The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test

Regulatory Toxicology and Pharmacology xxx (2015) 1e7

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The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test Ana Paula Ressetti Abud a, 1, Jaiesa Zych a, 1, 2, Thamile Luciane Reus a, 1, Crisciele Kuligovski a, Elizabeth de Moraes a, b, Bruno Dallagiovanna a, Alessandra Melo de Aguiar a, * a b


sica de C

rio de Biologia Ba Laborato elulas-tronco, Instituto Carlos Chagas, Fiocruz, Curitiba, PR, Brazil Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 July 2015 Received in revised form 13 August 2015 Accepted 10 September 2015 Available online xxx

Human adipose-derived stem cells (ADSC) were evaluated as cell culture model for cytotoxicity assay and toxicity prediction by using the neutral red uptake assay (NRU). In this study, we compared ADSC and the murine cell line BALB/c 3T3 clone A31 to predict the toxicity of 12 reference substances as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods. We predicted the LD50 for RC-rat-only weight and RC-rat-only millimole regressions for both cell culture models. For RC rat-only weight regression, both cells had the same accordance (50%), while for RC rat-only millimole regression, the accordance was 50% for ADSC and 42% for 3T3s. Thus, ADSC have similar capability for GHS class prediction as the 3T3 cell line for the evaluated reference substances. Therefore, ADSCs showed the potential to be considered a novel model for use in evaluating cytotoxicity in drug development and industry as well as for regulatory purposes to reduce or replace the use of laboratory animals with acceptable sensitivity for toxicity prediction in humans. These cells can be used to complete the results from other models, mainly because of its human origin. Moreover, it is less expensive in comparison with other existing models. © 2015 Elsevier Inc. All rights reserved.

Keywords: Cytotoxicity Stem cells Toxicity prediction

1. Introduction The use of animals for toxicity assessment is applied for both quality control and product development. However, the use of animals has been questioned worldwide resulting in several efforts to reduce, replace or refine their use, as first mentioned by Russell and Burch published in 1959 as the 3Rs strategy (Russell and Burch,

Abbreviations: ADSC, human adipose-derived stem cells; BRACVAM, Brazilian Centre for the Evaluation of Alternative Methods; DMEM, Dulbecco's Modified Eagle's Medium; DMSO, dimethyl sulfoxide; D-PBS, Dulbecco's Phosphate Buffered Saline; ECVAM, European Centre for the Evaluation of Alternative Methods; GHS, Globally Harmonized System of Classification and Labeling of Chemicals; IC50, the dose that could decrease in vitro endpoint by 50%; ICCVAM, The Interagency Coordinating Committee on the Validation of Alternative Methods; LD50, lethal dose 50%; NHK, normal human keratinocytes; NRU, neutral red uptake assay; OECD, The Organization for Economic Co-operation and Development; RC, Registry of Cytotoxicity.
, Rua Professor * Corresponding author. Instituto Carlos Chagas, Fiocruz-Parana Algacyr Munhoz Mader, 3775, Curitiba, PR 81350-010, Brazil. E-mail address: alessandra_aguiar@fiocruz.br (A.M. Aguiar). 1 Authors contributed equally to this work. 2
, TECPAR. Curitiba, PR, Brazil. Present address: Instituto de Tecnologia do Parana

1959), which has been extensively reviewed since then (Flecknell, 2002; Kandarova and Letasiova, 2011). Liebsch and colleagues also noted that it is very important to use human cells and tissues for toxicity prediction rather than animals (Liebsch et al., 2011). Reducing animal number has many implications, such as economic and social impact. For this purpose, international and national committees were created to organize the validation of alternative methods. Examples of such committees include ECVAM (European Centre for the Evaluation of Alternative Methods) in Europe, ICCVAM (The Interagency Coordinating Committee on the Validation of Alternative Methods) in USA (Stokes, 2002) and the recently created BRACVAM, (Brazilian Centre for the Evaluation of Alternative Methods) in Brazil (Eskes et al., 2009). Moreover, Brazil regulatory authorities have accepted 17 methods to animal testing in 2014. Laboratories that perform the correspondent animal test have to fully replace them in till 2019. Among these 17 methods, it has been recognized the use of the in vitro cytotoxicity test to estimate starting doses for acute oral systemic toxicity, using cell lines 3T3 (murine fibroblast) or NHK (normal human keratinocytes) as recommended by OECD (The Organization for Economic Cooperation and Development) as test guidance no. 129 (OECD,

