactions of sodium carboxy methyl cellulose after intraperitoneal application in rats

Abstract

Sodium carboxy methyl cellulose (SCMC) is an important absorbable biomaterial for anti-adhesion and hemostasis medical devices used in the abdominal cavity. However, the systemic toxicity of SCMC following intraperitoneal route has not been revealed sufficiently. Three SCMC solutions with gradient concentrations were intraperitoneally injected into 3 groups of rats with the doses of 50 mg/kg, 320 mg/kg and 2000 mg/kg respectively all at once to observe the dose-dependence of systemic reactions of SCMC and 10 rats (5 rats per sex) of each group were sacrificed 3 days, 7 days, 28 days and 90 days after injection to evaluate the time-dependence of the reactions. A range of adverse effects were shown in rats of the high-dose group which were found varied with time extending and virtually disappeared 90 days after injection. Slight reactions were observed in the medium-dose group while negligible effects were found in the low-dose group. The intraperitoneal application of SCMC can induce reversible systemic adverse effects to rats at the dose higher than 320 mg/kg and it is essential to take both dose- and time-dependent effects into account while designing a systemic toxicity study for absorbable biomaterials.

INTRODUCTION

As an absorbable macromolecular biomaterial, SCMC has been widely used for food additives and pharmaceutical excipients since it can form a gel when dissolved in water (Aisi and Gongmin, 1998; Mondal and Yeasmin, 2016; Bär et al., 1995). In the past decades, SCMC has been proven an effective material for anti-adhesion medical devices (Berdah et al., 2014; Corrales et al., 2008; YelimlieŞ et al., 2003; Vrijland et al., 2002; Wurster et al., 1995; Hemadeh et al., 1993; Sousa et al., 2001; Yaacobi et al., 1993) because the viscosity of body fluids such as synovial fluid and aqueous humor can be mimicked by SCMC solutions which possess the ability to separate serosal and peritoneal surfaces during epithelial regeneration (Sousa et al., 2001; Yaacobi et al., 1993). Furthermore, it is demonstrated that SCMC can be used as hemostasis materials due to its unique characteristics. Firstly, once it has contacted with blood, a gel can be formed which is able to block the ruptures in the capillaries. Meanwhile, the combination of acidic carboxy methyl of SCMC and Fe²⁺ in hemoglobin accelerates the adsorption of platelets and red blood cells. This allows the formation of thrombus. Additionally, the coagulation factors in the blood can be activated by SCMC, which enhances its hemostasis ability (Wei et al., 2019; Tomizawa, 2005; Mei, 1997; Galgut, 1990).

As a food additive and pharmaceutical excipient, SCMC is always ingested orally. Therefore, many studies have focused on its safety following oral route and it has been proven that no obvious adverse effects were detected (Mondal and Yeasmin, 2016; Bär et al., 1995; McElligott and Hurst, 1968). Apart from its oral applications, the SCMC, as aforementioned, would be intraperitoneally administrated into the human body as anti-adhesion and hemostasis materials. According to International Organization for Standardization (ISO) 10993-1: 2018 Biological evaluation of medical devices-Part 1: Evaluation and testing within a risk management process, extractables/leachables can be introduced to the systemic circulation, lymphatic system, and/or cerebrospinal fluid and, therefore, systemic toxicity should be considered. For the evaluation of systemic toxicity, in ISO10993-11: 2017 Biological evaluation of medical devices-Part 11: Tests for systemic toxicity A.2 to A.10, several routes of administration are listed including implantation, inhalation, intraperitoneal, intravenous et al. Particularly, in ISO10993-11, it is emphasized that the most clinically relevant route of administration shall be used. Hence, it has significant importance to evaluate the systemic toxicity risks of SCMC following intraperitoneal route. However, up till now, no related research works has been reported.

