toxicity of potential irritants in mammalian cellsin vitro

3
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 34, 56–58 (1996) ARTICLE NO. 0044 Toxicity of Potential Irritants in Mammalian Cells in Vitro A. SANTA MARI ´ A,* ,1 J. M. POZUELO,² A. L O ´ PEZ,* AND F. SANZ* *Dpt. Toxicologia, Instituto de Salud Carlos III, Majadahonda 28220 Madrid, Spain; and ² Universidad San Pablo (CEU), Madrid, Spain Received April 10, 1995 Sodium lauryl sulfate is well known to cause irritation of Four bioactive materials were tested for their ability to pro- the skin and to enhance the penetration of chemicals into duce toxicity in vitro in two cell lines: rabbit corneal cells the epidermis (Ikarashi et al., 1993) and sometimes has been (SIRC) and human fibroblasts. These compounds are widely chosen as a positive control to the Draize test (Basketter et used by industries, causing toxic effects to ecosystems and par- al., 1994). ticulary having an impact on human health. Different chemicals Lansdown (1991) demonstrated in vivo adverse effects of were chosen which were structurally unrelated: triethanol- zinc pyrithione in response to topical application as a mar- amine, zinc pyrithione, sodium lauryl sulfate, and oleamine. ginal epidermal hyperplasia. However, zinc pyrithione Cytotoxicity was evaluated measuring the total protein content lacked genotoxic activity when it was evaluated in vitro in (inhibition of cell growth). Results indicated that zinc pyrithi- the Salmonella test (Ames) (Skoulis et al., 1993). one produced the highest inhibition of cell growth when two The aim of this paper was to evaluate the ocular and skin cell lines were treated in the two doses tested. The lowest cyto- toxic effect was observed with oleamine. Triethanolamine and irritation in vitro of these products. Two cell lines were used: sodium lauryl sulfate produced a similar cytotoxic effect in Fc rabbit corneal cells (SIRC) as alternative to the Draize test cells, although sodium lauryl sulfate revealed higher inhibition and human fibroblasts (skin and muscle) to study skin irri- of cell growth than triethanolamine on SIRC cells. From these tancy. results, it can be concluded that some products as triethanol- amine and zinc pyrithione should be observed in future special MATERIAL AND METHODS toxicological studies. q 1996 Academic Press, Inc. Cell cultures. Human skin and muscle fibroblasts (Fc) derived from neonatal and SIRC were purchased from Flow INTRODUCTION laboratories (UK). Cells were routinely cultured at 377C in There are numerous products currently being marketed a 5% CO 2 atmosphere in minimal essential medium (MEM), that contain bioactive materials (e.g., hair conditioner, medi- modified with Earle’s salt and supplemented with 10% fetal cal shampoos, antiaging creams). But, they are marketed as calf serum. cosmetics because they avoid making drug claims. These Chemicals. Four products used as components of com- have been popularly called ‘‘cosmeceuticals.’’ An important mercial shampoos have been evaluated: triethanolamine aspect of the safety assesment process for these products is (TE), zinc pyrithione (Znpt), sodium lauryl sulfate (SLS), a determination of their capacity to produce adverse skin and oleamine (OA). These products were added to the culture effects, such as irritation and toxicity. An assessment of skin medium in two concentrations (5 and 10%), as these are and ocular irritation is required of these products in order relevant concentrations in cosmeceuticals. to help ensure consumer safety. These chemical pollulants are also present as discharges Inhibition of cell growth (protein method). The method in surface waters with an impact on the ecosystem. For this used was that of Knox et al. (1986), in which Kenacid blue reason, study was made of different chemicals, structurally is employed to stain the cell protein. Cells were harvested unrelated, as components of shampoos: triethanolamine, zinc and diluted to a final concentration of 15 1 10 3 cells/ml in pyrithione, sodium lauryl sulfate, and oleamine. MEM medium. The cell suspension was shaken for about Data from literature demonstrated the irritation potential 30 min and then 200-ml aliquots were added to each well of triethalonamine to human skin in vivo when the patients of a 96-well tissue culture plate. Plates were incubated for were tested with topical preparations (Pasche et al., 1994). 24 hr to allow the cells to attach. The medium covering the Schnuch et al. (1993) found that triethalonamine and other cells was then removed and 200-ml aliquots of the test chemi- chemicals produced adverse effects in vivo. cal solutions were added. Two dose levels were made up for each compound and 8 wells were used for each dose 1 To whom correspondence and reprint requests should be addressed. level. The remaining wells were controls and received 200 56 0147-6513/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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Page 1: Toxicity of Potential Irritants in Mammalian Cellsin Vitro

ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 34, 56–58 (1996)ARTICLE NO. 0044

Toxicity of Potential Irritants in Mammalian Cells in Vitro

A. SANTA MARIA,*,1 J. M. POZUELO,† A. LOPEZ,* AND F. SANZ*

*Dpt. Toxicologia, Instituto de Salud Carlos III, Majadahonda 28220 Madrid, Spain; and †Universidad San Pablo (CEU), Madrid, Spain

Received April 10, 1995

Sodium lauryl sulfate is well known to cause irritation ofFour bioactive materials were tested for their ability to pro- the skin and to enhance the penetration of chemicals into

duce toxicity in vitro in two cell lines: rabbit corneal cells the epidermis (Ikarashi et al., 1993) and sometimes has been(SIRC) and human fibroblasts. These compounds are widely chosen as a positive control to the Draize test (Basketter etused by industries, causing toxic effects to ecosystems and par- al., 1994).ticulary having an impact on human health. Different chemicals Lansdown (1991) demonstrated in vivo adverse effects ofwere chosen which were structurally unrelated: triethanol-

zinc pyrithione in response to topical application as a mar-amine, zinc pyrithione, sodium lauryl sulfate, and oleamine.ginal epidermal hyperplasia. However, zinc pyrithioneCytotoxicity was evaluated measuring the total protein contentlacked genotoxic activity when it was evaluated in vitro in(inhibition of cell growth). Results indicated that zinc pyrithi-the Salmonella test (Ames) (Skoulis et al., 1993).one produced the highest inhibition of cell growth when two

The aim of this paper was to evaluate the ocular and skincell lines were treated in the two doses tested. The lowest cyto-toxic effect was observed with oleamine. Triethanolamine and irritation in vitro of these products. Two cell lines were used:sodium lauryl sulfate produced a similar cytotoxic effect in Fc rabbit corneal cells (SIRC) as alternative to the Draize testcells, although sodium lauryl sulfate revealed higher inhibition and human fibroblasts (skin and muscle) to study skin irri-of cell growth than triethanolamine on SIRC cells. From these tancy.results, it can be concluded that some products as triethanol-amine and zinc pyrithione should be observed in future special

MATERIAL AND METHODStoxicological studies. q 1996 Academic Press, Inc.

Cell cultures. Human skin and muscle fibroblasts (Fc)derived from neonatal and SIRC were purchased from FlowINTRODUCTIONlaboratories (UK). Cells were routinely cultured at 377C in

There are numerous products currently being marketed a 5% CO2 atmosphere in minimal essential medium (MEM),that contain bioactive materials (e.g., hair conditioner, medi- modified with Earle’s salt and supplemented with 10% fetalcal shampoos, antiaging creams). But, they are marketed as calf serum.cosmetics because they avoid making drug claims. These

Chemicals. Four products used as components of com-have been popularly called ‘‘cosmeceuticals.’’ An importantmercial shampoos have been evaluated: triethanolamineaspect of the safety assesment process for these products is(TE), zinc pyrithione (Znpt), sodium lauryl sulfate (SLS),a determination of their capacity to produce adverse skinand oleamine (OA). These products were added to the cultureeffects, such as irritation and toxicity. An assessment of skinmedium in two concentrations (5 and 10%), as these areand ocular irritation is required of these products in orderrelevant concentrations in cosmeceuticals.to help ensure consumer safety.