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Please cite this article in press as: Abud, A.P.R., et al., The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test, Regulatory Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.yrtph.2015.09.015

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A.P.R. Abud et al. / Regulatory Toxicology and Pharmacology xxx (2015) 1e7

2010). In addition, many attempts have been developed that aimed to validate alternative methods of toxicity prediction. Special concern is about the poor correlation between in vitro data, which are expressed by IC50 (the dose that could decrease in vitro endpoint by 50%) values and in vivo data, which are expressed by LD50 (lethal dose 50%). This correlation was previously established by the Registry of Cytotoxicity (RC) (Halle, 2003) and it was further complemented by ICCVAM (ICCVAM and 2006a; ICCVAM and 2006b) to get a more reliable LD50 prediction for not only pure substances but also for mixtures (ICCVAM and 2006b). As mentioned before, 3T3 cells and NHKs are widely accepted as cell culture model for the neutral red uptake assay to determine starting doses for acute oral toxicity tests and correlation between IC50 and LD50 prediction (ICCVAM and 2006a; ICCVAM and 2006b; ICCVAM and 2006c; ICCVAM and 2006d; ICCVAM and 2006e). The main cellular model used in cytotoxicity assays are primary cell cultures and transformed strains. Each of the cell models has both advantages and disadvantages. The disadvantages are mainly related to the high cost and variability of primary cultures and genetic instability or physiological response of transformed strains; Additionally, these cell models are expected to be used for the toxicity prediction in animal models and not for the prediction of toxicity in humans, whereas there are no in vitro validated methodology for this aim. Stem cells play an important role in regenerative medicine therapies because of their main properties of self-renewal and differentiation into many cell types representing a great step for healing processes and cell therapy, as reviewed in (Nava et al., 2012). Self-renewal and differentiation process are also interesting for cytotoxicity assays (Hook, 2012; Kang and Trosko, 2011). Stem cells are a good model for predicting toxicity because they can be used to evaluate cytotoxic effects on cell viability as well as the cell differentiation process (Hook, 2012; Luttun and Verfaillie, 2005), and possibly more relevant for human toxicity prediction. Moreover, they represent a well established in vitro model for human multipotent cell population, they do not require as much supplements as NHK and finally, they are available from commercial sources or they can be isolated in house. The use of adult stem cells in cytotoxicity assays has also been previously discussed (Hook, 2012; Kang and Trosko, 2011). Although adult stem cells have more limited self-renewal and a more restricted differentiation potential than pluripotent stem cells (Hook, 2012; Luttun and Verfaillie, 2005) they have the advantage of physiological relevance, availability, reproducibility and scalability for toxicity prediction (Hook, 2012). Bone marrow mesenchymal stem cells were evaluated as good cell culture model for toxic class prediction by neutral red uptake compared with NHKs and murine 3T3 fibroblasts (Scanu et al., 2011). Though these results are promising, bone marrow-derived stem cells are not so easily obtained, so other sources of multipotent stem cells must be evaluated for cytotoxicity assays. There are no reports about the use of adipose tissue derived stem cells (ADSC) for toxicology evaluation by a validated methodology. ADSC are readily obtained from subcutaneous adipose tissue lipoaspirates. This tissue is normally discharged, which represents an available source for stem cell isolation (Gimble et al., 2007). The aim of this study is to evaluate the use of ADSC as cell culture model for in vitro toxicology assay in comparison with a validated murine model. 2. Methods 2.1. Cells and culture conditions Human Adipose-Derived Stem Cells (ADSC) from lipoaspirates were purchased from a commercial source (Lonza®, Walkersville,

