Safety evaluation of selenium sulfide antidandruff shampoos

TOXICOLOGY

Safety

AND

APPLIED

Evaluation

PHARMACOLOGY

20,89-96 (1971)

of Selenium

Sulfide

Antidandruff

Shampoos1

LAURENCEM. CUMMINSAND EUGENET. KIMURA Division of Experimental Pharmacology, Abbott Laboratories, North Chicago, Illinois 60064 Received November Id,1970

Safety Evaluation of Selenium Sulfide Antidandruff Shampoos. LAURENCE M., and KIMURA, EUGENE T. (1971). Toxicoi. Appl. Pharmacol. 20, 89-96. It is well documentedthat various seleniumcompoundsare highly toxic. However, it should be pointed out that there are alsocertain ionic formsof seleniumthat arenot astoxic whentaken orally. One suchcompoundis the water-insolubleseleniumsulfide(Se&). Studies were conducted, therefore, to presentthe following data to support the safety of seleniumsulfide and the seleniumsulfide-containingshampoos: (1) seleniumsulfide (Se&) should not be classifiedin the sametoxicity categoryas the highly toxic sodiumselenite,sinceSe& is20timeslesstoxic; (2) comparativeoral LD50 valuesin rats of Se& and active components from other leading antidandruff shampoosindicate that Se& should be classifiedin the sametoxicity category astheseother active ingredients.(3) the oral LD50 valuesfor 1% and 2.5 % Se& shampoosare about 10 times the emetic doses,thus suggestingthat a toxic amount of shampoocould not be ingestedwithout emesisoccurring; and (4) oral dosing of a 1% SeSzformulation to dogsindicatesessentiallyno absorption as indicated by the lack of an increasedseleniumblood level. CUMMINS,

Selenium was first discovered to be a toxicant when Robinson (1933) found that wheat administered to cattle. Similar studies in the ensuing years confirmed this early report, and a generalization was established that selenium was an extremely toxic element. Unfortunately this generalization has unfairly included all ionic forms of selenium, even though it was later discovered that all ionic species are not nearly as toxic as the soluble selenite and selenate salts. As a result of this generalization, many texts, such as those by Goodman and Gilman (1965), Drill (1965), and Beckman (1961), as well as numerous individual papers have cited this concept that selenium sulfide is as toxic as the soluble salts. The purpose of these studies, is, therefore, to present data that will demonstrate that selenium sulfide in shampoos should not be compared to those soluble selenium salts when referring to their toxicity.

containing lo-12 ppm selenium caused death when chronically

METHODS AND MATERIALS Oral toxicity of seleniumcompounds.Comparative oral toxicity studies were carried out in rats with the following selenium compounds: sodium selenite,? selenourea,3 ’ A portionof thisworkwaspresented at theAnnualMeetingof theFederationof AmericanSocieties for ExperimentalBiology in Atlantic City, New Jersey 1970. 2 Sodium selenite from Alfa Inorganic, Inc., stock No. SE-103. 3 Selenourea from K and K Laboratories, Inc., Lot 84028 DF. 89