These chemical pollulants are also present as discharges Inhibition of cell growth (protein method). The methodin surface waters with an impact on the ecosystem. For this used was that of Knox et al. (1986), in which Kenacid bluereason, study was made of different chemicals, structurally is employed to stain the cell protein. Cells were harvestedunrelated, as components of shampoos: triethanolamine, zinc and diluted to a final concentration of 15 1 103 cells/ml inpyrithione, sodium lauryl sulfate, and oleamine. MEM medium. The cell suspension was shaken for about

Data from literature demonstrated the irritation potential 30 min and then 200-ml aliquots were added to each wellof triethalonamine to human skin in vivo when the patients of a 96-well tissue culture plate. Plates were incubated forwere tested with topical preparations (Pasche et al., 1994). 24 hr to allow the cells to attach. The medium covering theSchnuch et al. (1993) found that triethalonamine and other cells was then removed and 200-ml aliquots of the test chemi-chemicals produced adverse effects in vivo. cal solutions were added. Two dose levels were made up

for each compound and 8 wells were used for each dose1 To whom correspondence and reprint requests should be addressed. level. The remaining wells were controls and received 200

560147-6513/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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Page 2: Toxicity of Potential Irritants in Mammalian Cellsin Vitro

57TOXICITY OF POTENTIAL IRRITANTS

TABLE 1Cell Density (%) on SIRC Cells

TE Znpt SLS OA

Replicates 5% 10% 5% 10% 5% 10% 5% 10%

A 89 79 76 69 85 77 98 95.5B 87 76.7 73 67 84.8 76.7 97 94.7C 88.5 77 74.5 70 85.4 75 99 95D 88 76 76.2 65 84 73 97.8 93.8E 87.7 75.8 72.8 66.8 84.7 74.5 98.8 94.8F 87.5 76.5 74.9 67.5 85 75 98 95

Mean { standard error 87.95 { 0.27 76.83 { 0.43 74.56 { 0.54 67.55 { 0.66 84.82 { 0.17 75.20 { 0.55 98.10 { 0.27 94.80 { 0.21

ml of medium only. Plates were then incubated for 24 hr. All experiments were replated six times (replicates) ontriplicate cultures.At the end of this period, the medium covering the cells was

removed and the cells were washed twice with phosphate- Statistical Analysis. A bidirectional analysis of variancebuffered salts without magnesium or calcium (PBS ‘‘A’’) at (ANOVA) with six replicates and two variation factorsphysiological pH. The final wash was removed and 200 ml (doses and treatments) was done with data. The least signifi-of fixative (3% glutaraldehyde in PBS ‘‘A’’) added to each cant difference (LSD) test was done when F was significant.well and left for 2 hr. On removal of the fixative, cells werestained for 30 min with a 200-ml well of the dye solution

RESULTS AND DISCUSSION(0.4 g of Kenacid blue was dissolved in 250 ml of ethanoland 630 ml of water). Immediately before use, 12 ml ofglacial acetic acid was added to each 88 ml of stain and the The results from treatments for SIRC cells and human

fibroblasts are presented in Tables 1 and 2, respectively.solution was filtered. The dye was then removed and 200ml of destain (ethanol:glacial acetic acid:water, in the ratio Table 3 reflects the results of variance analysis, F values,

and their significance. These results agree with some authors2:1:17) was added to each well. The plate was agitated for5 min and the destain removed. The destain procedure was (Igarashi, 1986; Batten et al., 1994).

The highest cytotoxicity was observed when the two cellrepeated, agitating for 15 min, until the destain no longertook up the blue coloration of the dye. lines were treated with zinc pyrithione, and the significance

was calculated at P õ 0.01 (LSD test).The final destain was removed and precisely 200 ml/wellof desorbing solution (1 M potassium acetate in 70% ethanol) Lansdown (1991) found the dermal irritancy of zinc com-

pounds as zinc pyrithione in animal models.added. The absorbance of the solution in each well wasmeasured at 620 nm using a microplate reader (EL 312 c). On the contrary, the lowest cytotoxic effect (P õ 0.01)

was observed with oleamine on two cell lines used.The average absorbance at each dose level was calculatedand plotted as the percentage absorbance of control against The cytotoxic effects of sodium lauryl sulfate and trietha-

nolamine were similar in Fc cells. However, when SIRCdose.