USA; catalog number PT-5006). ADSC from 2 different lots were cultured in accordance with datasheet instructions. In brief, cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco Invitrogen®, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Gibco Invitrogen®, Carlsbad, California, USA) and 4 mM L-glutamine (Gibco Invitrogen®, Carlsbad, California, USA), without antibiotics. The cells were maintained at 37
C in 5% CO2 atmosphere. 3T3 NIH Clone A31 cells (Rio de Janeiro Cell Bank e BCRJ, Rio de Janeiro, Brazil) were cultured in accordance with datasheet instructions. Culture conditions to the assay are the same as described above and the cell concentration was 3.5  104/ ml for ADSC and 2.5  104/ml for 3T3. 2.2. Test substances To evaluate cytotoxicity prediction, 12 reference substances with known toxicity effects according to the classification of the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) were selected for this study. According to ICCVAM (ICCVAM and 2006e), it is necessary to test at least 12 from thirty reference substances to evaluate the neutral red uptake (NRU) assay reliability in comparison with the validated 3T3 or NHK NRU test method. We chose two from each GHS hazard categories and two unclassified ones. Positive control, sodium dodecyl sulfate is included in this list (Table 1). All of the chemicals were purchased from SigmaeAldrich® (Saint Louis, USA). Test substance preparation was performed in accordance with ICCVAM recommendations (ICCVAM and 2006b). Solubility and diluent were appropriately determined. In brief, each drug was immediately solubilized, and serial dilutions were applied to cell culture plates that had been previously prepared. In total, six wells were used for each drug concentration. When the chemical was not soluble on cell medium, we used DMSO Dimethyl Sulfoxide, SigmaeAldrich® (Saint Louis, USA) and the same concentration of this solvent was used on control group medium. We used serial dilution of each drug using log-factor. Eight serial dilutions were made and the log-factor were used according to the cell type. We used ICCVAM recommendations (ICCVAM and 2006c) to these assays. All substances remained in culture for 48 h until cytotoxicity evaluation by neutral red uptake assay. 2.3. Neutral red uptake (NRU) For cytotoxicity evaluation, the dose of each test compound that could decrease cell viability by 50% (IC50) was determined. This value is used to determine the toxicity prediction compared with previous LD50 (lethal dose 50%) data, which is an in vivo test used to assess toxicity. For this purpose, the methodology of neutral red uptake (ICCVAM and 2006c) was applied. This is a quantitative cell viability assay in which the toxic effect of the test substance is correlated with the decay of the neutral red uptake by cells. The test is recommended by OECD (OECD, 2010) for determining the initial estimated doses for acute oral toxicity, among others. Briefly, after rinsing with D-PBS (Dulbecco's Phosphate Buffered Saline), cells were stained with 25 mg/mL neutral red (SigmaeAldrich®, Saint Louis, USA) in DMEM (Gibco Invitrogen®, Carlsbad, California, USA) and incubated for 3 h at 37
C. Wells were rinsed with D-PBS, and neutral red that was taken up by the cells was extracted with a solution composed of 50% ethanol and 1% acetic acid (Merck®, Whitehouse Station, New Jersey, USA). Plates were shaken for 20e45 min to extract NR from the cells and form a homogeneous solution. The optical density of the samples was measured at 540 nm on a Synergy H1 Multi-Mode reader (Biotek®,

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Table 1 Test substances. GHSa class

Test substance

CASb number

Diluent

LD50  5 mg/kg (class 1) LD50  5 mg/kg (class 1) 5 < LD50  50 mg/kg (class 2) 5 < LD50  50 mg/kg (class 2) 50 < LD50  300 mg/kg (class 3) 50 < LD50  300 mg/kg (class 3) 300 < LD50  2000 mg/kg (class 4) 300 < LD50  2000 mg/kg (class 4) 2000 < LD50  5000 mg/kg (class 5) 2000 < LD50  5000 mg/kg (class 5) LD50 > 5000 mg/kg (unclassified e class 6) LD50 > 5000 mg/kg (unclassified e class 6)

Cycloheximide Indomethacin Sodium arsenite Sodium dichromate dihydrate Sodium oxalate Hexachlorophene Propranolol HCl Sodium dodecyl sulfate Potassium chloride Trichloroacetic acid Glycerol Ethylene glycol

66-81-9 53-86-1 7784-46-5 7789-12-0 62-76-0 70-30-4 3506-09-0 151-21-3 7447-40-7 76-03-9 56-81-5 107-21-1

Culture DMSO Culture Culture Culture DMSO Culture Culture Culture Culture Culture Culture

a b

medium medium medium medium medium medium medium medium medium medium

Globally Harmonized System of Classification and Labeling of Chemicals. Chemical Abstract Service.

