Fd Chem. Toxic. Vol. 24, No. 6/7, pp. 477-479, 1986 0278-6915/86 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1986 Pergamon Journals Ltd

IN VITRO TOXICITY OF SOME COSMETIC INGREDIENTS

I. DE ANGELIS, L. GIUBILEI, A. STAMMATI, F. ZAMPAGLIONI, f . Zucco*, G. BARTOLINI and G. SALVATORE

Istituto Superiore di Sanit~i, Laboratorio di Tossieologia Comparata ed Ecotossicologia and *Istituto di Tecnologie Biomediche, CNR, Roma, Italy

Abstract--Interest concerning the possible adverse effects of cosmetic products is increasing and at the same time toxicologists are faced with the problems arising from in vivo testing and the growing development of alternative methods. A series of n-alkyl benzoates has been tested on a continuous cell line (HEp-2). Dose-effect correlations have been established for methyl, ethyl, n-propyl and n-butyl benzoates by determining the total protein content of the cells at 24 hr. The toxicity ranking of these benzoates according to the IC50 has been shown to be butyl > propyl > ethyl > methyl benzoate. Experiments carried out on time-effect correlation and on colony-forming ability seem to suggest that the damage does not increase with time and is probably caused mostly by an initial selective effect on the cell population.

Introduction

Because of the continually increasing use of cosmetics and toiletries, the control of these products from the toxicological point of view has become as important as for other chemicals. Since tests on animals are expensive and time-consuming and some of them are criticized for both scientific and ethical reasons, alter- native methods are being developed to assess the safe use of cosmetics.

A continuous cell line (HEp-2) has been used in a study of the effect of four n-alkyl benzoates on cell growth. This epithelial cell line, derived from a human carcinoma of the larynx, is well characterized, easy to handle and particularly suitable when changes in basal cell functions and structures have to be explored.

Methyl benzoate (MB), ethyl benzoate (EB), n- propyl benzoate (PB) and n-butyl benzoate (BB) were tested. They are commonly used as preservative and fragrance ingredients in cosmetics (Opdyke, 1974a, b; Opdyke & Letizia, 1983; Table 1) and are regulated by an EEC Directive (EEC, 1982), which specifies the maximum admissible level of benzoic acid, its salts and esters as 0.5% acid. These esters may also be added to cosmetic products in concentrations other than those laid down in the Annex if they are included for other specific purposes apparent from the presentation of the product, e.g. as deodorants in soaps or as anti-dandruff agents in shampoos.

Abbreviations: BB, EB, MB and PB=n-butyl, ethyl, methyl and n-propyl benzoates; EEC = European Eco- nomic Community,

Experimental

Cell cultures. HEp-2 cells, an epithelial cell line derived from a human carcinoma of the larynx, were routinely cultured in monolayers at 37°C in 5% COJair, in disposable plastic flasks (Falcon Plastics, Oxnard, CA, USA) containing minimum essential medium (MEM) supplemented with Earle salts, 5% foetal bovine serum (Flow Laboratories, McLean, VA, USA), 4 mu-glutamine, 0.2%0 sodium bicarbonate, 100 IU penicillin/ml and 100 #g streptomyein/ml.

Preparation of benzoate solutions. Benzoates were added to the complete medium, which after vigorous shaking was left overnight at 24°C. On the following day the solutions were checked by eye and by diffraction spectra observation for the presence of undispersed particles. To confirm the level of each benzoate, an aliquot of the culture medium was carefully dispensed into a special glass vial and the benzoate was determined by gas-chromatography headspace analysis. The operating conditions are to be described in a paper to be published by G. Salvatore.

Dose-effect correlations. HEp-2 cells were plated in 25-cm 2 Falcon flasks at 105cells/flask. On the next day, serial dilutions of a benzoate were added to the cells, beginning in each case with a concentration close to the saturation level--1000, 500, 250 and 120 mg/litre for MB, EB, PB and BB, respectively. Each concentration was tested in duplicate and the flasks were kept at 37°C with the screw plugs well closed. After exposure for 24 hr the cells were treated for protein determination.

Time-effect correlations. The cells were plated as

Table 1. Some available toxicological and usage data on benzoates

Benzoate

Oral (rat) LD~0 Skin (g/kg) irritation

Max conch (%) used in:

Soaps Detergents Creams Perfumes

Methyl Ethyl n-Butyl

3.4 Moderate 6.5 Moderate 5.1 Moderate

477

0.1 0.01 0.03 0.4 0.1 0.01 0.1 0.8 0.1 0.01 0.03 0.6

478 I. DE ANGELIS et al.

BB PB EB MB 100 ~ .o p . . . •

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iv / o I I l I l I I I I 100 2 0 0 3 0 0 4 0 0 500 600 700 8 0 0 900 I000 1100

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Fig. 1. Dose-dependent effects of ethyl, methyl, n-propyl and n-butyl benzoates (EB, MB, PB and BB) on HEp-2 cells exposed to the chemical for 24 hr, expressed as linear regression of growth-inhibition data

(means of three experiments).

above and after 24hr the IC50, statistically deter- mined from the previous experiment for each benzo- ate, was added to the flasks, the concentrations used being 495, 289, 122 and 61 mg/litre for MB, EB, PB and BB, respectively. The flasks were kept at 37°C with the screw plugs well closed for 7 days, two flasks for each benzoate being removed daily for protein determination. After 24 hr and at the end of the 7-day exposure period, the concentration of benzoate was determined as described above.

