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An in vitro screening test for the assessment of the irritant potential of parenteral formulations
Toxic. in Vitro Vol. 3, No. 4, pp. 335-339, 1989 Printed in Great Britain. All rights reserved
0887-2333/89 $3.00 + 0.00 Copyright © 1989 Pergamon Press pie
AN IN VITRO SCREENING TEST FOR THE ASSESSMENT OF THE IRRITANT POTENTIAL OF PARENTERAL FORMULATIONS S. A. WILSON, M. L. JASIEWICZ,F. W. BONNER, S. BROWN and C. J. POTTER Toxicology Department and Pharmaceutical Sciences Department, Sterling-Winthrop Research Centre, Alnwick, Northumberland NE66 2JH, UK (Received 17 October 1988; revisions received 3 February 1989)
Abstract--A HeLa cell/neutral red cytotoxicity assay has been investigated as an in vitro screen for assessing the irritancy potential of parenteral formulations. Five commercially available intramuscular injectables, three irritants and two non-irritants, were examined for cytotoxic effects in this system. The ranking of the injectables tested, in order of decreasing cytotoxic effects, correlated with both previously reported qualitative in vitro data and their reported irritancy when administered to man. The results show that the HeLa cell/neutral red cytotoxicity assay may provide an alternative to animal studies for the assessment of the irritancy potential of parenteral formulations during the preliminary stages of their development.
INTRODUCTION
When administered to man, intramuscular drug preparations can produce varying degrees of discomfort resulting in pain, irritation and damage to muscle tissue. It is important, therefore, that a test for local tissue tolerance is included in the safety assessment for such products. Currently, there are no regulatory guidelines for the conduct of such studies and within the pharmaceutical industry a number of different methods have been utilized (Cingolani et al., 1985; Comereski et al., 1986; Nelson et al., 1949; Wilkinson, 1970). The basic principle of these tests is that a drug is injected intramuscularly into an experimental animal, which is killed at a certain time after the injection. Muscular damage is subsequently quantified by determination of serum creatine kinase and/or by histopathology and microscopic examination of the lesion at the injection site. In recent years, public concern for animal welfare, together with the escalating costs incurred with the use of laboratory animals, have promoted the need for the establishment and validation of alternative assays using cells in culture. An additional limitation of animal studies is that they are not particularly well suited for the screening of the large numbers of parenteral formulations that may be produced during initial pre-formulation studies. For the assessment of irritant potential in the preliminary stages of development, an in vitro model predictive of in vivo muscle damage would provide an attractive adjunct to animal studies. Abbreviations: EMEM = Earle's modified Eagle's minimal
essential medium supplemented with 10% foetal calf serum, 292 #g L-glutamine/ml, I0 pg gentamicin/ml and 2 mg sodium bicarbonate/ml; MTD = maximum tolerated dose; NR = neutral red; NRs0 = concentration of test agent that reduces the test colour to 50% of the control absorbance; NR90 = concentration of test agent that reduces the test colour to 90% of the control absorbance.
A qualitative in vitro assay for the assessment of the irritant potential of injectable drug formulations has been described by Oshida et al. (1979). This assay was based on the visual assessment of neutral red (NR) uptake by HeLa cells following treatment with a wide variety of injectables. Quantitative N R uptake assays have subsequently been developed for use as first-order screening tests in safety assessment strategies. A wide variety of compounds has been tested, and these methods are considered to be reliable for predicting ocular irritancy potential (Borenfreund and Puerner, 1985b; Hockley and Baxter, 1986), for comparing relative toxicities (Riddell et al., 1986) and for determining structure-activity relationships (Babich and Borenfreund, 1987). The study described here was undertaken to evaluate the applicability of a quantitative HeLa cell/NR uptake assay to the irritancy testing of injectable formulations. MATERIALS AND METHODS
Cell culture
HeLa $3 cells, obtained from Huntingdon Research Centre, Huntingdon, UK, were grown in Earle's modified Eagle's minimal essential medium supplemented with 10% foetal calf serum, 292 #g L-glutamine/ml, 10pg gentamicin/ml and 2 m g sodium bicarbonate/ml. The media components were obtained from GIBCO Europe Ltd, Paisley, UK, and the serum was obtained from Sera Labs, Crawley, UK. Complete culture medium will subsequently be designated EMEM. The cells were maintained as monolayer cultures in a humidified 5% CO2 atmosphere at 37°C and were released from the flask surface with 0.25% trypsin solution. Test articles
The non-irritants hyoscine butylbromide 20 mg/ml injection (Boehringer Ingelheim, Brackneli, UK), and sodium chloride 0.9% (w/v) infusion (Boots Hospital
335
336
S.A. WILSON et al.