Winooski, Vermont, USA). Absorption was assessed using the blanks as reference. For each set of test substance plates used in an assay, a separate plate of SDS concentrations was used as a positive control. All of the acceptance criteria described in the BALB/c 3T3 NRU Cytotoxicity Test Method by ICCVAM (Test Acceptance Criteria for Positive Control and Test Acceptance Criteria for Test Substances) (ICCVAM and 2006c) were considered in the tests. 2.4. Data and statistical analysis Data were analyzed in accordance with ICCVAM recommendations (OECD, 2010; ICCVAM and 2006c; ICCVAM and 2006e; Scanu et al., 2011). In brief, data were analyzed in Microsoft Office Excel, 365® to determine cell viability and were converted into a percentage of the control. To calculate the IC50 value, data were transferred to GraphPad Prism® 6.0 to apply a Sigmoidal doseeresponse (variable slope) with four parameters. These parameters were used with rearranged Hill Function, which is considered to be the best mathematical model to fit in vitro dose response data (ICCVAM and 2006e; Scanu et al., 2011). Outliers were analyzed using Grubbs test (available online in http://graphpad.com/ quickcalcs/Grubbs1.cfm). IC50 data were expressed as the geometric mean ± standard deviation of three independent experiments for each test substance and for each cell model (6 replicates), 2 lot of ADSC were evaluated in comparison to one lot of 3T3. The calculated IC50 value and LD50 available data (ICCVAM and 2006c; ICCVAM and 2006e) were transformed to log values and plotted as a linear regression. Linear fits were compared with the F test, and p value and slope were also obtained. IC50 was also used to predict LD50 and GHS class for test substances. The IC50 geometric mean was used to predict the LD50 by RC-rat only weight regression using the formula log LD50 (mg/ kg) ¼ 0.372 log IC50 (mg/ml) þ 2.024 (R2 ¼ 0.325) or RC-rat only millimole regression using the formula log LD50 (mmol/kg) ¼ 0.439 log IC50 (mM) þ 0.621 (R2 ¼ 0.452) (ICCVAM and 2006b; Scanu et al., 2011). Both regressions can be used for LD50 prediction though RC rat-only weight regression is normally used for mixtures where test substances are in combination with others, while RC ratonly millimole regression is normally used for pure substances (ICCVAM and 2006e). 3. Results 3.1. ADSC is an adequate cell model for neutral red uptake (NRU) in cytotoxic assays The first question raised with this study was whether ADSC

could perform NRU with the same efficiency as 3T3 cells, which is the reference cell line. Thus, NRU was performed, and both cells took up the dye (Fig. 1A, control). The second step was to determine whether a viability doseeresponse curve could be obtained with ADSC compared with 3T3. For this aim, NRU was performed with sodium dodecyl sulfate (a GHS class 4 chemical), which is a NRU positive control that is recommended by ICCVAM (ICCVAM and 2006b). Morphological analysis (Fig. 1A) and dose response curves (Fig. 1B) based on Hill function demonstrated that ADSC respond in a dose-dependent manner to cytotoxic chemicals and demonstrated excellent correlation with Hill function as demonstrated by R2 above 0.95. Thus, ADSC were adequate as a cell culture model for NRU viability assays for cytotoxicity evaluation. 3.2. ADSC as cell model for IC50 calculation to predict LD50 Our primary aim was to determine whether ADSC were adequate cell culture model for cytotoxicity evaluation and LD50 prediction. For this purpose, we calculated IC50 values for the 12 chemicals (three assays per chemical) by the geometric mean as well as in mg/mL (Table 2). IC50 values expressed in mg/mL were convert to mM in order to predict LD50 by RC-rat only millimole regression (Table 3). To determine whether ADSC had similar capability for predicting LD50 as the 3T3 cell line, linear regressions were plotted using log IC50 values (mg/ml) and log LD50 values (mg/kg) (Fig. 2A) or log IC50 values (mM) and log LD50 values (mmol/kg) (Fig. 2B). Linear fits were compared by an F-test, and there was no difference between and 3T3 and ADSC1 (Fig. 2AeB). Therefore, ADSC have similar capability for GHS class toxicity prediction as the 3T3 cell line for the substances evaluated. Thus, IC50 values were used for LD50 calculation to predict the GHS class of toxicity (Table 3). Thus, prediction was correct for 50% of test substances in ADSC using both regression models, while for the 3T3 cell line, it was 50% correct with RC-rat only weight regression and 41.7% correct with RC-rat only millimole regression. Both ADSC and 3T3 cells were correct in prediction of GHS class of toxicity for less toxic test substances and both failed to predict the correct GHS class for more toxic chemicals. There was no overprediction of the GHS class of toxicity for ADSC using both regressions, while for 3T3, one test substance (8.3%) was overpredicted when RC rat-only weight regression was used, and two substances (16.7%) were overpredicted when RC rat-only millimole regression was used. Therefore, ADSC were as adequate cell culture model for the GHS class of toxicity prediction as 3T3 by the NRU assay. In contrast to very similar GHS class correct prediction, the absolute LD50 value predicted by ADSC was higher than 3T3 cells, especially for class 1 test substances (Table 3). The GHS

