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Dose- and Time-Dependent Systemic Adverse Reactions of Sodium Carboxy Methyl Cellulose after Intraperitoneal Application in Rats

Background: 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 suciently. Results: 3 SCMC solutions with gradient concentrations were intraperitoneally injected into 3 group rats respectively all at once to observe the dose-dependence of systemic reactions of SCMC and 10 rats of each group were sacriced 3d, 7d, 28d and 90d after injection to evaluate the time-dependence of the reactions. A range of adverse effects were shown in rats of high-dose(2000mg/kg) group which were found varied with time extending and virtually disappeared 90 days after injection. Slight reactions were observed in medium-dose(320mg/kg) group while negligible effects were found in low-dose(50mg/kg) group. Conclusion: The intraperitoneal application of SCMC can induce reversible systemic adverse effects to rats at the dose higher than 320mg/kg and it is essential to take both dose- and time-dependent effects into account while designing a systemic toxicity study for absorbable biomaterials.

Body weight data (mean ± SD, male, n = 5) 390.4 ± 29.0 † n = 4 because 1 rat died 2 days after injection; ‡ n = 3 because 2 rats died in the initial 2 days after administration; **P 0.01, compared to the control group; ↓ represents signi cant decrease. The data obtained between 5 to 13 weeks of 90d group are not presented as no signi cant effects are observed.

Clinical pathology
As for male rats in 3d group injected with the high-dose of 2000 mg/kg•BW, 7 hematology parameters of signi cant changes occurred, as shown in Table 2, 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 in ammatory 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. These results are further illustrated in Fig. 2, in which the parameter variations of high-dose group can be clearly observed. 7 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. 28 days after injection, slight changes occurred to parameters including RBC and APTT. These parameters, for rats in 90d group, were restored completely and thus are not listed in Table 2. Meanwhile, for medium and low dose groups, negligible SCMC-induced effects on the hematology parameters were observed. 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 3. It is clear that intense parameter variations were induced by high-dose SCMC at 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 3d 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 decreased, 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 Ca 2+ , IP, Na + and K + .
For medium-dose group, partial of the changed parameters in high-dose group including TP in 3d group as well as ALT, AST and CHOL in 7d group were also decreased compared to that of corresponding control group, indicating slight adverse effects caused by medium-dose of SCMC. It is worth noting, from Table 3, that signi cant statistical differences were observed between the medium-and high-dose groups for the parameters of TP (3d group), ALT (7d group) and AST (7d group), which further evidence the dose effects of injected SCMC. For easy comparison, the values of these parameters obtained from four groups are illustrated in Fig. 3. Furthermore, low-dose SCMC induced negligible clinical chemistry parameter variations. Tables 4 and 5 show the corresponding parameters of female rats, which demonstrate similar variation trends to that of male ones.   Gross pathology 3 days after injection, scanty ascites were observed from the rats of high-dose group. Figure 4 presents the morphological appearance of a liver obtained from high-dose group, which, compared with that of the control group, shows darker red and more obvious veins. SCMC-induced liver congestion is considered to be responsible for the change of liver color and veins. The livers in 28d group with high-dose injection were without any evident macroscopic lesion, suggesting the gradually recovering of livers with time extending. Similar reactions were also observed in spleen of high-dose group. Meanwhile, such phenomena were seldom observed in medium-and low-dose group. Moreover, the necropsies of the dead rats were conducted, showing large amount of scanty ascites and dark color of the livers and spleens. Table 6 compared some selected organ coe cients of four dose groups over time. As can be seen, high-dose SCMC induced the spleen coe cients increase of male rats and liver coe cients increase of female rats 28 days after injection while coe cients of liver, spleen, adrenal and kidneys of female rats were also increased in 90d group compared with that of the control group.  Table 7 presents the proportions of main histopathology ndings and corresponding severities obtained from four time groups injected with different doses of SCMC. It is clear that, for 3d 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 28d group, vacuolization reactions of kidneys and uterus were induced by high-dose SCMC and that of spleen had an alleviation. 90 days after injection, while insigni cant changes occurred to adrenal, kidneys and uterus, organs consist of spleen, liver and pancreas showed relieved reactions. Furthermore, when compared with 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 7. Meanwhile, no histopathology ndings of organs are induced by low-dose SCMC.
As the most important immune organ of mononuclear phagocyte system, spleen shows strong reaction with high-dose of SCMC and its time-dependence is demonstrated in Fig. 5, from which the most severe vacuolization was observed in 7d group, as shown in Fig. 5(b). The reactions of spleens in 7d group compared with the control group are exhibit in Fig. 6. As can be seen from Fig. 6(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. 6(c) and (d). A representative macrophage is marked by the red arrow in Fig. 6(d). Figure 7 and 8 show the histopathologic appearances of livers of 3d and 7d groups, respectively. As can be seen from Fig. 7, slight congestion was observed in the liver of rats in high-dose group 3 days after administration. Furthermore, the changes of livers are found to possess strongest dose dependence, as shown in Fig. 8. It is clear that, compared with the control group, the livers in high-dose group suffers from remarkable vacuolization. Along with the dosage of SCMC decreasing to medium, the vacuolization of livers signi cantly decreased and, eventually, intact livers are observed in low-dose group.
The high-dose SCMC related reaction of pancreas in 7d group are shown in Fig. 9(a), while that of adrenal, kidneys and uterus obtained from 90d groups are illustrated in Fig. 9(b)-9(c), respectively. It is found that, in these organs, vacuolization is also induced by absorbed SCMC.
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. 10. Comparing with the control group, severe apoptosis and decrease of lymphocytes were observed in thymus of dead rats, which are presented in Fig. 10(a) and 10(b). Meanwhile, as shown in Fig. 10(c) and 10(d), medium decrease of lymphocytes and increase of macrophages were noted in spleen. As for other organs, taking kidneys for instance (see Fig. 10(e) and 10(f)), while in ltration of macrophages and polymorphic nucleus WBC were observed in interstitium, light blue-stained substances considered to be absorbed SCMC were found emerged in these regions. These phenomena are probably responsible for the death of the rats. 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.

Conclusions
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 in ammatory 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 lowdose(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.

Materials And Methods
Animals 160 SD rats, 6-8 weeks old with half male and half female, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

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  Table 8. After euthanizing by exsanguination while being anesthetized, a gross necropsy was performed in terms of the external surface of the body, all ori ces, 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 coe cients (relative to body weight). These organs, along with stomach, intestine, pancreas, lung and thyroid gland were preserved in 10% formalin to form xed tissues, which, ultimately, were embedded, sectioned and stained with hematoxylin-eosin for pathology analyses.  Data from male and female were analyzed separately with SPSS17.0 statistical software (SPSS, US, IBM). Calculations resulting in probability (P) values less than 0.05 were considered to be statistically signi cant.

Declarations
Ethics approval and consent to participate All procedures for animal experiments were performed in accordance with Institutional Animal Care and Use Committee Protocols.

Consent for publication
All the authors of the paper approved the publication of the article.

Availability of data and material
Data related to the current study are available from the corresponding author on reasonable request.

Competing interests
All authors declare that they have no actual or potential competing of interest.