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Cytotoxicity tests of antibacterial agents on human fibroblasts cultures
PHARMACEUTICA ACTAHELVETIAE ELSEVIER
Pharmaceutica Acta Helvetiae 69 (1994) 159-162
Cytotoxicity tests of antibacterial agents on human fibroblasts cultures Pierre Rivalland *, Katell Vi6, Laurence Coiffard, Yannick De Roeck-Holtzhauer CAEC, Universitd de Nantes, 68 Bd Eugene Orieux, 44000 Nantes, France Received 5 April 1994; accepted 15 July 1994
Abstract
The authors tried to determine and compare the cytotoxicity of several primary substances used in cosmetic or pharmaceutic industry as antimicrobial agents, on single-layer cultures of human fibroblasts. The cytotoxic effect of Germall 115", Kathon CG* and Pentonium* was pointed out using the colorimetric method with MTT (3-[4-5-dimethyl thiazol 2-yl]-2,5 diphenyl tetrazolium bromide). For each one of these substances, we tested different concentrations with variable contact times. These trials allowed us to classify these products by increasing toxicity as follows: Kathon CG* and Pentonium*. In our experimental conditions, Germall 115" was not cytotoxic.
Keywords: Antibacterial agents; Cytotoxicity; Human fibroblasts cultures
1. Introduction
When faced with the recent problems raised by cytotoxicity tests realized on animals and with the legislation that will soon forbid them, a certain number of research teams started to study the possibility to use replacement techniques called "alternative methods" (Manzulli and Maibach, 1975; Hoh et al., 1987; Stephens, 1990; Bason et al., 1991; Ponec, 1992). In the last few years, several in vitro systems for predicting the effect of toxics as an alternative to animal tests have been described (Knox et al., 1986). Cell cultures were very useful for investigating the behaviour of structurally similar compounds such as surfactants and characterizing their relative irritancy compared to the in vivo test. We tried to set up cultures of human epidermic cells (Baudry, 1985; Rivalland, 1993) and then to test the cytotoxicity of different primary substances involved in the formulation of cosmetics. During this work, we
* Corresponding author. Elsevier Science B.V. SSDI 0 0 3 1 - 6 8 6 5 ( 9 4 ) 0 0 0 3 0 - 1
determined the cytotoxicity on single-layer human fibroblasts cultures, of antibacterial agents used as preservatives. We tried to determine the toxicity of a range of increasing concentrations with a contact time arbitrarly set at 2 minutes. We then chose a solution of toxic concentration to study the evolution of the toxicity with contact time.
2. Materials and methods
2.1. Cell cultures Cultures of normal human fibroblasts (NHF) were established from 1-mm diameter punch biopsies of human skin obtained from adult subjects during breast plastic surgery, after migration of the cells out of the explants in EMEM medium supplemented with 0.2 mM L-glutamine, 100 UI m1-1 penicillin G, 10 mg m1-1 streptomycin and 5% FCS (Fetal Calf Serum) at 37°C in a humidified atmosphere containing 95% air and 5% CO 2. The fibroblasts were subcultured after trypsin treatment (0.25%; Eurobio) in SFRI.