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Fig. 1. SDS cytotoxicity in the (NRU) assay. Murine Balb/c 3T3 clone A31 cells and ADSC were treated for 48 h with different SDS dilutions and were then incubated with neutral red solution for 3 h. SDS cytotoxicity is demonstrated by the absence or a reduction in neutral red uptake compared with the control (A). SDS cell viability by NRU for 3T3, ADSC1 and ADSC2 (B). A dose response curve that fits Hill Function was obtained for 3T3 (R2 ¼ 0.9894) and both ADSC samples (R2 ¼ 0.9759 and R2 ¼ 0.9699, respectively). Representative data from one experiment. Bar ¼ 30 mm.

Table 2 IC50 values in mg/mL as calculated from the geometric mean of three independent experiments and standard deviation. GHS class

Test substance

1 1 2 2 3 3 4 4 5 5 6 6

Cycloheximide Indomethacin Sodium arsenite Sodium dichromate dihydrate Sodium oxalate Hexachlorophene Propranolol HCl Sodium dodecyl sulfate Potassium chloride Trichloroacetic acid Glycerol Ethylene glycol

Geometric mean IC50 mg/mL

Standard deviation

3T3

ADSC1

ADSC2

3T3

ADSC1

ADSC2

0.040 103.9 1.6 0.61 204.6 4.0 19.1 43.8 4331.2 2096.5 43313.7 38293.5

20.7 1020.7 5.3 1.3 147.2 41,0 53.4 67.0 8254.8 6974.1 96937.2 79274.9

54.5 1085.9 7.5 0.2 251.0 33.3 43.5 70.7 7301.3 6182.7 112073.1 82772.3

0.001 16.5 0.9 0.2 27.8 2.3 8.6 4 1754.4 508.3 10669.2 7622.0

3.5 159.0 0.3 0.2 76.9 8.2 3.4 5.7 1766.6 226.3 16730.7 30468.1

12.6 427.6 0.9 0.2 66.2 3.7 2.7 5.0 436.5 613.7 8079.7 22804.1

class predicted by ADCS was underestimated by 3 or 4 classes, whereas it was underestimated by 1, 2 or 3 classes by 3T3 cells. These data indicates that ADSC are more resistant than 3T3 cells for tested substances. 4. Discussion In this study, the use of human ADSC was evaluated for toxicity prediction by applying a validated protocol (OECD, 2010; ICCVAM and 2006c). Our results suggest that ADSC could be used as a new cell culture model for predicting starting doses in acute toxicity assays. Moreover, ADSC are comparable to the reference cell line 3T3 clone A31 for GHS class prediction by NRU in accordance with a validated methodology. Among the 12 test substances evaluated in this study, 8 were tested previously with bone marrow-derived mesenchymal stem cells (Scanu et al., 2011). In this study, there was 50% agreement of

