Cytotoxic and genotoxic effects of leaking chemicals from serum infusion sets: an in-vitro study

Abstract

Safety concerns about medical devices playing important role in health sciences and bioengineering research are rising day by day. Although there are specific standards regarding disposable medical materials, the information is updating with the toxicological studies. In this study, cytotoxic/genotoxic effects of chemicals leaking from serum infusion sets that have an important place in the clinic were investigated. Media containing leakage chemicals were prepared from equal samples taken from the plastic line sections of 13 different brands of serum infusion sets containing phthalates and the effects on the cultured cells were compared with the untreated control groups. To obtain leaking chemicals, extracting period was selected as 72 h, a routine set-change time in the clinic as indicated in various publications. Neutral red uptake and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests were performed in L929 cells to determine cytotoxicity, and cytokinesis blocked micronucleus technique was performed in lymphocytes to determine genotoxicity. Cytotoxic and genotoxic damage levels were compared by evaluating cell-viability rates relative to control, micronucleus frequency, and nuclear division index values. The results showed that all sets caused a decrease in cell viability revealing the effects both on lysosomal and mitochondrial activity and increase in micronucleus frequencies in general. The number of similar studies is extremely limited, and in this study in addition to the short-term effects of using the serum infusion sets, the information about the sample tests to determine the biosecurity of disposable medical materials is given.

1. Introduction

Exposure to chemicals from medical devices has been one of the discussed medical issues recently. There are not enough studies about health threats of materials especially used in invasive procedures and contact directly with blood and leaching chemicals that can pass from these materials to the patient whom the product is applied. According to ISO 10993 supplements, for medical products and biomaterials, the extractable and leachable material detection is important for evaluating the possible toxicity. The term “extractable” means chemicals passing through any elastomeric, plastic, glass, stainless steel materials of any product, or coating materials under exaggerated time and temperature conditions while in contact with a suitable solvent. In normal conditions, these chemicals are “leachables”. These substances must be known using a biomedical device in physiological conditions for ensuring biosafety.^1,2

Disposable medical devices are essential materials in healthcare settings. Especially single-use plastic devices are consumed in every patient. Because of that, commercial plastics are highly demanded in this area. In addition to polymers such as polyethylene and polypropylene, one of the most used polymers is polyvinyl chloride (PVC). It is widely used due to its low cost, easy processing, and suitable properties for many application areas. According to many types of research, it is estimated that ~25% of all plastic medical products are PVC.³ PVC is used as a raw material for many medical materials such as infusion sets, enteral feeding bags, cannulas, which are used extensively in the medical field.^4–6 PVC is a rigid material on its own but it is made softer by combining with plasticizers, particularly phthalate esters.⁷

The phthalate derivatives such as dioctyl phthalate, di-n-decyl phthalate, and especially diethyl hexyl phthalate (DEHP) are widely used for the plasticization of PVC and have been in medical use since 1955.^4–8 Phthalates are semi-volatile compounds that are not chemically bonded to plastic polymer structures, so they can easily pass from the materials in to water, air, or other external environments.⁷ Understanding how they can affect general health and especially reproductive health when they are exposed and also the underlying mechanisms of their effects will improve the development of strategies to reduce the burden and negative consequences of environmental factors on humans. In many studies, toxic effects of phthalate derivatives are discussed.^4,8 DEHP levels were found to be increased in patients hospitalized in intensive care units, who received blood, thrombocyte transfusion, or total parenteral nutrition, were connected to a mechanical ventilator, and who underwent dialysis. In the literature, it has been reported that DEHP exposure has negative effects on the reproductive, cardiovascular, respiratory, gastrointestinal hematopoietic, urinary and neurological systems, and also immunological and lymphoreticular systems.^5,8,9 On the other hand, the literature about phthalates in medical materials used in the market is still very limited. The most recent study is an analysis by Kostic et al. in which they determined the amount of DEHP for a few medical supplies from hospitals. Plastic dialysis bag and tube, infusion bottle, and infusion set were used in that study. Plastic materials are divided into parts with an area of ~1 cm² and applied some extraction procedures. The samples were analyzed with gas chromatography–mass spectrometry without the clean-up phase. As a result of the study, the amount of DEHP leaked from PVC bags was found to be higher than low-density polyethylene (LDPE) bottles.¹⁰