160
P. Riralland et al. / Pharmaceutica Acta Heh,etiae 69 (1994) 159 162
2.2. Treatment with compounds
Fibroblasts used in these experiments performed in duplicate were between the fourth and eleventh passages. T h r e e commercialy available preservatives were tested: Germall 115" (imidazolidinylurea; ISP, Sutton); DL50 by oral passage for rats: 5.2 g kg-1. - Kathon CG* (mix of 5-chloro-2-methyl-4-isothiazoline-3-one and of 2-methyl-4-isothiazoline-3-one; R o h m and Haas); DL50 by oral passage for rats: 3.35 g k g - 1. Pentonium* (water solution titrating 80% in benzalkonium chloride; Pentagon Chemical); DL50 by oral passage for rats: 250 mg kg 1. The solutions of these three products, whose concentrations are expressed in percentages, were realized using phosphate buffer from Dulbecco (PBS; Eurobio). In preliminary experiments, the preservatives were incubated for 2 min at concentrations ranging from 0.0001% to 30%. For definitive assays, the compounds were tested at concentrations ranging from 0.0001% to 30% and for different incubation times (1 to 30 rain). All compounds were incubated with confluent fibroblasts cultures in M E M without FCS at 37°C in a humidified atmosphere containing 95% air and 5% CO 2. After the incubation period, the medium was removed and the monolayers were washed with phosphate buffered saline (PBS). 2.3. M T T test
The M T T test was chosen to study the preservative damage caused to the fibroblasts (Denizot and Lang, 1986; Triglia et al., 1991). We realized a solution of M T T (dimethylthiazol diphenyl tetrazolium bromide; Society ICN) with 0.5 mg ml-~ in phosphate buffer (buffer PBS; ICN). The solution was p r e p a r e d and sterilized by filtration; 250 ~I of solution of the product to be tested were put in each well. After a given contact time, the product was eliminated, then each well was rinsed with phosphate buffer and at last 250/zl of M T T solution were added in each well. After 4 h of stewing, the supernatant was eliminated and 250/zl of D M S O (dimethylsulfoxide; Society ICN) were added in each well in order to solubilize the blue cristals of formazan that were formed. The absorbance of different solutions was measured using a spectrophotometer Titertrek Uniskan II at 540 rim.
3.
Results
For each product, except Germall 115", we first determined the toxicity of a range of increasing concentrations, after 2 min of contact time (Table 1) and then we chose a concentration of medium toxicity for which we studied the toxicity as a function of contact time (Table 2). 3.1. Germall 115"
No toxicity could be determined with the solutions of concentrations varying from 0.5 to 30% and prepared at the last moment, after 2 rain and 30 rain of contact. We then prepared the solutions the day before, thinking that the release of formol was not happening in the solutions made at the last moment. The results obtained in this case also did not show any toxic phenomenon. The question that remained was whether this test was able to establish the toxicity of this product or not, or whether Germal 115" was simply absolutely not toxic for fibroblasts in culture. 3.2. Kathon CG*
The solution at 0.4% having a p H close to 6, we realized a standard solution of chloridic acid in PBS of p H = 6, in order to check if the acidity of the solution was a factor of toxicity. From the result obtained Table 1 Cytotoxity as a function of the concentration after 2 min of contact time Product Concentrations (%) Cytotoxicity(%) tested Germall 115 * 1.00 2.0 5.00 13.0 15.00 10.0 30.00 4.0 Pentonium *
0.0001 0.0005 0.001 0.005 0.01 0.10 0.25 0.50
1.0 4.0 15.5 37.5 50.4 55.3 54.3 57.3
Kathon CG *
0.01 0.02 0.10 0.20 0.40
4.5 19.0 33.0 40.0 52.3
P. Rivalland et al. / Pharmaceutica Acta Helvetiae 69 (1994) 159-162 Table 2 Cytotoxicity expressed as a percentage depending on the contact time Products tested
Contact time (min)
Cytotoxicity (%)
Germall 115 * (1%)
1 2 5 10 20 30
11.0 10.0 10.0 15.0 20.0 17.0
1 5 10 15 20
14.4 18.0 22.0 28.0 33.0
1 5 10 15 20
23.5 31.0 33.0 35.0 39.0
Pentonium * (0.001%)
Kathon CG* (0.02%)
(11.6% of toxicity), we concluded that the slight acidity of the solution did not induce any toxicity. The range of concentrations tested from 0.01 to 0.4% after 2 min of contact time showed that below 0.02% the cytotoxicity decreased a lot. At a concentration of 0.02% and as a function of contact time from 1 to 30 min, we observed a slight but continuous increase of the cytotoxicity from 23.5 to 39.0%.
3.3. Pentonium* The range of tested concentrations from 0.0001 to 0.5% after 2 min of contact time showed that the cytotoxicity decreased a lot below 0.001%. Depending
60-
40-
161
Cytotoxicity
[]
30.
Kathon CG ~ u ~ 0.02 % o .~.~" ~,~,~
a~,,. P e n t o n i u m ~ ~, ~-'~
O001 °/o
20.