GHS class prediction (4/8) for both RC-rat only weight regression or RC-rat only millimole regression, which was equivalent to that observed in our study. In contrast, 10 of 12 test substances were also evaluated using NHK cells (normal human keratinocytes), and in this case, there was only 30% agreement (3/10) for GHS class prediction using both mathematical regressions (ICCVAM and 2006c). NHK cells also demand much more culture media supplements than ADSC. Thus, ADSC are less expensive and more reliable than NHK for GHS class prediction by NRU. To our knowledge, this is the first time that ADSC were used for LD50 prediction following the NRU ICCVAM protocol. Although we have demonstrated promising data, the evaluated in vitro system for predicting starting doses for acute toxicity assays failed to determine the correct GHS class for the most toxic test substances in this and other studies (ICCVAM and 2006c; Scanu et al., 2011). Thus, this method could be used for an initial drug screening, albeit more accurate and sensitive methods need to be still developed. In

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Table 3 LD50 predictions. LD50 were predicted from the geometric mean of the IC50 by RC-rat only weight regression or rat only millimole regression for the experimental data. n ¼ 3.

Test substance

LD50

(Toxic Class)

mg/kg #

Cycloheximide (class 1) Indomethacin (class 1) Sodium arsenite (class 2) Sodium dichromate dihydrate (class 2) Sodium oxalate (class 3) Hexachlorophene (class 3) Propranolol HCl (class 4) Sodium dodecyl sulfate (class 4) Potassium chloride (class 5) Trichloroacetic acid (class 5) Glycerol (class 6) Ethylene glycol (class 6)

Predicted LD50 (mg/kg) by RC -rat only weight regression

Predicted LD50 (mg/kg) by RC- rat only millimole regression

3T3

ADSC1

ADSC2

3T3

ADSC1

ADSC2

2

30.6*

326.5***

467.7***

125.4**

374.2***

571.9***

2.42

594.4***

1390.9***

1423.3***

868.5***

2368.6****

2433.8****

41

124.6*

195.8*

223.6*

77.8*

132.7*

155.2*

50

87.8*

116.5*

106.9*

82.0*

114.5*

103.5*

155

765.0 *

676.9*

825.4*

674.3*

583.5*

737.5*

61

177

420.7*

389.4*

223.5

620.8*

566.6*

470

316.6

464.1

429.9

371.2

582.9

532.5

1288

431.1

504.9

515.2

526.7

634.7

650.0

b

2602

2381.3

3027

2891.9

1853.1

2459.7

2330.6

4999

1818.0b

2843

2718.4

2092.9

3547.4

3364.7

8567

5608.2

7567.9

7987.6

5733.3

8165.7

8702.7

b

5991.4

6106.0

12691

5357

7022.3

7136

4353.1

Color code: Right GHS class prediction (green), * underprediction for one class (yellow), ** underprediction for two classes (orange), ***underprediction for three classes (blue), ****underprediction for four classes (red), b overprediction (gray). # ICCVAM 2006c

Fig. 2. IC50 and LD50 correlation. The geometric mean of IC50 values obtained for 3T3 (continuous line) ADSC1 (dotted line) and ADSC2 (dashed line) for each test substance are expressed in log of concentration in mg/mL (A) or mM (B) in correlation with the LD50 value expressed in log of mg/kg (A) or millimol/kg (B). F test calculated p values for slope and intercept F test are shown for each ADSC donor compared with 3T3 (A and B).

addition to the previously described application of BM-MSCs in cytotoxicity assays (Scanu et al., 2011), we confirmed that ADSC can also be used for this purpose with similar predictive capability of 3T3 cells and had even better accuracy of GHS class prediction than NHK cells for the evaluated test substances as demonstrated in previous studies (ICCVAM and 2006c; Scanu et al., 2011). Furthermore, they are healthy human cells that can be easily isolated and characterized (Dominici et al., 2006). Moreover, adult stem cells from adipose tissue have become an accessible and reproducible model for several purposes, and there is increased availability from healthy donors undergoing elective aesthetic procedures from

whom adult stem cells can be obtained from disposed tissue (Baer, 2014). Different commercial sources of adult stem cells are also available. Both ADSC and 3T3 cells are comparable in prediction of GHS class of toxicity; however, both failed to predict the correct GHS class for more toxic test substances in this study. We also observed that ADSC were more resistant than 3T3 cells, especially for more toxic class 1 test substances, as the LD50 predicted by ADSC for these substances was underestimated by 3 or 4 classes. Thus, ADSC can survive higher concentrations than 3T3 cells. It is unknown whether the differentiation potential of ADSC would remain