Products, Nottingham, UK), were used as supplied by the manufacturers, as was the irritant gentamicin sulphate 40 mg/ml injection (Roussel Laboratories, Uxbridge, UK). The irritants tetracycline hydrochloride 50 mg/ml injection (Lederle Laboratories, Gosport, UK), and streptomycin sulphate 250 mg/ml injection (Evans Medical, Beaconsfield, UK), were reconstituted in water for injection according to the manufacturers' instructions immediately before use. Hyoscine butylbromide and streptomycin sulphate injections are solutions of the respective drugs in water for injection. When reconstituted, tetracycline hydrochloride injection contains 125 mg/ml ascorbic acid as a buffering agent. Gentamicin sulphate injection contains an unspecified, but 'suitable', preservative. All the test agents were diluted in E M E M before addition to the cells. Neutral Red medium A filter sterilized solution of N R (0.3%, w/v) was obtained from Sigma Chemical Co. Ltd, (Poole, Dorset, UK). An aliquot of this was aseptically added to E M E M to give a final N R concentration of 50/~ g/ml. The addition of N R to culture medium results in the formation of a fine precipitate. To prevent this from interfering with the assay, N R medium was incubated overnight at 37°C and centrifuged at 1500g for 10min before use. Cytotoxicity assay Each well of a 96-well microtitre plate was seeded with 9 × 103 H e L a $3 cells in 0.2 ml E M E M and the plate was incubated for 24 hr at 37°C in 5% CO2. The culture medium was then removed and replaced with 0.2 ml E M E M containing the appropriate concentration of injectable under test. Each of seven concentrations was added to eight wells of the plate (one lane). Two eight-well lanes were treated with 0.2 ml E M E M and two lanes were treated with 0.2ml E M E M containing water for injection at an equivalent volume to the highest concentration of test injectable (solvent control). To serve as a blank for subsequent absorbance measurements, the remaining lane was treated throughout all stages of each experiment with EMEM. Following incubation for 24 hr in the presence of the test injectable, the culture medium was removed and replaced with 0.2 ml N R medium. The plate was incubated for a further 3 hr at 37°C. The N R medium was then removed and the wells were rinsed twice with phosphate-buffered saline to remove unincorporated stain. The plate was allowed to drain and 0.2 ml 1% acetic acid-50% ethanol was added to each well to extract the dye. The plate was left for 20 rain at r o o m temperature, then shaken gently before the absorbance of each well was measured at 540nm on a microtitre plate-reading spectrophotometer (Microtek Multiscan MC, Flow Laboratories, Irvine, UK). In preliminary dose-ranging studies, each injectable was tested at seven concentrations over the range 0.0001 to 100/11 injectable/ml E M E M . During these preliminary studies, cytotoxicity was also assessed microscopically prior to the determination of N R uptake. F r o m microscopic examination it was
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possible to determine the m a x i m u m tolerated dose (MTD), the concentration causing only minimal morphological changes. Such changes were manifested as a reduction in cell density or by a change in cell shape. For a more accurate evaluation of relative cytotoxicity, each injectable was subsequently tested at seven concentrations over a narrow dose range (0.25 to 10 x MTD). For these studies at least one independent repeat assay was performed. To standardize the assay, the relationship between absorbance at 540 nm and H e L a $3 cell number was investigated. After allowing for cell attachment, various densitities of cells (103 to 4 × 104 per well; eight wells per density) were incubated with 0.2ml N R medium for 3 hr. The dye was then extracted as indicated above and the absorbance of each well measured at 540 nm. RESULTS The relationship between the number of H e L a $3 cells seeded and the amount of N R extracted is shown in Fig. 1. Each point represents the mean results from two independent experiments. There is a good linearity from 1 × 103 to 4 x 104 cells per well (linear regression coefficient 0.997). F o r H e L a $3 cells seeded at a density of 9 × 103 cells per well, 48-hr growth in culture medium under the assay conditions yielded absorbances at 540 nm between 0.4 and 0.5. Table 1. Comparison of MTD and NRgovalues determined during dose-ranging studies in the HeLa cell neutral red assay MTD* NRg0t Injectable (,uI/ml) (# l/ml) Sodium chloride 0.9% (w/v) > 100 > 100 infusion Hyoscine butylbromide 100 39.8 20 mg/ml injection Gentamicin sulphate 10.0 2.8 40 mg/ml injection Streptomycin sulphate 10.0 2.6 250 mg/ml injection Tetracycline hydrochloride 1.0 1.1 50 mg/ml injection *Maximum tolerated dose, concentration that causes only minimal morphological alteration on microscopic examination. fConcentration that reduces the absorbance at 540 nm to 90% of the control.
lrritancy screen for parenteral formulations 120 •
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Fig. 2. Effects of irritant injectables on the uptake of NR by HeLa $3 cells: (a) tetracycline hydrochloride 50 mg/ml injection; (b) streptomycin sulphate 250 mg/ml injection; and (c) gentamicin sulphate 40 mg/ml injection. The first part of the cytotoxicity assay consisted of the screening of a wide range of concentrations (0.0001-100pl injectable/ml EMEM). From these dose-ranging studies, two parameters, the MTD and an approximate NRg0 value, were determined and are presented in Table 1. The NR90 value is the concentration of test agent that reduces the test colour to 90% of the control absorbance. For both irritant and non-irritant injectables there is close agreement between the visually designated MTD and the measured decrease in dye absorption. The MTD values ranged from 1 #l/ml for the irritant injectable tetracycline hydrochloride 50 mg/ml injection, to > 100/d/ml for the non-irritant sodium chloride 0.9% (w/v) infusion. The NR90 values for these injectables, determined following microscopic examination of the wells in the dose-ranging studies, were 1.1 and >100/A/ml, respectively. Poorer agreement between the MTD and NRg0 values was observed for gentamicin sulphate 40 mg/ml injection, streptomycin sulphate 250 mg/ml injection, and hyoscine butylbromide 20 mg/ml injection (Table 1). This is probably due to the dilution series used in preliminary studies. The second part of the assay involved the screening of a narrower range of concentrations (0.25 to 10 x MTD). The dose-response curves for the three irritant injectables, gentamicin sulphate, tetracycline hydrochloride and streptomycin sulphate, are shown in Fig. 2. The reproducibility of the cytotoxic effects of tetracycline hydrochloride injection in HeLa $3 cells is illustrated in Fig. 2a, which shows the curves obtained from four independent determinations. Figures 2b and c show the mean dose-response curves for streptomycin sulphate injection and gentamicin sulphate injection, respectively. For these injectables similar reproducible cytotoxic effects were obtained, as indicated by the error bars. The mean doseresponse curves for the non-irritant injectables
hyoscine butylbromide and sodium chloride are given in Fig. 3, together with error bars that represent the reproducibility of their cytotoxic effects. A narrower range of concentrations was not tested for either of these injectables since the wide-ranging studies were sufficient to indicate their lack of cytotoxicity. The mean NRg0 and NRso values, where obtainable, determined from these dose-response curves are given in Table 2. The NRs0 value is the concentration of test agent that reduces the absorbance to 50% of the control value. Differences in NRg0 values were observed in the two parts of the assay, particularly for streptomycin sulphate injection. This is probably due
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Fig. 3. Effects of non-irritant injectables on the uptake of NR by HeLa $3 cells: (a) sodium chloride 0.9% (w/v) infusion; and (b) hyoscine butylbromide 20 mg/ml injection.