According to ISO10993-11, which is applicable to a broad range of medical devices, the systemic toxicity tests for absorbable biomaterials are designed to be limit tests accompanied with single treatment duration. However, such a study design has disadvantages. From the perspective of limit tests, which are uses of a single group treated at a suitable dosage of test sample in order to delineate the presence or absence of a toxic hazard, it is not able to obtain the detailed information of dose-response or dose-effect of SCMC. Furthermore, the treatment duration of the systemic toxicity test is generally fixed to, for example, 90 days for subchronic test in rodents following intraperitoneal route. Under this situation, the variation of toxic reactions over time cannot be investigated. Unlike systemic toxicity reactions of traditional non-absorbable materials, the reactions of absorbable biomaterials are closely related to its absorption, distribution, metabolism and excretion when applied to the body, which, in fact, may possess strong dose- and time-dependence. Therefore, proper study design has to be uniquely tailored to the nature of these absorbable materials and their intended clinical applications.

In this work, we designed a scientific and reasonable test to fully reveal the systemic toxicity of SCMC including its dose- and time-dependence following intraperitoneal route and, simultaneously, to provide an important reference for the clinical use of SCMC and for study design of absorbable biomaterials.

MATERIALS AND METHODS

Animals

160 SPF Sprague Dawley rats (80 per sex), 6-8 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. Rat feed purchased from Beijing Keao Xieli Feed Co., Ltd., Beijing, China and purified water obtained from water purifier were supplied to the rats. Quality tests have been carried out on the feed and water and the results met the criteria for SPF animals used in the study (certificate number for feed: GMAXKCAK40862508, certificate number for water: SOPANI062I). The protocol and any amendments or procedures involving the care or use of animals on this study were reviewed and approved by the Institutional Animal Care and Use Committee Laboratory Animals (IACUC) of our laboratory.

Materials

It is well known that, for practical applications, most of the SCMC intraperitoneal anti-adhesion and hemostasis medical devices were made with low viscosity SCMC. Therefore, low viscosity SCMC powder (CAS: 9004-32-4, viscosity: 100, degree of substitution: 0.81, sodium content: 7.4%, Lot No.: SLB × 2402) purchased from Sigma, Saint Louis, MO, USA was used in our work.

Experimental procedures

According to their weight, rats were divided at random into 4 groups with each group composed of 5 males and 5 females, represented as control group (C), low-dose group (L), medium-dose group (M) and high-dose group (H). Rats of each dose group were then randomly divided into 4 time groups: 3 days, 7 days, 28 days and 90 days groups. In the pre-experiments, 4 fixed doses, comprising 5 mg/kg, 50 mg/kg, 500 mg/kg, and 2000 mg/kg, were set following the “fixed dose procedure”. According to the results, 50 mg/kg, which did not lead to obvious adverse reactions in the rats, was used as the low-dose while 2000 mg/kg, which led to severe reactions, was used as the high-dose. 320 mg/kg, which is the geometric mean of the two doses, served as the medium-dose. SCMC powder was dissolved in sterile water for injection, forming the test solutions with the concentrations of 5 mg/mL, 32 mg/mL and 200 mg/mL, respectively. These test solutions were intraperitoneally injected into the rats with the dose volume of 10 mL/kg•body weight (BW), corresponding to the doses of 50 mg/kg for L, 320 mg/kg for M and 2000 mg/kg for H. Meanwhile, the control group was injected with sterile water for injection with the same dose volume. In order to simulate the clinic use, the SCMC solutions were intraperitoneally injected into rats all at once. During the clinical observation procedure, the rats were weighed weekly and observed daily for general health. Subsequently, 10 rats (5 males and 5 females) of each dose group were sacrificed respectively 3 days, 7 days, 28 days and 90 days after injection in order to evaluate the time-dependence of systemic toxicity reactions of SCMC. At the day before necropsy, rats of each group were weighed and fasted overnight, and the anesthetization was implemented following intraperitoneal route with pentobarbital sodium at the dosage of 40 mg/kg•BW. Blood samples for clinical pathology analyses were collected from abdominal aorta. Hematology and clinical chemistry parameters investigated in our work and corresponding abbreviations are shown in Table 1. After euthanizing by exsanguination while being anesthetized, a gross necropsy was performed in terms of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. Organs, including testes/ovaries, uterus/epididymis, spleen, liver, adrenals, kidneys, thymus, heart and brain, were weighed wet as soon as possible after dissection to obtain the organ coefficients (relative to body weight). These organs, along with stomach, intestine, pancreas, lung and thyroid gland were preserved in 10% formalin to form fixed tissues, which, ultimately, were embedded, sectioned and stained with hematoxylin-eosin for pathology analyses.