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unaffected in these conditions. The effect of low triclosan concentrations on adipogenic human mesenchymal stem cell differentiation was reported even in non-toxic concentrations for cell viability or lactate dehydrogenase release (Guo et al., 2012). For future works, the differentiation potential of adult stem cells would be investigated as a more accurate endpoint for toxicity evaluation than viability. Stem cells are also very promising as an alternative to animal testing for toxicity prediction of drugs and chemicals. While numerous somatic cell lines (i.e., human keratinocytes and 3T3 mouse fibroblasts) are useful in regard to testing toxicity, adult stem cells have represented a new cell model capable of predicting GHS class (Scanu et al., 2011). These cells have important advantages because they are human and easily obtained, and they are able to self-renew and differentiate into many cell types (Luttun and Verfaillie, 2005). Another advantage of using these cells is the possibility to analyze the mode of action of a drug on a patient's own cells, which provides unprecedented human models to study both disease pathology in different genetic backgrounds as well as their response to drugs during their development (Bellin et al., 2012). So far, there is a validated methodology by ECVAM (European Committee for the Validation of Alternative Methods) that uses murine embryonic stem cells in a co-culture system with 3T3 cells to assess embryotoxicity. Despite the high accuracy of this methodology, the test alone is not sufficient for regulatory purposes and should be used within an integrated testing strategy (ECVAM, 2010). Moreover, it consists of an assay that uses murine cells (non-human system), which may impact the prediction of toxicity in humans; therefore, assays using human embryonic stem cells are being developed (Behar et al., 2012; Rajamohan et al., 2013; Coleman, 2014), although they still need to be validated. The main concern nowadays is to develop and validate alternative methods to laboratory animals. In Brazil, the Brazilian Centre for the Validation of Alternative Methods (BraCVAM) is helding the validation of alternative methods in interlaboratorial studies (Eskes et al., 2009; Presgarve, 2008). Additionally, the methodology developed in this work should be evaluated in a collaborative study in different laboratories in order to complete its validation process. We expect in the future, possibly with the development of new researches with human cells, specially stem cells for cytotoxicity assays, the statement, about the prediction of the acute toxicity not only in animals but also in humans from cytotoxicity data become a reality (Halle, 2003).

5. Conclusion In summary, ADSC represent an interesting cell model for alternative assays to animal tests with great relevance for toxicity prediction that represent important potential for industry applications and for regulatory purposes. They can be used to evaluate chemical compound cytotoxicity including products that are already established or are still in development. Therefore, they can bring a new perspective to cytotoxicity tests by reducing or replacing the use of laboratory animals. In summary, ADSC represents an interesting cellular model for alternative tests to animal testing. Such cells show comparable results for the 3T3 cell line, but with the advantage of being of human origin. Thus, it is concluded that this model could be used to complete the data provided by existing models, with great relevance for predicting toxicity. Our results suggest that they can be used to evaluate the cytotoxicity of chemical compounds, including products that are already established or are under development.

Conflicts of interest We report no potential conflicts of interest or financial interests. Acknowledgments The authors would like to thank Dr. Alejandro Correa and Dr. Marco Stimamiglio for review and very useful suggestions and Dr. ^go Barros Caruso for advice on statistical analysis. Rodrigo Re The authors thank the Program for Technological Development in Tools for Health-PDTISFIOCRUZ for the use of its facilities. ~o Oswaldo Cruz e FIOCRUZ This work was supported by Fundaça ~o Arauca
ria. and CAPES/Fundaça ~o A.P.R.A. and J.Z. received a fellowship from CAPES (Coordenaça ~o Arauca
ria, de Aperfeiçoamento de Pessoal de Nível Superior)/Fundaça and T.L.R. received a fellowship from CAPES. B.D received a fellowship (grant number: 309935/2012-1) from Conselho Nacional
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Please cite this article in press as: Abud, A.P.R., et al., The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test, Regulatory Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.yrtph.2015.09.015