338
S.A. Wmso~ et al.
Table 2. Comparison of in vitro and in vivo testing data for parenteral formulations tested in the HeLa cell/NR cytotoxicity assay NR5o NRg0 Cytotoxic (#l/ml) (,ul/ml) effects*
Injectable Sodium chloride 0.9% (w/v) infusion
>100
Hyoscine butylbromide 20 mg/ml injection
> 100
> 100
In vivo
effectst
--
ND
45.2
--
--
Streptomycin sulphate 250 mg/ml injection
78.0
51.8
+ +
--
Gentamicin sulphate 40 mg/ml injection
36.4
13.8
+ +
+ + +
2.4
1.2
+ + +
+ + + +
Tetracycline hydrochloride 50 mg/ml injection
N D = not determined *0.1 ml injectable dropped onto HeLa monolayer, treatment time 30min, stained with NR; cell attachment, morphology and cellular staining assessed (reported by Oshida et al., 1979). tRabbit, 0.5 ml intramuscular injection, lesions assessed on day 7 post-injection (reported by Oshida et al., 1979).
to the shape of the dose-response curve for this injectable (Fig. 2b), which shows a substantial shoulder up to a concentration of 50 pl/ml EMEM followed by a sharp decline over the range 50 to 100 #l/ml EMEM. Estimation of NRg0 values in the first part of the assay assumed a steady decline between concentrations exhibiting cytotoxic effects. The ranking of the injectables in order of decreasing cytotoxicity effect on HeLa $3 cells was similar whether NRg0 or NRs0 values were used: tetracycline hydrochloride injection > gentamicin sulphate injection > streptomycin sulphate injection > hyoscine butylbromide injection > sodium chloride infusion. However, the order for streptomycin sulphate and hyoscine butylbromide injections was reversed when NR90 values were considered. Again, this may be due to the difference in the concentrations tested for these injectables and, hence, the accuracy of interpolating NR90 and NRs0 values from their dose-response curves. DISCUSSION
Local tissue irritancy has been associated with the administration of parenteral drugs to man, and a test for local tissue tolerance is usually included in the safety assessment of such products. In the pharmaceutical industry, these tests are routinely performed using whole animals (Comereski et al., 1986). However, during the development of parenteral formulations it is possible that a large number of preparations, of equivalent pharmacological activity, may be produced. Differences in local toxicity profiles might then be an important consideration in the progression of one particular formulation to clinical trial. Since animal tests are time consuming, and animal and labour intensive, they are not well suited to this preliminary screening role. In addition, there are a number of technical problems inherent in these in vivo tests for parenteral drugs. For example, there can be a wide variation in the tissue response of individual animals to the injection, and if only small lesions are induced they may be difficult to identify at autopsy. These can make the results obtained difficult to interpret, particularly when closely related formu-
lations are being evaluated. At the preclinical stage of parenteral formulation development, an in vitro model, predictive of local irritancy potential in vivo, would therefore provide a useful screening test for new drugs. A reliable in vitro assay might also provide a means of evaluating modifications to marketed formulations without recourse to further animal studies. For use in either of these roles the assay must therefore be both versatile and sensitive to even slight formulation changes. The N R uptake assay is based on the incorporation of the vital dye, NR, into the lysosomes of viable uninjured cells after the incubation of the cultures with toxic chemicals. NR is a weakly cationic dye that penetrates the cell membrane by non-ionic diffusion and it binds intracellularly to anionic carboxylic and/or phosphate groups of the lysomal matrix (Nemes et al., 1979). Xenobiotics that injure the plasma or lysosomal membranes decrease the uptake and subsequent retention of the dye. After extraction from the lysosomes, the amount of N R is quantified spectrophotometrically and can be compared with the amount of dye from control cell cultures (Borenfreund and Puerner, 1985a,b). Cytotoxicity assays of this type are, therefore, simple to perform, rapid and relatively cheap, all potential advantages over animal tests in a preliminary screening role. NR assays have been used to evaluate the toxicities of a wide range of chemicals including surfactants (Borenfreund and Puerner, 1985b), bacteriostatic agents (Borenfreund and Shopsis, 1985) and inorganic salts (Borenfreund and Puerner, 1986; Jenssen and Syversen, 1987). For ocular irritants, the results obtained in vitro showed a good correlation with Draize test data (Borenfreund and Puerner, 1985b; Hockley and Baxter, 1986), and for other compounds these methods are considered to be reliable for comparing their relative toxicities (Riddell et al., 1986). These reports demonstrate the N R assay to be versatile and reliable as a screening method for ranking chemicals according to their acute toxicities. The sensitivity of the NR assay has been established by Babich and Borenfreund (1987), who have used this endpoint in the determination of the structureactivity relationship for a homologous series of compounds. The ranking of injectables in our study (Table 2) is in good agreement with both the qualitative in vitro and in vivo rabbit data reported by Oshida et al. (1979). Hence our results clearly demonstrate that the applications of NR eytotoxicity assays could be extended to include the assessment of parenteral formulations for irritant potential. An N R assay has been used previously to determine the cytotoxic effects of a wide variety of injectable formulations on HeLa $3 cells (Oshida et al., 1979). These workers, however, assessed cytotoxicity by microscopic examination of the treated cultures for changes in cellular morphology and detachment, and uptake of the dye, in comparison with control cultures. From this assessment, a ranking was then prepared by dividing the injectables tested into four categories ranging from no changes observed ( - ) to cellular detachment observed ( + + + ). The quantitative N R cytoxicity assay that we used has a number of advantages over this qualitative method, particularly as our ultimate aim is to use this system in the