DEHP toxicity may be more dangerous especially in newborn patients due to low metabolism and detoxification capacities and in premature infants low body weights.¹¹ Numerous studies have been carried out to evaluate neonates exposed to DEHP through medical equipment. In a study by Loff et al.¹², it is stated that the amount of DEHP leaking from PVC serum infusion lines plasticized with DEHP additive poses a risk especially for sensitive patients.

Bouattour et al.¹³, purposed an ex-vivo model for determining DEHP levels released from medical devices for evaluating DEHP exposure on newborns. In this study they detected the amount of DEHP in the air passing through PVC respiratory assistance medical devices. This is one of the important studies on DEHP toxicity of medical devices because there is no study to determine DEHP migration to respiratory medical devices. They stated that respiratory DEHP exposure is not the main source compared with other medical devices.¹³ Considering that medical equipment that comes into contact with blood is the main source of exposure, the serum infusion set, which is one of the most frequently used medical equipment in medical practice was chosen in our study. The serum infusion sets are also one of the most used PVC (mostly DEHP containing) medical supplies in hospitals. But, to the best of our knowledge according to literature research, there are limited number of studies about serum infusion sets. Evaluating the biocompatibility of these medical devices is a major concern in this area. The material used in the device must be assessed for cytotoxicity and genotoxicity to understand organism’s behavior toward it. In-vitro tests are used for imitating reactions to the device after replacing it in the organism.¹⁴

In this study, assessing the possible cytotoxic/genotoxic effects of leaching chemicals from 13 different brands of DEHP containing PVC serum infusion sets used in hospitals in all over our country was planned. For this purpose, the plastic line parts of serum infusion sets were extracted 72 h and cytotoxic effects were investigated by neutral red uptake (NRU) and 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity tests in L929 mouse fibroblast cells according to the ISO 10993-5 rule.¹⁵ Genotoxic effects were investigated in human peripheral blood lymphocyte cells using the cytokinesis blocked micronucleus (CBMN) assay according to ISO 10993 Part 3 about tests for genotoxicity.¹⁶

2. Materials and methods

2.1. Chemicals

Neutral red (NR) (3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin–streptomycin, trypan blue, ethanol, methanol, potassium dihydrogen phosphate, disodium hydrogen phosphate dihydrate, potassium chloride, acetic acid, nitric acid, formaldehyde, and Giemsa dye were purchased from Sigma; chromosome medium B, dimethyl sulfoxide (DMSO) from Merck; triton X-100, trypsin–EDTA, Dulbecco’s phosphate buffered saline (DPBS) from Gibco.

2.2. Serum infusion sets as test materials

In this study, the cytotoxic and genotoxic effects of serum infusion sets from different companies were investigated. The names of different brands offered for sale by companies operating ethically for commercial purposes are not mentioned in the content. The company information of these products was listed, and a sample number was given to all sets. Samples of equal length taken from these sets, coded with numbers 1–13, were used under the same conditions and simultaneously in cytotoxicity and genotoxicity experiments. In the study, the effects of leakage chemicals of 13 different brands of serum infusion sets were examined. Plastic line (hose) part of each set was used to obtain leakage chemicals. For the experiments, equal samples in the form of 2.5 cm sections were taken from the tubing sections of the serum sets, weighed on a precision balance and re-sterilized by ultraviolet light for 1 h to keep the structural properties of the material.

All sample extracts were prepared by method suggested in the ISO 10993-12 (International Organization for Standardization, 2012) and suitable for the PVC line parts of serum sets as biomaterials.¹ All samples were extracted 72 h in sterile tubes containing 2.5 ml of culture media (for cytotoxicity tests: 99% RPMI 1640 and 1% penicillin–streptomycin, without FCS to prevent protein interaction; for micronucleus test chromosome medium B). All the media containing the samples were kept in the incubator at 37 ° C for 72 h. The planned case is to examine whether the leakages that pass from the serum sets into the media will have a cytotoxic or genotoxic effect on the cells growing in this environment (Fig. 1).