10 O
4
i
~
10
15 '
2b
T i m e (min)
Fig. 2. Cytotoxicity as a function of contact time.
on contact time from 1 to 20 min and at a concentration of 0.001% we observed a continuous increase of the toxicity (from 14.4 to 33.0%). Putting aside Germall 115" that seemed to be nontoxic in our experimental conditions, it was possible to compare the toxicity of the two other products. The toxicity as a function of the concentration varied the same way (Fig. 1). We noted a low toxicity at low concentrations, a fast increase of the toxicity as a function of the concentration of toxic substance and we reached a maximum toxicity with high concentrations. By order of decreasing concentration, the following classification can be proposed: - Pentonium* - Kathon CG*. The evolution of the toxicity as a function of contact time showed, for the two products, a constant increase of toxicity with contact time (Fig. 2). It would have been interesting to lengthen the contact times, keeping in mind that the cells can damage spontaneously, due to the non suitable environment but independently of the toxic action.
T o x i c i t y (°Io) ~-~- ~, .,~..
Pentonium*
4. Discussion and conclusion
50-
I',Kathon CG ~
I
a\
4030-
I "% "~o
~
.q
20-
I
10-
M
?O/o
o~oio
Concentration
o&o/o
00C)1 o/o 0 0 0 0 1 ° f .
Log(c)
Fig. 1. Toxicity as a function of concentration.
The toxicity tests on single layers of fibroblasts are interesting as they allow the comparison of the cellular toxicity of different products. In our study, Germall 115" seemed to be the less toxic when Pentonium* was, on the opposite, the most agressive component. These results were expected, knowing the physicochemical properties of each product. Anyway, it is difficult to reach a definite conclusion, since the quantities that seemed non-toxic on single layers of fibroblasts are much below the maximum ones allowed in cosmetic products. It is necessary to have reference
162
P. Rivalland et al. / Pharmaceutica A cta Heh,etiae 69 (1994) 159-162
substances for which toxicity on single layers of fibroblasts would be related to a toxicity in vivo. The method developed here is a reproducible one, simple to set up, but can only be used for water-soluble products. Among all the tests in vitro available today, the determination of cytotoxicity using M T T is very common. It can be applied to other types of substrates such as keratinocytes in single layer or in pluristratified epithelium, as well as in any system of equivalent skin.
References Bason, M., Gordon, V. and Maibach, H. (1991) Skin irritation - in vitro assays. Int. J. Dermatol. 30, 623-626. Baudry, G. (1985) Mise au point d'une m6thode de culture ~pidermiques humaines dans le cadre des m&hodes alternatives. Th~se Doct. Pharm., Nantes. Denizot, F. and Lang, R. (1986) Rapid colorimetric assay for cell growth and survival: modifications to tetrazolium dye procedure
giving improved sensitivity and reliability. J. lmmunol. Meth. 7, 71-89. Hoh, A., Maier, K. and Dreher, R. (1987) Multilayered keratinocyte culture used for in vitro toxicology. Mol. Toxicol. 1,537-546. Knox, P., Uphill, P.F., Fry, J.R., Benford, J. and Balls, M. (1986). The Fram multicentre project on in vitro cytotoxicology. Food Cosmet. Toxicol. 24, 457-463. Manzulli, F.N. and Maibach, H.I. (1975) The rabbit as a model for evaluating skin irritants. A comparaison of results obtained on animals and man using skin exposure. Food Cosmet. Toxicol. 13, 533-543. Ponec, S. (1992) In vitro, cultured human skin cells as alterations to animals for skin irritancy screening. Int. J. Cosmet. Sci. 14, 245-264. Rivalland, P. (1993) Donn6es sur la cytotoxicit6 de diff&ents actifs et additifs cosm&iques sur fibroblastes humains. Th~se Doct. SVN, Nantes. Stephens, M. (1990) Test Skin. An in vitro test for detecting cytotoxicity and inflammation. Toxicologist 10, 78-83. Triglia, D., Braa, S., Yonana, C. and Naughton, G. (1991) Cytotoxicity testing a neutral red and MTT assays on a three dimensional human skin substrate. Toxicol. In Vitro 5, 573-578.