339
Irritancy screen for parenteral formulations screening of closely related formulations, perhaps exerting only slightly different cytotoxic effects, which would be difficult to determine by visual assessment. That this might be a realistic use of this assay is demonstrated by its ability to distinguish between the two injectables gentamicin sulphate and streptomycin sulphate on the basis of their NRgo and NRs0 values (Table 2), These injectables were placed in the same cytotoxicity category, ( + + ), by Oshida et al. (1979) and visually assigned the same MTD value, 10 pl/ml, in this study (Table 1). However, differences in the in vivo effects of these injections were recorded 7 days post-injection with, moderate changes ( + + + ) being observed for gentamicin sulphate injection and no change ( - ) observed for streptomycin sulphate injection (Oshida et al., 1979). Our method should, at least, provide a ranking for a series of injectables of unknown in vivo irritancy, with those of least cytotoxic potential progressing to further investigation in vivo. Additional information might also be extrapolated, without recourse to animal studies, if NRg0 and NRso values and the shapes of the dose-response curves obtained for unknowns are compared with those obtained previously, or concurrently, for injectables of known in vivo irritancy. It is apparent that for non-irritant injectables, which exert little toxicity, the NR90 value is the more useful parameter to consider when assigning rank order. Indeed, for the non-irritant injectables tested in this study, hyoscine butylbromide injection and sodium chloride infusion, the NR90 provided the only means of distinguishing the two, since both had NRs0 values in excess of 100 pl/ml (Table 2), the highest concentration tested in each case. The usefulness of the NRg0 value for distinguishing between chemicals that exert little toxicity has been noted previously (Babich and Borenfreund, 1987). The use of cells of non-muscle origin for in vitro local tolerance assessment has been questioned because the results obtained reflect a general cytotoxicity potential rather than a target-specific response (Williams et al., 1987). Williams et al. (1987) have, therefore, proposed a system using a rat skeletal muscle cell line (L6) and the depletion of muscleassociated creatine kinase as the index of cellular damage. However, since both toxicity to cell cultures and tissue irritation may be viewed as an expression of cellular damage, the use of HeLa $3 cells in our
TAN. 3/4---F
study would seem acceptable. The uptake of NR also presents a more easily quantifiable endpoint than creatine kinase assessment, and one that has the potential for a high degree of automation. REFERENCES
Babich H. and Borenfreund E. (1987) Structure-activity relationship (SAR) models established in vitro with the neutral red cytotoxicity assay. Toxic. in Vitro 1, 3-9. Borenfreund E. and Puerner J. A. (1985a) A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Cult. Meth. 9, 7-9. Borenfreund E. and Puerner J. A. (1985b) Toxicity determination in vitro by morphological alterations and neutral red absorption. Toxicology Lett. 24, 119-124. Borenfreund E. and Puerner J. A. (1986) Cytoxicity of metals, metal-metal and metal-chelator combinations in vitro. Toxicology 39, 121 134. Borenfreund E. and Shopsis C. (1985) Toxicity monitored with a correlated set of cell culture assays. Xenobiotica 15, 705-711. Cingolani E., Cingolani G., Mosconi P. and Coppi G. (1985) Local tolerance evaluation of intramuscular drug preparations. Farmaco Ed. Prat. 41, 89-97. Comereski C. R., Williams P. D., Bregman C. L. and Hoffendorf G. H. (1986) Pain on injection and muscle irritation: a comparison of animal models for assessing parenteral antibiotics. Fund. appl. Toxic. 6, 335-338. Hockley K. and Baxter D. (1986) Use of the 3T3 cell-neutral red uptake assay for irritants as an alternative to the rabbit (Draize) test. Fd Chem. Toxic. 24, 473-475. Jenssen J. and Syversen T. (1987) Cytotoxicity of vanadate on Chinese hamster ovary cells. A T L A 14, 152-155. Nelson A. A., Price C. W. and Welch H. (1949) Muscle irritation following the injection of various penicillin preparations in rabbits. J. Am. pharm. Ass. 38, 237-239. Nemes Z., Dietz R., Luth J. B., Gomba F., Hackenthal F. and Gross F. (1979) The pharmacological relevance of vital staining with neutral red. Experientia 35, 1475-1476. Oshida S., Degawa K., Takahashi Y. and Akishi S. (1979) Physico-chemical properties and local toxic effects of injectables. Tnhoku J. exp. Med. 127, 301-316. Riddell R. J., Clothier R. H. and Balls M. (1986) An evaluation of three in vitro cytotoxicity assays. Fd Chem. Toxic. 24, 469-471. Wilkinson J. H. (1970) Clinical significance of enzyme activity measurements. Clin. Chem. 16, 882-890. Williams P. D., Masters B. G., Evans L. D., Laska D. A. and Hoffendorf G. H. (1987) An in vitro model for assessing muscle irritation due to parenteral antibiotics. Fund. appL Toxic. 9, I 0 17.