Fig. 1.

Schematic description of the study.

A total of 15 groups, including 13 different set samples, and negative control and positive control (1% triton X-100 or 0.2 μg/ml MMC), were studied in 3 repetitions in all experiments applied to determine cytotoxicity and genotoxicity.

2.3. Cell culture

For both cytotoxicity assays L929 cell lines (mouse fibroblast) as a reference cell line for determination of cytotoxic effects of medical devices and materials, in accordance with ISO 10993-5 rule were used. L929 cells were reproduced in RPMI 1640 supplemented with 10% FBS and 1% penicillin streptomycin in 75 cm² culture flasks at 37 °C and 5% CO₂ atmosphere. Cells were checked daily by using an inverted microscope with phase contrast attachment (Olympus CKX41) and subcultures were performed when reaches 80% confluency.

2.4. Determination of the cytotoxicity by neutral red uptake assay

The cytotoxicity of serum sets was performed in L929 cells by NRU assay following the protocols described in Parın et al.¹⁷ and Taner et al.^18,13,14 L929 cells were seeded in 96 well plates and incubated for 24 h. Then cells were treated for additional 24 h with the sample solutions. After incubation period, medium was discarded, cells were washed with PBS, and incubated for 3 h with 50 μg/ml NR dye containing medium. Then medium was discarded again and cells were washed with warm PBS to remove the nonincorporated dye and fixed by destain solution (50% ethanol, 1% acetic acid, and 49% distilled water). The plates were shaken for 20 min, and the absorbance was measured at 540 nm. Results were expressed as the mean percentage of cell growth from 3 independent experiments comparing all data with untreated negative control cells.

2.5. Determination of the cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

MTT assay was applied as described in Parın et al.¹⁷ and Taner et al.¹⁸ Following disaggregation with trypsin/EDTA and resuspension of cells in medium, a total of 5 x 10⁴ cells/wells were plated in 96 well tissue-culture plates. After 24 h incubation, cells were treated with the medium containing leaching chemicals of serum sets for 24 h at 37 °C. Then the medium was removed and 10 μl MTT from 5 mg/ml of stock solution was added to each well and cells were incubated for an additional 4 h with MTT dye. At the end of incubation period, then medium discarded carefully and 100 μl of DMSO was added to each well. The absorbance of the solution in each well was measured at 570 nm. Results were expressed as the mean percentage of cell growth from 3 independent experiments.

2.6. Determination of the genotoxicity of serum sets by CBMN assay

Human peripheral blood cultures were prepared to examine the genotoxic effects of leakage chemicals from each serum infusion set. About 5 ml of blood samples were taken from different donors (aged of 27–30, who had any health problems and no history of exposure to drugs, genotoxic agents and also non-smokers, non-alcoholics) in heparin coated tubes. And 0.25 ml of blood was added to tubes containing 2.5 ml of chromosome medium and leakage chemicals of serum sets. In all sets of experiments, an untreated negative control, as well as a positive control (0.2 μg/ml MMC) was also prepared. After 44 h of culture initiation cytochalasin B (Cyt-B) was added to all cultures at a final concentration of 6 μg/ml. When a total of 72 h incubation period completed, culture tubes were centrifuged at 1,000 rpm for 10 min. After supernatant was discarded, an ice-cold hypotonic solution (0.075 M KCl) was added and tubes were held 5 min at 37 °C. Then cells were fixed with cold methanol:glacial acetic acid (3:1, v/v) for 15 min and fixation procedure was repeated twice. 1% formaldehyde was added to last fixative was. The fixed cells were dropped on nitric acid cleaned slides. After preparation, air-dried slides were stained by 5% Giemsa dye for 13 min. Total 3,000 binucleated cells (1,000 from each donor) per concentration were scored for MN index. Furthermore, total 1,500 lymphocytes (500 lymphocytes from each donor) were scored for detecting the cytokinesis-block proliferation index (CBPI) and calculated according to Surrales et al.¹⁹ and as explained in Taner et al.¹⁸

2.7. Statistical analysis

For cytotoxicity tests, each sample was compared with the control group and determined as % cell viability. For the CBMN test data, sample groups were compared statistically with the untreated negative control and positive control groups with Z test.