Pharmaceutica Acta Helvetiae 69 (1994) 159-162
Cytotoxicity tests of antibacterial agents on human fibroblasts cultures Pierre Rivalland *, Katell Vi6, Laurence Coiffard, Yannick De Roeck-Holtzhauer CAEC, Universitd de Nantes, 68 Bd Eugene Orieux, 44000 Nantes, France Received 5 April 1994; accepted 15 July 1994
Abstract
The authors tried to determine and compare the cytotoxicity of several primary substances used in cosmetic or pharmaceutic industry as antimicrobial agents, on single-layer cultures of human fibroblasts. The cytotoxic effect of Germall 115", Kathon CG* and Pentonium* was pointed out using the colorimetric method with MTT (3-[4-5-dimethyl thiazol 2-yl]-2,5 diphenyl tetrazolium bromide). For each one of these substances, we tested different concentrations with variable contact times. These trials allowed us to classify these products by increasing toxicity as follows: Kathon CG* and Pentonium*. In our experimental conditions, Germall 115" was not cytotoxic.
Keywords: Antibacterial agents; Cytotoxicity; Human fibroblasts cultures
1. Introduction
When faced with the recent problems raised by cytotoxicity tests realized on animals and with the legislation that will soon forbid them, a certain number of research teams started to study the possibility to use replacement techniques called "alternative methods" (Manzulli and Maibach, 1975; Hoh et al., 1987; Stephens, 1990; Bason et al., 1991; Ponec, 1992). In the last few years, several in vitro systems for predicting the effect of toxics as an alternative to animal tests have been described (Knox et al., 1986). Cell cultures were very useful for investigating the behaviour of structurally similar compounds such as surfactants and characterizing their relative irritancy compared to the in vivo test. We tried to set up cultures of human epidermic cells (Baudry, 1985; Rivalland, 1993) and then to test the cytotoxicity of different primary substances involved in the formulation of cosmetics. During this work, we
* Corresponding author. Elsevier Science B.V. SSDI 0 0 3 1 - 6 8 6 5 ( 9 4 ) 0 0 0 3 0 - 1
determined the cytotoxicity on single-layer human fibroblasts cultures, of antibacterial agents used as preservatives. We tried to determine the toxicity of a range of increasing concentrations with a contact time arbitrarly set at 2 minutes. We then chose a solution of toxic concentration to study the evolution of the toxicity with contact time.
2. Materials and methods
2.1. Cell cultures Cultures of normal human fibroblasts (NHF) were established from 1-mm diameter punch biopsies of human skin obtained from adult subjects during breast plastic surgery, after migration of the cells out of the explants in EMEM medium supplemented with 0.2 mM L-glutamine, 100 UI m1-1 penicillin G, 10 mg m1-1 streptomycin and 5% FCS (Fetal Calf Serum) at 37°C in a humidified atmosphere containing 95% air and 5% CO 2. The fibroblasts were subcultured after trypsin treatment (0.25%; Eurobio) in SFRI.