Table 1. Parameters and abbreviations of clinical pathology analyses.

Data analyses

Data from male and female were analyzed separately with SPSS17.0 statistical software (SPSS, US, IBM, New York City, NY, USA). Analysis of Variance (ANOVA) was used to compare the differences for each parameter among the 4 groups. Calculations resulting in probability (P) values less than 0.05 were considered to be statistically significant.

RESULTS

Clinical observation and mortality

One day after intraperitoneal injection, one male rat in the 28 days group of the high-dose group was found dead. One male rat in the 7 days group and another one in the 28 days group of the high-dose group died 2 days after injection. No toxic symptoms under clinical observation or mortality were observed in the rest of the animals during the study. Therefore, the mortality of rats of the high-dose group was 7.5%. Moreover, the necropsies of the dead rats were conducted, showing large amount of ascites and dark color of the livers and spleens.

Body weight

The mean body weights (MBWs) of male rats in each group are presented in Table 2, from which it is clear that, for all time groups, high-dose SCMC slowed weight gain significantly in the first week just after injection. Over the subsequent weeks, despite the fact that the MBWs of the high-dose group remained at a relatively low level, comparable growth rate to the control group was observed, indicating the reduction of adverse effects over time. Meanwhile, medium and low dose of SCMC showed no significant effects on the MBW, as evidenced in Table 2. As for female rats, the MBWs of each test group kept the same as that of the control group during the experimental procedure, showing rather weak dependence on dose and time.

Table 2. Body weight data (mean ± standard deviation (SD), male, n = 5).

Clinical pathology

As for male rats in the 3 days group injected with the high-dose of 2000 mg/kg•BW, 7 hematology parameters of significant changes occurred, as shown in Table 3, indicating strong effects of high-dose SCMC. Among these changes, the parameter of WBC was increased compared with control group, which was related to the SCMC-induced acute inflammatory response, while RBC, Hgb, Hct% and PLT were decreased, suggesting hypersplenism occurred. Furthermore, adverse effect on coagulation function was considered to be responsible for the increase of PT and APTT. Seven days after injection, the level of WBC of high-dose group was found to be comparable to that of the control group, while the variation amplitude of 6 other parameters decreased rapidly. Twenty-eight days after injection, slight changes occurred to parameters including RBC and APTT. These parameters, for rats in the 90 days group, were restored completely and thus are not listed in Table 3. Meanwhile, for the medium and low dose groups, negligible SCMC-induced effects on the hematology parameters were observed.

Table 3. Summary of hematology data (mean ± SD, male, n = 5).

Further investigations on the change of clinical chemistry parameters of male rats over dose and time were performed, the results of which are shown in Table 4. It is clear that intense parameter variations were induced by high-dose SCMC in the initial 3 days, while, with the time extending, these parameters gradually returned to their normal levels. Such a variation trend is coincident with that obtained from hematology parameters. It was found that, for the 3 days group, the mean values of chemistry parameters including ALT, AST, ALP, γ-GT, TP, CHOL and TG were dramatically decreased, suggesting the high-dose SCMC induced liver function damage. The value of GLU was increased, implying the adverse effects of glucose metabolism. Furthermore, electrolyte disturbance was induced by the high-dose SCMC, which can be evidenced by the content change of ions such as Ca²⁺, IP, Na⁺ and K⁺.

Table 4. Summary of clinical chemistry data (mean ± SD, male, n = 5).