0887-2333/89 $3.00 + 0.00 Copyright © 1989 Pergamon Press pie
AN IN VITRO SCREENING TEST FOR THE ASSESSMENT OF THE IRRITANT POTENTIAL OF PARENTERAL FORMULATIONS S. A. WILSON, M. L. JASIEWICZ,F. W. BONNER, S. BROWN and C. J. POTTER Toxicology Department and Pharmaceutical Sciences Department, Sterling-Winthrop Research Centre, Alnwick, Northumberland NE66 2JH, UK (Received 17 October 1988; revisions received 3 February 1989)
Abstract--A HeLa cell/neutral red cytotoxicity assay has been investigated as an in vitro screen for assessing the irritancy potential of parenteral formulations. Five commercially available intramuscular injectables, three irritants and two non-irritants, were examined for cytotoxic effects in this system. The ranking of the injectables tested, in order of decreasing cytotoxic effects, correlated with both previously reported qualitative in vitro data and their reported irritancy when administered to man. The results show that the HeLa cell/neutral red cytotoxicity assay may provide an alternative to animal studies for the assessment of the irritancy potential of parenteral formulations during the preliminary stages of their development.
INTRODUCTION
When administered to man, intramuscular drug preparations can produce varying degrees of discomfort resulting in pain, irritation and damage to muscle tissue. It is important, therefore, that a test for local tissue tolerance is included in the safety assessment for such products. Currently, there are no regulatory guidelines for the conduct of such studies and within the pharmaceutical industry a number of different methods have been utilized (Cingolani et al., 1985; Comereski et al., 1986; Nelson et al., 1949; Wilkinson, 1970). The basic principle of these tests is that a drug is injected intramuscularly into an experimental animal, which is killed at a certain time after the injection. Muscular damage is subsequently quantified by determination of serum creatine kinase and/or by histopathology and microscopic examination of the lesion at the injection site. In recent years, public concern for animal welfare, together with the escalating costs incurred with the use of laboratory animals, have promoted the need for the establishment and validation of alternative assays using cells in culture. An additional limitation of animal studies is that they are not particularly well suited for the screening of the large numbers of parenteral formulations that may be produced during initial pre-formulation studies. For the assessment of irritant potential in the preliminary stages of development, an in vitro model predictive of in vivo muscle damage would provide an attractive adjunct to animal studies. Abbreviations: EMEM = Earle's modified Eagle's minimal
essential medium supplemented with 10% foetal calf serum, 292 #g L-glutamine/ml, I0 pg gentamicin/ml and 2 mg sodium bicarbonate/ml; MTD = maximum tolerated dose; NR = neutral red; NRs0 = concentration of test agent that reduces the test colour to 50% of the control absorbance; NR90 = concentration of test agent that reduces the test colour to 90% of the control absorbance.
A qualitative in vitro assay for the assessment of the irritant potential of injectable drug formulations has been described by Oshida et al. (1979). This assay was based on the visual assessment of neutral red (NR) uptake by HeLa cells following treatment with a wide variety of injectables. Quantitative N R uptake assays have subsequently been developed for use as first-order screening tests in safety assessment strategies. A wide variety of compounds has been tested, and these methods are considered to be reliable for predicting ocular irritancy potential (Borenfreund and Puerner, 1985b; Hockley and Baxter, 1986), for comparing relative toxicities (Riddell et al., 1986) and for determining structure-activity relationships (Babich and Borenfreund, 1987). The study described here was undertaken to evaluate the applicability of a quantitative HeLa cell/NR uptake assay to the irritancy testing of injectable formulations. MATERIALS AND METHODS
Cell culture
HeLa $3 cells, obtained from Huntingdon Research Centre, Huntingdon, UK, were grown in Earle's modified Eagle's minimal essential medium supplemented with 10% foetal calf serum, 292 #g L-glutamine/ml, 10pg gentamicin/ml and 2 m g sodium bicarbonate/ml. The media components were obtained from GIBCO Europe Ltd, Paisley, UK, and the serum was obtained from Sera Labs, Crawley, UK. Complete culture medium will subsequently be designated EMEM. The cells were maintained as monolayer cultures in a humidified 5% CO2 atmosphere at 37°C and were released from the flask surface with 0.25% trypsin solution. Test articles
The non-irritants hyoscine butylbromide 20 mg/ml injection (Boehringer Ingelheim, Brackneli, UK), and sodium chloride 0.9% (w/v) infusion (Boots Hospital
335
336
S.A. WILSON et al.