3. Results

3.1. Cytotoxic effects of serum infusion sets by NRU assay

According to the results of NRU test (Fig. 2) in L929 cells, the groups treated with leakages of materials; cell viability in all groups decreased compared with the negative control group. In addition, below 70% cell viability was observed, which is accepted as the cytotoxic effect limit. According to the test results among 13 different serum infusion set samples used in the study, it was determined that the serum infusion set sample numbers 1 and 9 were the samples with the highest cytotoxic effect with values below 50% (44.87% and 42.34% as respectively) percentage of viable cells compared with the control. In the NRU method, which is based on the measurement principle of lysosomal activity, it is thought that the leaching chemicals of serum infusion sets may cause toxic effects by interacting with the cell membrane or enter the cell through the membrane and accumulating in organelles and cytoplasm.

Fig. 2.

Effects of serum set leakages on cell viability of L929 cells by NRU assay. Results were expressed as the mean percentage of cell growth inhibition from 3 independent experiments. Cell viability was plotted as percent of control (assuming data obtained from the absence of nanofiber as 100%).

3.2. Cytotoxic effects of serum infusion sets by MTT assay

The results of the cytotoxic effect of the leaching chemicals of 13 different serum sets evaluated by MTT assay in L929 cells are given in Figure 3. According to MTT test results, the groups treated with leaching chemicals of serum sets caused some decreases in cell viability in all groups compared with the negative control group. However, because viability did not fall below 70% except the samples 7 and 8, it was not evaluated as cytotoxic by MTT assay. According to the results of the MTT test, it was determined that the 8th sample showed the highest cytotoxic effect.

Fig. 3.

Effects of the serum sets leakage on cell viability of L929 cells by MTT assay. Results were expressed as the mean percentage of cell growth inhibition from 3 independent experiments. Cell viability was plotted as percent of control (assuming data obtained from the absence of serum set as 100%).

3.3. Genotoxic effects of serum infusion sets by the CBMN assay

Genotoxic effect of the leaking chemicals of 13 different brand serum infusion sets was evaluated using CBMN method in human peripheral blood lymphocyte cells. The MN values, frequencies, and nuclear division indices determined in the 45 groups (which were studied with 3 replicates in cultures prepared from different donors, each of 15 groups prepared as 13 serum infusion sets, control group, and positive control) are shown in Table 1. When the groups treated with chemicals leaking from serum sets were compared with the negative control group, it was observed that it caused an increase in MN formation in samples 1–10, but not in 11–13 samples (Table 1). The increase in only 1 serum set (serum set 6) was statistically significant (P ˂ 0.05). When these increases were compared with the positive control, it was also determined that there was no increase as positive control level. When NDI values were compared with the control group, it was seen that serum sets did not cause a statistically significant change.

Table 1.

Effects of the leaching chemicals of serum infusion sets on the frequency of micronucleus and CBPI in cultured human lymphocytes.