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biphenyl selenium,4 selenium sulfide’ (l-30 Al.particle size), and elemental selenium6 (l-30 p particle size). In this study male Sprague-Dawley rats, weighing between 50 and 100 g, were gavaged in groups of 6 with various doses of the above chemicals, prepared as 0.1-20 “6 suspensions in 0.5 y,; methylcellulose. A total of 30-36 rats were used per compound. All these animals and those used in subsequent studies were observed for 7-10 days following treatment. The LD50 values and the associated confidence limits, in this and in subsequent studies, were calculated according to the method of Litchfield and Wilcoxon (1949). Selenium blood levels in dogs following oral ingestion of selenium compounds. The selenium blood levels produced by oral administration of the above selenium compounds were obtained in dogs so that correlations between toxicity, aqueous solubilities, and blood selenium levels could be made. All dogs were dosed with each compound so that they received 2.0 mg Se/kg. The five compounds tested were prepared as 0.1-0.3 % suspensions in 0.5 y; methylcellulose. Two male mongrel dogs were used for each compound, and blood samples were taken at 0, 1, 2, 4, 6, 8, 12, 24, and 32 hr after dosing. Two milliliters of whole blood were analyzed according to the procedure of Watkinson (1966), and the average selenium blood levels of the two dogs on each compound are presented in Fig. 1 Comparative LD.50 values of various active antidandrufl compounds. The oral LD50 values for the following compounds were obtained in the same manner as previously described: sulfur, benzalkonium chloride, selenium sulfide, zinc pyrithione, and hexachlorophene. The oral LD50 of these active ingredients and their percent (w/v) concentrations in their clinical formulations are presented in Table 2. Establishment of emetic dose 100 of selenium sulfide shampoo formulations. Mongrel dogs of both sexes, weighing between 5.7 and 12.5 kg, were gavaged with either 1 yi7 or 2.5 “/<” selenium sulfide shampoos at 0.25, 1.O, 2.0, and 5.0 ml/kg, 3 animals per dose. This emetic dose was established from experiments carried out in 40-50 dogs over a period of several years. These results are reported in Table 3. Determination of the LD.50 of selenium sulfide shampoo formulations. Male SpragueDawley rats, weighing between 50 and 100 g, in groups of 6, and female Swiss-Webster mice, 20-24 g in groups of 10, were gavaged with increasing doses of the formulations (2.5Tis and 1 “/i’ selenium sulfide detergent suspensions). A total of 30-36 rats and 50-60 mice were used per compound. These results are shown in Table 3. Selenium blood levels produced by oral ingestion of a selenium sulfide shampoo formulation in dogs. Figure 2 gives the average selenium blood levels of 2 groups of 4 mongrel dogs ranging in weight from 7.6 to 11.O kg. Four dogs in one group were given a 0.1 ml/kg po dose of a 1 “/,’ selenium sulfide shampoo. 5 This formulation contained 5.52 mg of Se/ml as Se&. Four additional dogs were also given by gavage a 0.1 ml/kg dose of a specially formulated shampoo. The latter shampoo was identical to the previous selenium sulfide supplemented detergent, with the exception that sodium selenite was substituted for seleniumdisulfide 4 Biphenyl selenium from K and K Laboratories, Inc., Lot 6276. 5 Selenium sulfide from Abbott Laboratories Lot 807-7491. 6 Elemental selenium from Abbott Laboratories Lot 3619-110. 7 Selsun@ Suspension, containing 2.5% Se&. 8 Selsun BlueTm Anti-Dandruff Lotion Shampoo, containing 1% Se%

SAFETY

OF SELENIUM

91

SULFIDE

This formulation contained 5.52 mg of Se/ml as sodium selenite. Blood samples were obtained at 0,2,4, 8, 12, and 24 hr after dosing. The blood samples were assayed for selenium as previously mentioned according to the Watkinson (1966) procedure. In the group of dogs receiving the sodium selenite supplemented formulation, emesis occurred in all 4 dogs within 45 min after dosing. However, there was no emesis in the group of dogs given selenium sulfide shampoo. RESULTS

AND

DISCUSSION

The oral LD50 values in rats for a number of selenium compounds are presented in Table 1. These five different selenium compounds are chosen to demonstrate the large differences in LD50 values that occur depending on both (1) the oxidation state of selenium and (2) its aqueous solubility. The aqueous solubilities were carried out in 0.01 N HCI to more closely simulate acidic conditions in the stomach. TABLE SELENIUM

Compound

COMPOUNDS

TESTED,

1

THE SOLUBILITIESAND LD50 VALUES

Solubility in 0.01 N HCl

Rat oral LD50 (95 % CL) (w/kg)

Na,SeO,

700 mg/ml

Se II H*N-C-NH2

30 mg/ml

Se&

Insoluble (4 mg/ml)

138 (110-172)

5 mg/ml

360(308421)

Elemental Se

Insoluble (4 mg/ml)

7 (4.4-l 1.2)

50 (35.7-70.0)

6700(6OOtL7300)

The least toxic was the insoluble elemental selenium with an LD50 of 6.7 g/kg. Signs of drug action included pilomotor activity, decreased body activity, dyspnea, diarrhea, anorexia, and cachexia. Fatalities occurred within 18-72 hr; survivors appeared normal, outwardly, at the end of the 7-day observation period. The most toxic of the selenium compounds was the highly soluble sodium selenite with an oral LD50 of 7 mg/kg. Signs of drug action were similar to those seen with high doses of the elemental selenium. The much less soluble (30 mg/ml) selenourea showed an LD50 of 50 mg/kg. Selenium sulfide, by comparison with sodium selenite, was about 20 times less toxic, its calculated LD50 being 138 mg/kg. The biphenyl selenium compound gave an oral LD50 of 360 mg/kg. Again, signs of drug action were essentially similar to those seen from the other selenium compounds. Figure 1 shows the selenium blood levels in dogs of five selenium compounds administered po at a dose of 2 mg/kg. Acute oral LD50 values were previously determined in rats for these compounds. The purpose of this experiment was to see whether toxicity