TABLE 2Cell Density (%) on Human Fibroblasts

Te Znpt SLS OA

Replicates 5% 10% 5% 10% 5% 10% 5% 10%

A 85 75 72 65 84.5 72 98.8 92B 83.2 72.6 69.8 63 86.5 75.6 99 96C 84.5 73.2 70.5 66.2 83.5 71.8 98.1 95D 83.9 71.8 72.3 61.8 84.5 72.6 97.9 95.5E 83.4 70.5 68.8 62 86 74.3 98.5 96F 82.9 72.6 70.9 63.4 85.2 73.9 97.5 94.3

Mean { standard error 83.82 { 0.30 72.62 { 0.56 70.72 { 0.49 63.57 { 0.64 85.03 { 0.41 73.34 { 0.55 98.30 { 0.21 94.80 { 0.57

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Page 3: Toxicity of Potential Irritants in Mammalian Cellsin Vitro

58 SANTA MARIA ET AL.

TABLE 3 in vitro and that they can cause damage in the ecosystemStatistical Analysis of ANOVA Data: F Values with an impact on human safety.

Cell linesREFERENCES

Variable df SIRC FcBasketter, D. A., Whittle, E., Griffiths, H. A., and York, H. (1994). The

identification and classification of skin irritation hazard by a human patchTreatment 3 162.58** 1060.85**test. Food Chem. Toxicol. 32, 769–775.Dose 1 66.13** 416.97**

Batten, T. L., Wakeel, R. A., Douglas, W. S., Evans, C., White, M. I.,Interaction 3 3.87* 8.33**Moody, R., and Ornerod, A. D. (1994). Contact dermatitis from the oldformula E45 cream. Contact Dermatitis 30/3, 159–161.Note. Significance indicated by *P õ 0.05; **P õ 0.01.

Igarashi, H. (1986). With which component of the bovine isolated corneadoes sodium lauryl sulphate react to produce opacity? Toxicol. Lett. 32,157–162.cells were used, the cytotoxicity of SLS was higher (P õ

Igarashi, H. (1987). The opacification of the bovine isolated cornea by0.05) than that of TE.surfactants and other chemicals. A process of protein denaturation? AtlaIgarashi (1987) demonstrated that some TE compounds15, 8–19.

were more of irritants than SLS compounds when they wereIkarashi, Y., Tsukamoto, Y., Tsuchiya, T., and Nakamura, A. (1993). Influ-

tested on corneal epithelium. Igarashi (1986) also suggested ence of irritants on lymph node cell proliferation and the detection ofthat SLS compounds caused irritancy by precipitating pro- contact sensitivity to metal salts in the murine local lymph node assay.

Contact Dermatitis 3, 128–132.teins.Knox, P., Uphill, P. F., Fry, J. R., Benford, D. J., and Balls, M. (1986).On the other hand, Batten et al. (1994) demonstrated that

The FRAME multicentre project on in vitro cytotoxicity. Food Chem.TE compounds produced allergical reactions in clinical ex-Toxicol. 24, 457–463.periments.

Lansdown, A. B. (1991). Interspecies variations in response to topical appli-These authors suggest that TE should be controlled whencation of selected zinc compounds. Food Chem. Toxicol. 29, 57–64.

it is used as component in cosmetics. These results agreePasche, K. F., Piletta, P. A., Hunziker, N., and Hauser, C. (1994). High

with this consideration. sensitizacion rate to emulsifiers in patients with chronic leg ulcers. Con-In all cases (TE, Znpt, SLS, and OA) the highest concen- tact Dermatitis 31, 226–228.

tration (10%) used indicated the lowest cell growth in both Schnuch, A., Arnold, R., Bahner, F., Brasch, J., Diepgen, T. L., Enders,F., Frosch, P. J., Fuchs, T., Geier, J., Henseler, T., Muller, S., Peters,cell lines. These results indicate that in vitro tests usingK. P., Schulze-Dirks, A., Stary, A., Uter, W., and Zimmer-Mann, J.cell cultures are valuable tools for evaluating toxicity of(1993). Epicutaneous patch testing with an emulsifier, additive and vehi-chemicals widely used in the industries.cle series. Results of the Information Network of Dermatological Clinics(INDK). Dermatosen Beruf Unwelt 41, 176–183.

CONCLUSION Skoulis, N. P., Barbee, S. J., Jacobson-Kram, D., Putman, D. L., and San,R. M. (1993). Evaluation of the genotoxic potential of zinc pyrithione in

It can be concluded that these results support the facts the Salmonella (Ames) assay, CHO/HGPRT gene mutation assay andmouse micronucleus assay. J. Appl. Toxicol. 13, 283–289.that TE, Znpt, and SLS bioactive materials exhibit toxicity

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