Experimental Group	Scored BN cells	Number of MN			Total MN	MN (%) ± SE	CBPI ± SE
		Donor1	Donor2	Donor3
(−) Control	3,000	2	2	2	6	0.2 ± 0.08	1.17 ± 0.28
(+) Control	3,000	12	18	20	50	1.67 ± 0.23	1.14 ± 0.27
Sample 1	3,000	6	3	3	12	0.4 ± 0.12	1.11 ± 0.27
Sample 2	3,000	4	4	1	9	0.3 ± 0.10	1.14 ± 0.27
Sample 3	3,000	6	4	4	14	0.47 ± 0.12	1.20 ± 0.28
Sample 4	3,000	6	1	1	8	0.27 ± 0.09	1.11 ± 0.27
Sample 5	3,000	2	2	5	9	0.3 ± 0.10	1.17 ± 0.28
Sample 6	3,000	10	5	3	18	0.6 ± 0.14a	1.20 ± 0.28
Sample 7	3,000	5	2	3	10	0.33 ± 0.11	1.20 ± 0.28
Sample 8	3,000	4	3	3	10	0.33 ± 0.11	1.20 ± 0.28
Sample 9	3,000	4	5	4	13	0.43 ± 0.12	1.29 ± 0.29
Sample 10	3,000	3	3	1	7	0.23 ± 0.09	1.35 ± 0.30
Sample 11	3,000	1	3	2	6	0.2 ± 0.08	1.20 ± 0.28
Sample 12	3,000	1	0	0	1	0.03 ± 0.03	1.50 ± 0.31
Sample 13	3,000	1	1	1	3	0.1 ± 0.06	1.29 ± 0.29

^aSignificantly different at P ≤ 0.05 when compared with the negative control (z test). BN: binucleate, MN: micronucleus, CBPI: cytokinesis block proliferation index, SE: standard error.

4. Discussion

Biosafety determination of medical materials and devices is an important affair because of their enormous uses. The most important feature of these medical materials in terms of clinical suitability is a high-level biocompatibility. Biocompatibility is the quality of a material to function without causing any undesirable local or systemic effects on the host.^2,20 There are some test procedures for safety evaluations of these medical materials. In particular, the ISO 10993 standard determines the basic criteria for evaluating the compatibility.¹

In this study, 13 different brands of DEHP containing PVC serum infusion sets were investigated in terms of their cytotoxic and genotoxic effects as a part of biocompatibility testing to assess the general evaluation of this medical material. Cytotoxic effects were investigated by NRU and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity tests and genotoxic effects were investigated using the cytokinesis block technique and the micronucleus test. These tests are recommended in ISO 10993 biocompatibility testing protocols. ISO 10993-5 (in-vitro cytotoxicity tests) criteria were used for cytotoxicity studies.¹⁵

Serum infusion set is a medical consumable that provides the connection between the serum bag and the patient. All serum sets consist of different parts such as the needle entering the patient’s vascular tract at one end, a plastic line section in the middle, a drip chamber connected to this line, and a piercing pin. In this study, it was planned to investigate the possible effects of leaching chemicals from the line sections of serum infusion sets.

PVC is one of the most frequently used polymers for many disposable materials and it is plasticized before use because when not plasticized it has a harder and more rigid structure. For the plasticization process, phthalate derivatives such as dioctyl phthalate, di-n-decyl phthalate, trioctyltrimellytate (TOTM), DEHP are widely used. Phthalates (phthalate esters) are dialkyl or alkyl aryl esters of phthalic acid. Phthalates, which were first produced in the 1920s, were introduced to the market with large-scale commercial production of PVC softened with phthalate esters, which was first developed in the 1950s.²¹ Phthalates are a member of industrial chemicals used for many purposes, such as plasticizers that give PVC products flexibility and durability. It is also used in solvents, motor oils, fixing agents, and personal care products such as detergents. When phthalates are added to PVC, they are not covalently bound, so they are easily released to the environment, so humans and animals are also exposed to these chemicals.²²

Our study based on examining possible chemicals that may leak from serum infusion sets, which are connected to the patient’s vascular access up to 72 h, in terms of biomaterial safety and to investigate whether there will be differences between brands. Because the line (tubing) part of the serum sets is predominantly made of PVC raw material, it was thought that there would be a leakage of chemical exposure, especially phthalate derivatives, during the planning of the study. Although no quantitative analysis has been made for material components in this, Bernard et al.²³, it is stated that the main leakage from PVC-sourced products are phthalates. According to this information, we have considered that investigating a possible cytotoxic and genotoxic effect of chemical leaks and extracts of plastic materials of serum sets on mammalian cells is an important issue.