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could be correlated with blood selenium levels. With the exception of the inconsistency seen in the high blood level resulting from the biphenyl selenium compound, the blood levels of the remaining 4 compounds correlated well with the degree of toxicity. That is, sodium selenite being the most toxic gave the highest blood level, followed in order by selenourea, selenium sulfide and elemental selenium. Possibly the best explanation for the high blood selenium level produced by the relatively nontoxic biphenyl selenium compound is that the covalently bound selenium compound is the most lipophilic compound tested, thus leading to better absorption.

I 4

I 8

1 12

I 16 Time

I 20

I 24

1 28

I 32

(Hours)

FIG. 1. Selenium blood levels in dogs after oral ingestion of the above compounds (dosed at 2.0 mg Se1kg).

However, a more plausible explanation is that this biphenyl covalently bound selenium compound resisted catabolism and apparently circulates as parent compound. This statement is supported by gas chromatographic studies which demonstrated that approximateIy 80% of the selenium blood levels could be accounted for as biphenyl selenium. This supports the popular theory that selenium compounds are only highly toxic when the selenium is metabolized to the Se4+ oxidation state (Rosenfeld and Beath, 1948; Potter and Elvehjem, 1937). Once the selenium compound is metabolized to this ionic species, it has the ability to bind very tenaciously to proteins (Cummins and Martin, 1967) thus probably acting as a toxicant by some means of enzyme inhibition. Henschler and Kirschner (1969), in their recent report on the absorption and toxicity of selenium disulfide, also presented evidence in mice to question the concept carried over in the literature from author to author that the insoluble sulfide of selenium was equally toxic and as easily absorbed from the gastrointestinal tract as the soluble selenium comnounds. The narticle sizes of the chemical specimen ranged from .

SAFETY

OF SELENIUM

93

SULFIDE

approximately 1 to 50 p in diameter with a peak at 5-l 5 CL,and were determined microphotographically. They obtained an oral LD50 in mice of 3.69 g/kg (95 % CL 3.154.28). Our studies with a sample of selenium sulfide of l-30 p in diameter with a peak (75%) of 5-10 /* showed an oral LD50 of 142 mg/kg (95% CL 123.5-163.5) in mice while rats showed an oral LD50 of 138 mg/kg. Therefore, their results indicate that sodium selenite is about 77 times more toxic than selenium sulfide, whereas we find it to be about 20 times more toxic. This difference may be explained by the different particle size used in each study. Table 2 shows the acute oral LD50 in rats of the active ingredients of some commonly used shampoo formulations. Sulfur was the least toxic of the compounds tested, being nonlethal at a dose of 50 ml/kg of a 20 % suspension, equivalent to 10 g/kg. Dyspnea, decreased activity and diarrhea were the only side effects seen at this massive dose. By contrast, hexachlorophene and zinc pyrithione showed LD50 values of 90 and 92 mg/kg, respectively. Selenium disulfide, as shown in Table 1, had an oral LD50 of 138 mg/kg, while benzalkonium chloride showed an LD50 of 400 mg/kg. TABLE

2

COMPARATIVE ORAL LD50 VALUES IN RATS OF THE ACTIVE INGREDIENTS IN COMMONLY USED SHAMPOO FORMULATIONS

Compound Sulfur Benzalkoniumchloride Seleniumdisulfide Zinc pyrithione Hexachlorophene

LD50 (95 % CL) bm/kg) Greater than 10,000 400(342469) 138(110-172) 92 (73-l 17) 90 (85-96)

o/0 Active (w/v) ingredient commonly found in shampoo formulations 2.0-8.0 2.0-3.0 1.o-2.5 2.0 3.0-5.0