Products, Nottingham, UK), were used as supplied by the manufacturers, as was the irritant gentamicin sulphate 40 mg/ml injection (Roussel Laboratories, Uxbridge, UK). The irritants tetracycline hydrochloride 50 mg/ml injection (Lederle Laboratories, Gosport, UK), and streptomycin sulphate 250 mg/ml injection (Evans Medical, Beaconsfield, UK), were reconstituted in water for injection according to the manufacturers' instructions immediately before use. Hyoscine butylbromide and streptomycin sulphate injections are solutions of the respective drugs in water for injection. When reconstituted, tetracycline hydrochloride injection contains 125 mg/ml ascorbic acid as a buffering agent. Gentamicin sulphate injection contains an unspecified, but 'suitable', preservative. All the test agents were diluted in E M E M before addition to the cells. Neutral Red medium A filter sterilized solution of N R (0.3%, w/v) was obtained from Sigma Chemical Co. Ltd, (Poole, Dorset, UK). An aliquot of this was aseptically added to E M E M to give a final N R concentration of 50/~ g/ml. The addition of N R to culture medium results in the formation of a fine precipitate. To prevent this from interfering with the assay, N R medium was incubated overnight at 37°C and centrifuged at 1500g for 10min before use. Cytotoxicity assay Each well of a 96-well microtitre plate was seeded with 9 × 103 H e L a $3 cells in 0.2 ml E M E M and the plate was incubated for 24 hr at 37°C in 5% CO2. The culture medium was then removed and replaced with 0.2 ml E M E M containing the appropriate concentration of injectable under test. Each of seven concentrations was added to eight wells of the plate (one lane). Two eight-well lanes were treated with 0.2 ml E M E M and two lanes were treated with 0.2ml E M E M containing water for injection at an equivalent volume to the highest concentration of test injectable (solvent control). To serve as a blank for subsequent absorbance measurements, the remaining lane was treated throughout all stages of each experiment with EMEM. Following incubation for 24 hr in the presence of the test injectable, the culture medium was removed and replaced with 0.2 ml N R medium. The plate was incubated for a further 3 hr at 37°C. The N R medium was then removed and the wells were rinsed twice with phosphate-buffered saline to remove unincorporated stain. The plate was allowed to drain and 0.2 ml 1% acetic acid-50% ethanol was added to each well to extract the dye. The plate was left for 20 rain at r o o m temperature, then shaken gently before the absorbance of each well was measured at 540nm on a microtitre plate-reading spectrophotometer (Microtek Multiscan MC, Flow Laboratories, Irvine, UK). In preliminary dose-ranging studies, each injectable was tested at seven concentrations over the range 0.0001 to 100/11 injectable/ml E M E M . During these preliminary studies, cytotoxicity was also assessed microscopically prior to the determination of N R uptake. F r o m microscopic examination it was
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0
Number
I
I
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10
20
30
40
of HeLa
$3 ceLLs per weLL (x dO00)
Fig. 1. Effect of cell number on the uptake of NR by HeLa $3 cells incubated in the presence of 50,ug NR/ml.
possible to determine the m a x i m u m tolerated dose (MTD), the concentration causing only minimal morphological changes. Such changes were manifested as a reduction in cell density or by a change in cell shape. For a more accurate evaluation of relative cytotoxicity, each injectable was subsequently tested at seven concentrations over a narrow dose range (0.25 to 10 x MTD). For these studies at least one independent repeat assay was performed. To standardize the assay, the relationship between absorbance at 540 nm and H e L a $3 cell number was investigated. After allowing for cell attachment, various densitities of cells (103 to 4 × 104 per well; eight wells per density) were incubated with 0.2ml N R medium for 3 hr. The dye was then extracted as indicated above and the absorbance of each well measured at 540 nm. RESULTS The relationship between the number of H e L a $3 cells seeded and the amount of N R extracted is shown in Fig. 1. Each point represents the mean results from two independent experiments. There is a good linearity from 1 × 103 to 4 x 104 cells per well (linear regression coefficient 0.997). F o r H e L a $3 cells seeded at a density of 9 × 103 cells per well, 48-hr growth in culture medium under the assay conditions yielded absorbances at 540 nm between 0.4 and 0.5. Table 1. Comparison of MTD and NRgovalues determined during dose-ranging studies in the HeLa cell neutral red assay MTD* NRg0t Injectable (,uI/ml) (# l/ml) Sodium chloride 0.9% (w/v) > 100 > 100 infusion Hyoscine butylbromide 100 39.8 20 mg/ml injection Gentamicin sulphate 10.0 2.8 40 mg/ml injection Streptomycin sulphate 10.0 2.6 250 mg/ml injection Tetracycline hydrochloride 1.0 1.1 50 mg/ml injection *Maximum tolerated dose, concentration that causes only minimal morphological alteration on microscopic examination. fConcentration that reduces the absorbance at 540 nm to 90% of the control.