160
P. Riralland et al. / Pharmaceutica Acta Heh,etiae 69 (1994) 159 162
2.2. Treatment with compounds
Fibroblasts used in these experiments performed in duplicate were between the fourth and eleventh passages. T h r e e commercialy available preservatives were tested: Germall 115" (imidazolidinylurea; ISP, Sutton); DL50 by oral passage for rats: 5.2 g kg-1. - Kathon CG* (mix of 5-chloro-2-methyl-4-isothiazoline-3-one and of 2-methyl-4-isothiazoline-3-one; R o h m and Haas); DL50 by oral passage for rats: 3.35 g k g - 1. Pentonium* (water solution titrating 80% in benzalkonium chloride; Pentagon Chemical); DL50 by oral passage for rats: 250 mg kg 1. The solutions of these three products, whose concentrations are expressed in percentages, were realized using phosphate buffer from Dulbecco (PBS; Eurobio). In preliminary experiments, the preservatives were incubated for 2 min at concentrations ranging from 0.0001% to 30%. For definitive assays, the compounds were tested at concentrations ranging from 0.0001% to 30% and for different incubation times (1 to 30 rain). All compounds were incubated with confluent fibroblasts cultures in M E M without FCS at 37°C in a humidified atmosphere containing 95% air and 5% CO 2. After the incubation period, the medium was removed and the monolayers were washed with phosphate buffered saline (PBS). 2.3. M T T test
The M T T test was chosen to study the preservative damage caused to the fibroblasts (Denizot and Lang, 1986; Triglia et al., 1991). We realized a solution of M T T (dimethylthiazol diphenyl tetrazolium bromide; Society ICN) with 0.5 mg ml-~ in phosphate buffer (buffer PBS; ICN). The solution was p r e p a r e d and sterilized by filtration; 250 ~I of solution of the product to be tested were put in each well. After a given contact time, the product was eliminated, then each well was rinsed with phosphate buffer and at last 250/zl of M T T solution were added in each well. After 4 h of stewing, the supernatant was eliminated and 250/zl of D M S O (dimethylsulfoxide; Society ICN) were added in each well in order to solubilize the blue cristals of formazan that were formed. The absorbance of different solutions was measured using a spectrophotometer Titertrek Uniskan II at 540 rim.
3.
Results
For each product, except Germall 115", we first determined the toxicity of a range of increasing concentrations, after 2 min of contact time (Table 1) and then we chose a concentration of medium toxicity for which we studied the toxicity as a function of contact time (Table 2). 3.1. Germall 115"
No toxicity could be determined with the solutions of concentrations varying from 0.5 to 30% and prepared at the last moment, after 2 rain and 30 rain of contact. We then prepared the solutions the day before, thinking that the release of formol was not happening in the solutions made at the last moment. The results obtained in this case also did not show any toxic phenomenon. The question that remained was whether this test was able to establish the toxicity of this product or not, or whether Germal 115" was simply absolutely not toxic for fibroblasts in culture. 3.2. Kathon CG*
The solution at 0.4% having a p H close to 6, we realized a standard solution of chloridic acid in PBS of p H = 6, in order to check if the acidity of the solution was a factor of toxicity. From the result obtained Table 1 Cytotoxity as a function of the concentration after 2 min of contact time Product Concentrations (%) Cytotoxicity(%) tested Germall 115 * 1.00 2.0 5.00 13.0 15.00 10.0 30.00 4.0 Pentonium *
0.0001 0.0005 0.001 0.005 0.01 0.10 0.25 0.50
1.0 4.0 15.5 37.5 50.4 55.3 54.3 57.3
Kathon CG *
0.01 0.02 0.10 0.20 0.40
4.5 19.0 33.0 40.0 52.3
P. Rivalland et al. / Pharmaceutica Acta Helvetiae 69 (1994) 159-162 Table 2 Cytotoxicity expressed as a percentage depending on the contact time Products tested
Contact time (min)
Cytotoxicity (%)
Germall 115 * (1%)
1 2 5 10 20 30
11.0 10.0 10.0 15.0 20.0 17.0
1 5 10 15 20
14.4 18.0 22.0 28.0 33.0
1 5 10 15 20
23.5 31.0 33.0 35.0 39.0
Pentonium * (0.001%)
Kathon CG* (0.02%)
(11.6% of toxicity), we concluded that the slight acidity of the solution did not induce any toxicity. The range of concentrations tested from 0.01 to 0.4% after 2 min of contact time showed that below 0.02% the cytotoxicity decreased a lot. At a concentration of 0.02% and as a function of contact time from 1 to 30 min, we observed a slight but continuous increase of the cytotoxicity from 23.5 to 39.0%.
3.3. Pentonium* The range of tested concentrations from 0.0001 to 0.5% after 2 min of contact time showed that the cytotoxicity decreased a lot below 0.001%. Depending
60-
40-
161
Cytotoxicity
[]
30.
Kathon CG ~ u ~ 0.02 % o .~.~" ~,~,~
a~,,. P e n t o n i u m ~ ~, ~-'~
O001 °/o
20.