For medium-dose group, part of the changed parameters in high-dose group including TP in the 3 days group as well as ALT, AST and CHOL in the 7 days group were also decreased compared to that of corresponding control group, indicating milder adverse effects caused by medium-dose of SCMC. It is worthy noting, from Table 4, that significant statistical differences were observed between the medium- and high-dose groups for the parameters of TP (3 days group), ALT (7 days group) and AST (7 days group), which further evidence the dose effects of injected SCMC. Furthermore, low-dose SCMC induced negligible clinical chemistry parameter variations.

Tables 5 and 6 show the corresponding parameters of female rats, which demonstrate similar variation trends to that of male ones.

Table 5. Summary of hematology data (mean ± SD, female, n = 5).

Table 6. Summary of clinical chemistry data (mean ± SD, female, n = 5).

Gross pathology

Three days after injection, a small amount of ascites were observed from the rats of the high-dose group. Figure 1 presents the morphological appearance of a liver obtained from the high-dose group, which, compared with that of the control group, shows darker red and obvious veins. SCMC-induced liver congestion is considered to be responsible for the change of liver color and veins. The livers in the 28 days group with high-dose injection were without any evident macroscopic lesion, suggesting the gradual recovering of livers with time extending. Similar reactions were also observed in spleen of the high-dose group. Meanwhile, such phenomena were seldom observed in the medium- and low-dose group.

Fig. 1

Morphological appearance of the livers obtained from the rats in the high-dose group (left) and the control group (right). 1371 is the ID of the rat in high-dose group and 1012 is the ID of the rat in control group.

Table 7 compares some selected organ coefficients of the four dose groups over time. As can be seen, high-dose SCMC induced the spleen coefficients increase of male rats and liver coefficients increase of female rats 28 days after injection while coefficients of liver, spleen, adrenal and kidneys of female rats were also increased in the 90 days group compared with that of the control group.

Table 7. Summary of organ coefficients (mean ± SD, n = 5).

Histopathology

Table 8 presents the proportions of main histopathology findings and corresponding severities obtained from four time groups injected with different doses of SCMC. It is clear that, for the 3 days group with high-dose injection, vacuolization was observed in multiple organs including spleen, liver and pancreas, whereas, 7 days after injection, vacuolated adrenal was noted and the reaction severities of spleen and liver increased. As for the 28 days group, vacuolization reactions of kidneys and uterus were induced by high-dose SCMC and that of spleen had an alleviation. Ninety days after injection, while insignificant changes occurred to adrenal, kidneys and uterus, organs consist of spleen, liver and pancreas showed relieved reactions. Furthermore, when compared with the high-dose group, the organs with medium dose of SCMC were not apparently changed except that, 7 days after injection, slight reactions were occurred in livers and adrenals, as shown in Table 8. Meanwhile, no histopathology findings of organs are induced by low-dose SCMC.

Table 8. Summary of the incidence proportion and severities of vacuolization.

As the most important immune organ of mononuclear phagocyte system, the spleen shows strong reaction with high-dose of SCMC and its time-dependence is demonstrated in Fig. 2, from which the most severe vacuolization was observed in the 7 days group, as shown in Fig. 2(b). The reactions of spleens in the 7 days group compared with the control group are exhibited in Fig. 3. As can be seen from Fig. 3(a) and (b) diffuse reaction occurred in red pulp compared with the control group. Zooming-in further evidences that vacuoles are macrophages that have phagocytized foreign particles, as shown in Fig. 3(c) and (d). A representative macrophage is marked by the red arrow in Fig. 3(d).

Fig. 2

Time-dependence of reaction in spleen of the high-dose group; mild, medium, mild and slight reactions are observed from a-d respectively.

Fig. 3

Histopathologic appearances of spleens in control and high-dose group 7 days after injection; vacuolization can be observed from b and d; representative macrophage is shown by the red arrow in d.

Figures 4 and 5 show the histopathologic appearances of livers of the 3 days and 7 days groups, respectively. As can be seen from Fig. 4, slight congestion in the portal region was observed in the liver of rats in the high-dose group 3 days after administration. Furthermore, the changes of livers are found to possess strongest dose dependence, as shown in Fig. 5. It is clear that, compared with the control group, the livers in the high-dose group suffer from remarkable vacuolization in Kupffer cells. Along with the dosage of SCMC decreasing to medium, the vacuolization of livers significantly decreased and, eventually, intact livers are observed in low-dose group.