One of the objectives of this study is to compare different brands of products used in hospitals and sold in the market in terms of their toxic effects. There is no detailed information about the contents on the packages of 13 different brands of serum infusion sets provided for this study. Equal length samples taken from these sets coded with numbers 1–13 were used simultaneously in the cytotoxicity and genotoxicity tests under the same conditions. Because a visual scoring was made in the micronucleus test in the experiments, blind scoring was made by closing the sample names in the prepared preparations, and comparisons between negative and positive controls and sets were made later.

There are very few studies on this subject in the literature. In the study by Dakwar et al.²⁴, the deterioration in the qualitative properties of the infusion sets (such as color change, the release of potentially toxic chemicals) was investigated, and the relative toxicities of the leaking chemicals in different parts of each infusion LOT series were investigated. The sensitivity of selected different cell lines (HeLa and CEND cells) has been characterized and it has been shown that cEND brain endothelial cell lines can be used in in-vitro toxicity analyzes of disposable medical supplies leaks. After this study, the analysis of mechanisms in the toxicity of infusion set leaks in some cell lines (L-929 and bEnd.3 cells) was performed by in-vitro methods. In the study conducted by Kozlovskaya et al.²⁵, it was reported that the latex parts were the most toxic part, and it was shown that cell death was induced by oncosis due to the increased permeability of the cell membrane. They also investigated the in-vitro toxicities of infusion set leaks in a similar study, analyzing samples that did not show comparative toxicities from the same or different manufacturers in a single lot. They also stated that these cell lines are suitable for the in-vitro evaluation of disposable medical equipment toxicity in determining the sensitivity of toxic effects induced by the infusion set leaks on some cell lines (L-929, cEND, and bEnd).

Kozlovskaya et al.²⁶ investigated the in-vitro toxicities of infusion sets commonly used in Israel. Experimental results show that there is a significant level of toxicity induced by the analyzed samples, and they reported that they failed to meet the safety requirements (<30% reduction in metabolic activity in the MTT test, no significant changes in cell amount, proliferation, colony formation, etc.). These findings were also consistent with findings from previous in-vitro toxicity studies.²⁴

Within the scope of the study, the material components of the sets have not been analyzed. Kozlovskaya et al.²⁶ reported that natural and synthetic rubber material is the most toxic part of infusion sets, while the line and droplet chamber are less toxic. Therefore, the toxicity of infusion sets is considered depending on their design. There is no toxicity in the sets without flash ball and injection part (because it does not contain rubber part), and it is compatible with the recommendations of the regulatory bodies. On the other hand, parts containing rubber induced cell death due to toxic leaks. When the toxicity mechanism was investigated in detail, it was shown that infiltrates increase cell membrane permeability and induce cell death by oncosis via apoptotic intracellular pathways.²⁵ In the toxicity comparison of the rubber parts in the analyzed infusion sets, it was observed that the raw materials prepared from the flash ball and injection part were dependent on the material itself. For example, sets containing synthetic rubber flash balls were found to be more toxic than sets using natural rubber in the same part. However, because the storage period of the products is quite different from each other (2.5 and 13 years), it is thought that the storage conditions and duration also affect the toxicity. It is difficult to determine the safety impact of differences between raw material components and storage conditions.

We have observed leaking from serum infusion sets that occurred for 72 h were investigated in cell cultures in terms of their cytotoxic effects at 24 h of exposure. More detailed studies are needed with different cytotoxicity tests as well as NRU and MTT tests. However, it is thought that different results can be obtained by both hardenings the working conditions (such as temperature and pressure) at the stage of obtaining leaks and obtaining leaks at different durations and by allowing the media containing leaks to interact with the cells for different periods such as 48, 72, 96 h. In the micronucleus test, blood cells were planted in a medium containing leakage chemical, and a 72-h culture was applied with the target of cell divisions as recommended in the test protocol. It is thought that these effects can be investigated comparatively with genotoxicity tests such as the Comet test, where the effects of different exposure periods can be investigated. Considering that serum infusion sets stay in contact with the body for a maximum of 72 h in routine applications, testing of leaks occurring in interaction for 72 h in our study is the sufficient time to determine cytotoxic and genotoxic effects. However, toxicity should be examined both acutely and chronically, and the effects of repeated exposures should be investigated by other study plans.