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Fig. 2. Effects of irritant injectables on the uptake of NR by HeLa $3 cells: (a) tetracycline hydrochloride 50 mg/ml injection; (b) streptomycin sulphate 250 mg/ml injection; and (c) gentamicin sulphate 40 mg/ml injection. The first part of the cytotoxicity assay consisted of the screening of a wide range of concentrations (0.0001-100pl injectable/ml EMEM). From these dose-ranging studies, two parameters, the MTD and an approximate NRg0 value, were determined and are presented in Table 1. The NR90 value is the concentration of test agent that reduces the test colour to 90% of the control absorbance. For both irritant and non-irritant injectables there is close agreement between the visually designated MTD and the measured decrease in dye absorption. The MTD values ranged from 1 #l/ml for the irritant injectable tetracycline hydrochloride 50 mg/ml injection, to > 100/d/ml for the non-irritant sodium chloride 0.9% (w/v) infusion. The NR90 values for these injectables, determined following microscopic examination of the wells in the dose-ranging studies, were 1.1 and >100/A/ml, respectively. Poorer agreement between the MTD and NRg0 values was observed for gentamicin sulphate 40 mg/ml injection, streptomycin sulphate 250 mg/ml injection, and hyoscine butylbromide 20 mg/ml injection (Table 1). This is probably due to the dilution series used in preliminary studies. The second part of the assay involved the screening of a narrower range of concentrations (0.25 to 10 x MTD). The dose-response curves for the three irritant injectables, gentamicin sulphate, tetracycline hydrochloride and streptomycin sulphate, are shown in Fig. 2. The reproducibility of the cytotoxic effects of tetracycline hydrochloride injection in HeLa $3 cells is illustrated in Fig. 2a, which shows the curves obtained from four independent determinations. Figures 2b and c show the mean dose-response curves for streptomycin sulphate injection and gentamicin sulphate injection, respectively. For these injectables similar reproducible cytotoxic effects were obtained, as indicated by the error bars. The mean doseresponse curves for the non-irritant injectables
hyoscine butylbromide and sodium chloride are given in Fig. 3, together with error bars that represent the reproducibility of their cytotoxic effects. A narrower range of concentrations was not tested for either of these injectables since the wide-ranging studies were sufficient to indicate their lack of cytotoxicity. The mean NRg0 and NRso values, where obtainable, determined from these dose-response curves are given in Table 2. The NRs0 value is the concentration of test agent that reduces the absorbance to 50% of the control value. Differences in NRg0 values were observed in the two parts of the assay, particularly for streptomycin sulphate injection. This is probably due
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338
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Table 2. Comparison of in vitro and in vivo testing data for parenteral formulations tested in the HeLa cell/NR cytotoxicity assay NR5o NRg0 Cytotoxic (#l/ml) (,ul/ml) effects*
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>100
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N D = not determined *0.1 ml injectable dropped onto HeLa monolayer, treatment time 30min, stained with NR; cell attachment, morphology and cellular staining assessed (reported by Oshida et al., 1979). tRabbit, 0.5 ml intramuscular injection, lesions assessed on day 7 post-injection (reported by Oshida et al., 1979).
to the shape of the dose-response curve for this injectable (Fig. 2b), which shows a substantial shoulder up to a concentration of 50 pl/ml EMEM followed by a sharp decline over the range 50 to 100 #l/ml EMEM. Estimation of NRg0 values in the first part of the assay assumed a steady decline between concentrations exhibiting cytotoxic effects. The ranking of the injectables in order of decreasing cytotoxicity effect on HeLa $3 cells was similar whether NRg0 or NRs0 values were used: tetracycline hydrochloride injection > gentamicin sulphate injection > streptomycin sulphate injection > hyoscine butylbromide injection > sodium chloride infusion. However, the order for streptomycin sulphate and hyoscine butylbromide injections was reversed when NR90 values were considered. Again, this may be due to the difference in the concentrations tested for these injectables and, hence, the accuracy of interpolating NR90 and NRs0 values from their dose-response curves. DISCUSSION
Local tissue irritancy has been associated with the administration of parenteral drugs to man, and a test for local tissue tolerance is usually included in the safety assessment of such products. In the pharmaceutical industry, these tests are routinely performed using whole animals (Comereski et al., 1986). However, during the development of parenteral formulations it is possible that a large number of preparations, of equivalent pharmacological activity, may be produced. Differences in local toxicity profiles might then be an important consideration in the progression of one particular formulation to clinical trial. Since animal tests are time consuming, and animal and labour intensive, they are not well suited to this preliminary screening role. In addition, there are a number of technical problems inherent in these in vivo tests for parenteral drugs. For example, there can be a wide variation in the tissue response of individual animals to the injection, and if only small lesions are induced they may be difficult to identify at autopsy. These can make the results obtained difficult to interpret, particularly when closely related formu-
lations are being evaluated. At the preclinical stage of parenteral formulation development, an in vitro model, predictive of local irritancy potential in vivo, would therefore provide a useful screening test for new drugs. A reliable in vitro assay might also provide a means of evaluating modifications to marketed formulations without recourse to further animal studies. For use in either of these roles the assay must therefore be both versatile and sensitive to even slight formulation changes. The N R uptake assay is based on the incorporation of the vital dye, NR, into the lysosomes of viable uninjured cells after the incubation of the cultures with toxic chemicals. NR is a weakly cationic dye that penetrates the cell membrane by non-ionic diffusion and it binds intracellularly to anionic carboxylic and/or phosphate groups of the lysomal matrix (Nemes et al., 1979). Xenobiotics that injure the plasma or lysosomal membranes decrease the uptake and subsequent retention of the dye. After extraction from the lysosomes, the amount of N R is quantified spectrophotometrically and can be compared with the amount of dye from control cell cultures (Borenfreund and Puerner, 1985a,b). Cytotoxicity assays of this type are, therefore, simple to perform, rapid and relatively cheap, all potential advantages over