Fig. 4

Histopathologic appearances of livers in control and high-dose group 3 days after injection; congestion in the portal region can be observed in b.

Fig. 5

Dose-response of liver reactions 7 days after injection; a: normal liver of control group, b: mild reactions of high-dose group, c: slight reactions of medium-dose group, d: normal liver of low-dose group.

The high-dose-SCMC related reactions of pancreas in the 7 days group are shown in Fig. 6(b), while those of adrenal, kidneys and uterus obtained from the 90 days groups are illustrated in Fig. 6, respectively. It is found that, in these organs, vacuoles can be identified as vacuolated macrophages, which is also induced by absorbed SCMC.

Fig. 6

Reactions in multiple organs of high-dose group compared with the control.

As aforementioned, 3 rats died in the initial 2 days after high-dose injection of SCMC, whose organs including thymus, spleen and kidneys are shown in Fig. 7. Comparing with the control group, severe apoptosis and decrease of lymphocytes were observed in thymus of dead rats, which are presented in Fig. 7(a) and 7(b). Meanwhile, as shown in Fig. 7(c) and 7(d), medium decrease of lymphocytes and increase of macrophages were noted in spleen. The acute failures of these immune organs caused by the absorbed SCMC may be the main reason for the death of the rats. As for other organs, taking spleen for instance (see Fig. 7(e) and 7(f)), while increase of macrophages was observed in interstitium, light blue-stained substances considered to be SCMC were found on the visceral peritoneum of the organ.

Fig. 7

Histopathological appearances in some organs of dead rats compared with the control; a, c and e: normal organs of control group, b: severe lymphocytes apoptosis and decrease in cortex of thymus of dead rats, d: mild lymphocytes decrease in spleen of dead rats, f: increase of macrophages in interstitium, light blue-stained substances considered to be SCMC on the visceral peritoneum of the organ is shown by the red arrow.

DISCUSSION

Systemic toxicity evaluation for SCMC following intraperitoneal route

In our work, the rats showed adverse reactions to SCMC at the dose of 320 mg/kg and several rats (less than 50% of the total) died at the dose of 2000 mg/kg, which demonstrates that, for intraperitoneally applied SCMC, the minimal lethal dose (MLD) is higher than 320 mg/kg and the lethal dose 50% (LD50) is higher than 2000 mg/kg. These results show obvious differences when compared with the previous studies in which SCMC was orally applied and no visible reactions of animals were observed at the dose as high as 10-20 g/kg/day (Mondal and Yeasmin, 2016; Bär et al., 1995; McElligott and Hurst, 1968). Furthermore, although no irreversible injury to organ tissues or cells occurred during the experiments, it has significant importance to control the dose of SCMC below that can induce adverse effects when used as anti-adhesion or hemostatic material in the abdominal cavity. The results of our work provide important toxicology information for the clinical use of SCMC.

Mechanism behind the adverse effects

As is well known, the same as humans, due to the absence of β-lactamases in the body, rats are unable to digest macromolecule SCMC into glucose. Therefore, after being administrated into the peritoneal cavity of the rat, SCMC can be absorbed as macromolecule into peritoneal capillaries and then transported into the interstitium of spleen, liver and pancreas. In these organs, along with the increase of SCMC, owing to the viscosity of SCMC, blood circulation and substances transportation are blocked. Meanwhile, the SCMC in these organs is recognized as a foreign body by mononuclear phagocyte system (Sage et al., 2009; den Haan and Kraal, 2012) which triggers a nonspecific immune response. During this process, monocyte-macrophages proliferate and gather to these organs to phagocytize and dispose SCMC, which might aggravate the blocking phenomena. Further, along with the absorption of SCMC into blood, plasma osmotic pressure can be increased, leading to the increase of blood volume. As a result, slight congestion is induced in hepatic sinusoids while liver synthesized and secreted substances including clotting factors, liver enzymes, proteins and lipids are decreased (Gowda et al., 2009). In spleen, these aforementioned phenomena enhance its retention effects to blood cells, which induce the decreases of RBC and platelets (den Haan and Kraal, 2012; Mebius and Kraal, 2005). Similarly, in pancreas, the synthesis and secretion of insulin are inhibited, inducing the increase of blood glucose. Additionally, Na⁺ is absorbed into the blood along with the absorption of SCMC, which subsequently causes the changes of other ions including Ca²⁺, IP and K⁺. With time extending, SCMC is completely absorbed into blood and decomposed gradually into smaller particles by macrophages until they can be excreted by kidneys and other organs, which is coincident with the reduction of adverse reactions over time observed in our work.