It is reported in the literature that leaking chemicals are generally caused by plasticizing chemicals such as phthalate, especially DEHP. To determine the factor causing toxicity, toxicity mechanisms can be elucidated in detail by analyzing the material components. More research is needed to determine the toxic leakages from each part of the sets and to reveal the toxicity risks during clinical use. As a result, it has been shown that serum sets, which are used extensively in health applications, have weak cytotoxic and genotoxic effects in short-term infusions. Especially for short-term infusions, our study contributes to the answer to an important question in medical practice as well as the scientific field to know the risk of leakage chemicals such as DEHP for the patient.

Due to the harmful effects of phthalates, research on alternative non-PVC materials has become one of the preferential area of material engineering all over the world. As a result, 2 different approaches, using plasticizers non-DEHP or the use of different polymers instead of PVC were found appropriate. But lack of the data for toxicological tests of alternative DEHP-free materials is another problem of these area.²⁷

It is reported that the level of toxicity determined in the studies is affected by factors such as the cells studied, the media components in which the cells are grown, the exposure time, and the analysis method for toxicity assessment. In the mentioned study, researchers recommend the use of cEND or bEnd capillary endothelial cells. However, they stated that the MTT test is not safe enough for in-vitro toxicity evaluations of medical consumables and may give misleading results. In this study, 2 separate cytotoxicity tests, NRU and MTT test, were performed for cytotoxicity analyzes of serum sets, and the results were compared, and it was seen that the NRU test gave much more consistent results compared with the MTT test for in vitro toxicity evaluations of disposable medical consumables. In-vitro toxicity analysis methods (MTT test, microscope-based analysis, etc.) are not harmonized by different regulatory bodies. Set exposure with random toxic leaks does not particularly reflect conditions during clinical use of medical consumables (L-929 cells, 24-h exposure period, composition of extraction medium). It is thought that the MTT test is not very suitable for medical products, as stated, and may give misleading and incorrect results. Although cell viability decreased according to the NRU test and CBPI results, results such as no toxic effect in MTT test and increased cell viability in cells exposed to leakage chemicals compared with the negative control. Microscope-based analyzes and neutral red tests gave more consistent results. These results are supporting the use of in-vitro toxicity studies on the evaluation of disposable medical devices.

On the other hand, the MTT test is based on the measurement of mitochondrial activity that is metabolic activity. In the NRU method, which is based on the principle of measuring lysosomal activity, cell viability is demonstrated by a different mechanism than MTT. It is thought that the extract of the chemical material in the medium may interact with the cell membrane or enter the cell passing the membrane and accumulating in the organelles and cytoplasm so causing toxic effects.

In this study, the genotoxic effects of chemicals leaking from serum sets were also investigated in human lymphocytes by micronucleus test. Increases in micronuclei frequency also support the results that leaching chemicals may cause direct clastogenic damage to DNA or aneugenic damage during cell divisions.

5. Conclusion

In this study, we aimed to determine the possible cytotoxic and genotoxic effects of leakage chemicals from serum infusion sets in mammalian cells in vitro. For cytotoxicity evaluation, NRU and MTT tests were performed on L929 mouse fibroblast cells, and for the genotoxicity assessment, micronucleus test was applied on human peripheral blood lymphocyte cells. Considering the data obtained in this study all sets cause a decrease in cell viability and an increase in micronucleus frequencies. In-vitro tests in cytotoxicity and genotoxicity studies are considered first-line and preliminary biomarker studies. Therefore, in this study, it is thought that in-vivo tests should be performed to investigate the effects of chemicals leaking from serum sets in more detail. For more meaningful and clear results, in-vivo studies as well as different genetic tests are recommended.

Authors’ contributions

This study was completed by Ayşegül Özlü as a master’s thesis. Study experiments, data evaluations, and manuscript writing were done jointly by both authors. Conceptualization, funding acquisition, methodology, and writing—original draft was done by Gökçe Taner.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Conflict of interest: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

The data underlying the results are availableas part of the article and no additional data source data are required.