animal tests in a preliminary screening role. NR assays have been used to evaluate the toxicities of a wide range of chemicals including surfactants (Borenfreund and Puerner, 1985b), bacteriostatic agents (Borenfreund and Shopsis, 1985) and inorganic salts (Borenfreund and Puerner, 1986; Jenssen and Syversen, 1987). For ocular irritants, the results obtained in vitro showed a good correlation with Draize test data (Borenfreund and Puerner, 1985b; Hockley and Baxter, 1986), and for other compounds these methods are considered to be reliable for comparing their relative toxicities (Riddell et al., 1986). These reports demonstrate the N R assay to be versatile and reliable as a screening method for ranking chemicals according to their acute toxicities. The sensitivity of the NR assay has been established by Babich and Borenfreund (1987), who have used this endpoint in the determination of the structureactivity relationship for a homologous series of compounds. The ranking of injectables in our study (Table 2) is in good agreement with both the qualitative in vitro and in vivo rabbit data reported by Oshida et al. (1979). Hence our results clearly demonstrate that the applications of NR eytotoxicity assays could be extended to include the assessment of parenteral formulations for irritant potential. An N R assay has been used previously to determine the cytotoxic effects of a wide variety of injectable formulations on HeLa $3 cells (Oshida et al., 1979). These workers, however, assessed cytotoxicity by microscopic examination of the treated cultures for changes in cellular morphology and detachment, and uptake of the dye, in comparison with control cultures. From this assessment, a ranking was then prepared by dividing the injectables tested into four categories ranging from no changes observed ( - ) to cellular detachment observed ( + + + ). The quantitative N R cytoxicity assay that we used has a number of advantages over this qualitative method, particularly as our ultimate aim is to use this system in the
339
Irritancy screen for parenteral formulations screening of closely related formulations, perhaps exerting only slightly different cytotoxic effects, which would be difficult to determine by visual assessment. That this might be a realistic use of this assay is demonstrated by its ability to distinguish between the two injectables gentamicin sulphate and streptomycin sulphate on the basis of their NRgo and NRs0 values (Table 2), These injectables were placed in the same cytotoxicity category, ( + + ), by Oshida et al. (1979) and visually assigned the same MTD value, 10 pl/ml, in this study (Table 1). However, differences in the in vivo effects of these injections were recorded 7 days post-injection with, moderate changes ( + + + ) being observed for gentamicin sulphate injection and no change ( - ) observed for streptomycin sulphate injection (Oshida et al., 1979). Our method should, at least, provide a ranking for a series of injectables of unknown in vivo irritancy, with those of least cytotoxic potential progressing to further investigation in vivo. Additional information might also be extrapolated, without recourse to animal studies, if NRg0 and NRso values and the shapes of the dose-response curves obtained for unknowns are compared with those obtained previously, or concurrently, for injectables of known in vivo irritancy. It is apparent that for non-irritant injectables, which exert little toxicity, the NR90 value is the more useful parameter to consider when assigning rank order. Indeed, for the non-irritant injectables tested in this study, hyoscine butylbromide injection and sodium chloride infusion, the NR90 provided the only means of distinguishing the two, since both had NRs0 values in excess of 100 pl/ml (Table 2), the highest concentration tested in each case. The usefulness of the NRg0 value for distinguishing between chemicals that exert little toxicity has been noted previously (Babich and Borenfreund, 1987). The use of cells of non-muscle origin for in vitro local tolerance assessment has been questioned because the results obtained reflect a general cytotoxicity potential rather than a target-specific response (Williams et al., 1987). Williams et al. (1987) have, therefore, proposed a system using a rat skeletal muscle cell line (L6) and the depletion of muscleassociated creatine kinase as the index of cellular damage. However, since both toxicity to cell cultures and tissue irritation may be viewed as an expression of cellular damage, the use of HeLa $3 cells in our
TAN. 3/4---F
study would seem acceptable. The uptake of NR also presents a more easily quantifiable endpoint than creatine kinase assessment, and one that has the potential for a high degree of automation. REFERENCES
Babich H. and Borenfreund E. (1987) Structure-activity relationship (SAR) models established in vitro with the neutral red cytotoxicity assay. Toxic. in Vitro 1, 3-9. Borenfreund E. and Puerner J. A. (1985a) A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Cult. Meth. 9, 7-9. Borenfreund E. and Puerner J. A. (1985b) Toxicity determination in vitro by morphological alterations and neutral red absorption. Toxicology Lett. 24, 119-124. Borenfreund E. and Puerner J. A. (1986) Cytoxicity of metals, metal-metal and metal-chelator combinations in vitro. Toxicology 39, 121 134. Borenfreund E. and Shopsis C. (1985) Toxicity monitored with a correlated set of cell culture assays. Xenobiotica 15, 705-711. Cingolani E., Cingolani G., Mosconi P. and Coppi G. (1985) Local tolerance evaluation of intramuscular drug preparations. Farmaco Ed. Prat. 41, 89-97. Comereski C. R., Williams P. D., Bregman C. L. and Hoffendorf G. H. (1986) Pain on injection and muscle irritation: a comparison of animal models for assessing parenteral antibiotics. Fund. appl. Toxic. 6, 335-338. Hockley K. and Baxter D. (1986) Use of the 3T3 cell-neutral red uptake assay for irritants as an alternative to the rabbit (Draize) test. Fd Chem. Toxic. 24, 473-475. Jenssen J. and Syversen T. (1987) Cytotoxicity of vanadate on Chinese hamster ovary cells. A T L A 14, 152-155. Nelson A. A., Price C. W. and Welch H. (1949) Muscle irritation following the injection of various penicillin preparations in rabbits. J. Am. pharm. Ass. 38, 237-239. Nemes Z., Dietz R., Luth J. B., Gomba F., Hackenthal F. and Gross F. (1979) The pharmacological relevance of vital staining with neutral red. Experientia 35, 1475-1476. Oshida S., Degawa K., Takahashi Y. and Akishi S. (1979) Physico-chemical properties and local toxic effects of injectables. Tnhoku J. exp. Med. 127, 301-316. Riddell R. J., Clothier R. H. and Balls M. (1986) An evaluation of three in vitro cytotoxicity assays. Fd Chem. Toxic. 24, 469-471. Wilkinson J. H. (1970) Clinical significance of enzyme activity measurements. Clin. Chem. 16, 882-890. Williams P. D., Masters B. G., Evans L. D., Laska D. A. and Hoffendorf G. H. (1987) An in vitro model for assessing muscle irritation due to parenteral antibiotics. Fund. appL Toxic. 9, I 0 17.