Reason for different systemic toxicity profile of SCMC applied following different routes

According to ISO10993-11, absorption, distribution and metabolism of leachates from the device or its materials to parts of the body are inevitable processes that induce generalized effects, as well as organ and organ system effects (ISO 10993-11: 2017 Biological evaluation of medical devices-Part 11: Tests for systemic toxicity). As a macromolecule, SCMC can hardly be absorbed by the stomach and intestine into blood (Bär et al., 1995; McElligott and Hurst, 1968). Hence, following oral route, even at the dose as high as 10-20 g/kg/day, animals show no visible reactions to SCMC, which has been proved by previous studies (Mondal and Yeasmin, 2016; Bär et al., 1995; McElligott and Hurst, 1968). Along with the change of administration route to intraperitoneal injection, SCMC is exposed directly in the body and the body would absorb it into blood, distribute it to organs, metabolize and finally excrete it out. As dose increases to 320 mg/kg, SCMC can not be disposed in time, which leads to an accumulation of SCMC in blood and organs, resulting in the occurrence of a series of adverse effects. These results demonstrate that, for certain material, the systemic reactions might be different when its administration routes are changed, for instance, from oral to intraperitoneal route. In this regard, the systemic toxicity reactions should be sufficiently considered when the applications of biomaterials are extended.

Systemic toxicity test design for absorbable biomaterials

In our work, it is evidenced that the systemic reactions of the absorbable materials may possess remarkable dose dependence, which is expressed in adverse effects of body weight, clinical pathology and histopathology with high-dose injection and negligible effects induced by low-dose SCMC. Such a dose effect is particularly evident in livers, for which medium dose leads to slight reactions. Furthermore, the investigations of systemic toxicity over time shows that the most significant reactions of clinical pathology and histopathology appear 3 and 7 days after injection, respectively, whereas 90 days after injection the reactions were not obvious, which indicates the strong time dependence of the reactions and, hence, the irrationality of the aforementioned single treatment duration. Therefore, for the purpose of study design of the absorbable biomaterials such as SCMC, it is essential to take both dose and time effects into account, which pave a way for the establishment of guidelines for the systemic toxicity tests related to these materials.

In conclusion, following intraperitoneal route, the systemic toxicity of SCMC are investigated thoroughly over dose and time, the results of which lead to the conclusion that intraperitoneal injection of SCMC at the dose of 2000 mg/kg can induce obvious reactions including acute inflammatory response, decreases of RBC and platelets, adverse effects on liver function and glucose metabolism, electrolyte disturbance as well as vacuolization in multiple organs. These reactions are found varied with time extending and virtually disappeared 90 days after injection. The medium-dose (320 mg/kg) group shows slight reactions while low-dose (50 mg/kg) SCMC induces negligible effects. Therefore, SCMC should be used as anti-adhesion or hemostatic material in the abdominal cavity below the dose that can induce adverse effects. Additionally, these facts indicate that, when design a systemic toxicity study for absorbable materials, it is essential to take both dose- and time-dependent effects into account while simulating the clinical use in terms of administration route and dose frequency.

ACKNOWLEDGMENTS

The authors are grateful to the financial support of National Key Research and Development Program (2016YFC1103205) and Science and Technology Fund of Shandong Province (2017GSF218004).

Conflict of interest

The authors declare that there is no conflict of interest.

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