Plating efficiency. The cells were plated in 25-cm 2 Falcon flasks at a density of 200 cells/flask in the presence of a benzoate at the ICs0 level. The cultures were incubated at 37°C for 8 days and then the cells were stained with May-Grunwald and Giemsa and colonies of at least ten cells were counted.

Protein determinations. The medium was removed and cells were washed three times with phosphate- buffered saline before being detached with 2% Na2CO 3 in 0.1 N-NaOH. The proteins were deter- mined by a slight modification of the method of Lowry, Rosebrough, Farr & Randall (1951).

R e s u l t s

An increase in toxicity with a rise in the benzoate concentration in the medium was observed and the ranking of the four substances according to their IC50 has shown that the harmful effects rise from methyl to butyl benzoate (Fig. 1). Regarding the length of exposure, no great differences were apparent up to day 4 of culture, with the growth inhibit ion persisting at around 50% for all the esters. However, on day 7, cells exposed to PB and BB seemed to recover, whereas MB-treated cells showed greater inhibition and EB-exposed cells did not show any variation from the estimated 50% growth inhibition (Fig. 2),

Benzoate concentrations in the medium showed a decrease of about 40-50% both in the presence and absence of the cells, as early as day 1 of incubation. A further decrease was observed on day 7 of culture particularly with PB and BB.

The monolayers of the treated cells are disturbed within 24 hr, and MB and EB, particularly, affect the morphology of the cells, which lose their typical epithelial shape and become elongated.

D i s c u s s i o n

Our in vitro results show that the ICs0 values of the four benzoates are between 10 3 and 10-4M and correlate quite well with the few available in vivo data, which indicate that these substances are of low toxicity (Sandmeyer & Kirwin, 1978). Furthermore, an in vitro study on HeLa cells gave an ICs0 of

2

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2000f

1000-

750

500

250

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Durat ion of culture (days)

Fig. 2. Time-dependent effects of (methyl (...-), ethyl ( . . . . . . ). n-propyl ( . . . . ) and n-butyl ( ) benzoates on the growth of HEp-2 cells, up to day 7 of culture, in comparison with the effect of time on untreated control cultures (---). The estimated 50% growth inhibition (50% of control values) is also shown (--O). Values are means + SEM for three experiments and those asterisked differ significantly from the 50% growth inhibition (P < 0.001 by analysis of

variance).

In vitro toxicity of some cosmetic ingredients 479

5.10 -4 M for BB (Freese, Levin, Pearce et al. 1979). As far as the ranking of the benzoates is concerned, it is notable that other studies using different in vitro systems and testing other compounds (phthalate es- ters and triorganotin chlorides) have shown a cor- relation between increasing toxicity and the length of the aliphatic chain (Lake, Lewis, Gray et al. 1985; Snoeij, Bol-Schoenmakers, Penninks & Seinen, 1986). In our case the mechanism involved seems to be attributable to a lethal effect occurring in the early phase of exposure. This was confirmed by experi- ments on colony-forming ability, studies in which total inhibition was observed for the four benzoates. This latter observation may indicate that the cell/compound ratio can also be critical for the toxicity determination.

The time-effect curves show different behaviour for the four benzoates. The partial recovery of the cells treated with PB and BB can be explained by the almost total disappearance of these compounds from the medium on day 7 of culture. Because of the physico-chemical characteristics of the benzoates, further studies on their stability in solution are needed.

These results suggest that such an in vitro system can be helpful in toxicity testing. Since these tests are sensitive enough to distinguish between compounds with low and similar IC50 values, and are also rela- tively simple, rapid and economic, the furtherance of their use as a partial substitute for animal experi- mentat ion is suggested.

Acknowledgements--The authors are grateful to Dr G. Zapponi for the statistical elaboration of the data. This

research was supported by Grant No. 84.01296.95 from the National Research Council Project "'Chimica fine e secondaria".

REFERENCES

EEC (1982). Council Directive 82/368/EEC, 17 May 1982. Annex VI, part I. Off. J. Eur. Commun. 25, (L167), 1.

Freese E., Levin B. C., Pearce R., Sreevalsan T., Kaufman J. J., Koski W. S. & Semo M. N. (1979). Correlation between the growth inhibitory effects, partition coefficients and teratogenic effects of lipophilic acid. Teratology 20, 413.

Lake B. G., Lewis D. F. V., Gray T. J. B., Beamand J. A., Hodder K. D., Purchase R. & Gangolli S. D. (1985). Induction of peroxisomal enzyme activities in primary rat hepatocyte cultures by phthalate monoesters: structure activity relationships. Poster presentation; International Conference on Practical In Vitro Toxicology. Session 6. Book of abstracts, p. 84.

Lowry O. H., Rosebrough N. J., Farr A. L. & Randall R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265.

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Snoeij N. J., van lersel A. A. J., Penninks A. H. & Seinen W. (1986). Triorganotin-induced cytotoxicity to rat thymocytes. Fd Chem. Toxic. 24, 599.