10 O
4
i
~
10
15 '
2b
T i m e (min)
Fig. 2. Cytotoxicity as a function of contact time.
on contact time from 1 to 20 min and at a concentration of 0.001% we observed a continuous increase of the toxicity (from 14.4 to 33.0%). Putting aside Germall 115" that seemed to be nontoxic in our experimental conditions, it was possible to compare the toxicity of the two other products. The toxicity as a function of the concentration varied the same way (Fig. 1). We noted a low toxicity at low concentrations, a fast increase of the toxicity as a function of the concentration of toxic substance and we reached a maximum toxicity with high concentrations. By order of decreasing concentration, the following classification can be proposed: - Pentonium* - Kathon CG*. The evolution of the toxicity as a function of contact time showed, for the two products, a constant increase of toxicity with contact time (Fig. 2). It would have been interesting to lengthen the contact times, keeping in mind that the cells can damage spontaneously, due to the non suitable environment but independently of the toxic action.
T o x i c i t y (°Io) ~-~- ~, .,~..
Pentonium*
4. Discussion and conclusion
50-
I',Kathon CG ~
I
a\
4030-
I "% "~o
~
.q
20-
I
10-
M
?O/o
o~oio
Concentration
o&o/o
00C)1 o/o 0 0 0 0 1 ° f .
Log(c)
Fig. 1. Toxicity as a function of concentration.
The toxicity tests on single layers of fibroblasts are interesting as they allow the comparison of the cellular toxicity of different products. In our study, Germall 115" seemed to be the less toxic when Pentonium* was, on the opposite, the most agressive component. These results were expected, knowing the physicochemical properties of each product. Anyway, it is difficult to reach a definite conclusion, since the quantities that seemed non-toxic on single layers of fibroblasts are much below the maximum ones allowed in cosmetic products. It is necessary to have reference
162
P. Rivalland et al. / Pharmaceutica A cta Heh,etiae 69 (1994) 159-162
substances for which toxicity on single layers of fibroblasts would be related to a toxicity in vivo. The method developed here is a reproducible one, simple to set up, but can only be used for water-soluble products. Among all the tests in vitro available today, the determination of cytotoxicity using M T T is very common. It can be applied to other types of substrates such as keratinocytes in single layer or in pluristratified epithelium, as well as in any system of equivalent skin.
References Bason, M., Gordon, V. and Maibach, H. (1991) Skin irritation - in vitro assays. Int. J. Dermatol. 30, 623-626. Baudry, G. (1985) Mise au point d'une m6thode de culture ~pidermiques humaines dans le cadre des m&hodes alternatives. Th~se Doct. Pharm., Nantes. Denizot, F. and Lang, R. (1986) Rapid colorimetric assay for cell growth and survival: modifications to tetrazolium dye procedure
giving improved sensitivity and reliability. J. lmmunol. Meth. 7, 71-89. Hoh, A., Maier, K. and Dreher, R. (1987) Multilayered keratinocyte culture used for in vitro toxicology. Mol. Toxicol. 1,537-546. Knox, P., Uphill, P.F., Fry, J.R., Benford, J. and Balls, M. (1986). The Fram multicentre project on in vitro cytotoxicology. Food Cosmet. Toxicol. 24, 457-463. Manzulli, F.N. and Maibach, H.I. (1975) The rabbit as a model for evaluating skin irritants. A comparaison of results obtained on animals and man using skin exposure. Food Cosmet. Toxicol. 13, 533-543. Ponec, S. (1992) In vitro, cultured human skin cells as alterations to animals for skin irritancy screening. Int. J. Cosmet. Sci. 14, 245-264. Rivalland, P. (1993) Donn6es sur la cytotoxicit6 de diff&ents actifs et additifs cosm&iques sur fibroblastes humains. Th~se Doct. SVN, Nantes. Stephens, M. (1990) Test Skin. An in vitro test for detecting cytotoxicity and inflammation. Toxicologist 10, 78-83. Triglia, D., Braa, S., Yonana, C. and Naughton, G. (1991) Cytotoxicity testing a neutral red and MTT assays on a three dimensional human skin substrate. Toxicol. In Vitro 5, 573-578.