A more precise picture of the relative potential clinical toxicity of these formulations is seen if the LD50 values for these active ingredients are now related to their actual percent concentrations in their respective formulations. For example, Se& in the 1 y/, SeSz formulation; zinc pyrithione, at 2 % ; and hexachlorophene has been used at up to 3 %. Thus, the safety factor for the formulation containing 1 ‘A SeS, is greater than is apparent by the LD50 value per se of the active ingredient alone. The LD50 values of the active ingredients must be viewed in the light of the concentrations in the final formulations. The results of the emetic dose studies in dogs and the margin of safety derived from dog and rat studies are shown in Table 3. The emetic dose 100 was found to be 1 ml/kg for the 1% selenium sulfide preparation and 0.5 ml/kg for the 2.5 7: selenium sulfide. Only slight diarrhea was seen with both preparations at 0.5 ml/kg. Emesis occurred in dogs within 3-20 min at high doses and within 2.8 hr at the 0.5 and 1 ml/kg doses. The LD50 values shown for mice were averaged from 5-6 samples tested; those for rats, from 3-4 samples tested. Although aware of the pharmacologic inconsistency of combining rodent and dog

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RELATIONSHIP BETWEEN ORAL EMETIC DOSE (DOGS) AND ORAL LD50 (RODENTS) OF SELENIUM SULFIDE SHAMPOO FORMULATIONS Acute LD50 (ml/kg) Emetic dose, Dogs

(ml/kg)

Mice

Rats

Margin of safety

Se!&, 1%

1.0

14.2

Se&, 2.5 %”

0.5

7.8 4.9

8to 14 lot0 II

Formulation

5.3

’ Selsum Blue’“’ Anti-Dandruff Lotion Shampoo, containing 1% Se&. b Selsun@ Suspension, containing 2.5 % SeS,.

data, we have, of necessity, compared the oral LD50 values obtained from rodents and related them to the emetic dose values obtained from dogs. This inability of rodents to vomit coupled with the easewith which dogs vomited when dosed with the 1% and 2.5 x, selenium sulfide detergent suspensionspresented a dilemma in arriving at a margin of safety. The therapeutic efficacy and, of course, the therapeutically effective doses could not be determined in either rodents or dogs. While the total amount of administered drug was not retained by the dog due to emetic action, rats, lacking this physiologic defensemechanism, retained the entire dose and continued to be exposed to the administered drug formulation. In spite of this forced retention of drug by the rat, the insoluble nature of the selenium sulfide apparently prevented any great absorption of the drug as seen by the comparative LD50 of the insoluble selenium sulfide with an LD50 of 138 mg/kg as compared with the soluble sodium selenitewith an LD50 of 7 mg/kg. The acute oral LD50 values in mice and rats of the seleniumsulfide detergent suspensions (Table 3), expressedin ml/kg (14.2 and 5.3 ml/kg for mice and 7.8 and 4.9 ml/kg for rats) are roughly equivalent in selenium sulfide content to 142, 133, 78, and 123 mg/kg, respectively. This, therefore, provides a margin of safety ranging from 8 to 14 for the 1% suspensionand from 10 to 11 for the 2.5% suspension. Figure 2 presents further evidence that a selenium sulfide supplemented shampoo has a relatively low degree of toxicity since the po administration of the shampoo to dogs resulted in essentially no increase in the normal selenium blood level. The high blood levels produced by the sodiumselenitesupplementedshampoowould undoubtedly have been much greater had emesisnot occurred in all 4 dogs on this formulation. Attempts were made to conduct a successfulstudy so that there would be 4 animals on each formulation without the occurrence of emesis.This objective was not achieved, even though 12 dogs were dosed with the sodium seleniteformulation, and in all cases the 0.1 mg/kg doseinduced emesiswithin 1 hr. Therefore, the 4 dogs that retained the shampoo for at least 30 min were selectedfor comparative purposes. Additional data (unpublished) that provide further support regarding the safety of the 1;i selenium sulfide shampoo were developed from a 1 yr shampoo study in man. This study involved 16 subjectswho shampooedweekly and each month provided a complete 24-hr urine specimen in that period immediately following a shampoo treatment. Eight control subjectswere also carried through the entire study, so that a

SAFETYOFSELENIUMSULFIDE

L

4I

6I

I

I

12

Time

16

95

I

20

i 24

(Hours)

FIG. 2. Selenium blood levels in dogs after oral ingestion of Se& and Na,SeOJ supplemented shampoos.

normal urinary selenium level could be established. The results from this study indicated no significant difference between the control and shampoo group at the 95 % confidence level in the amount of selenium excreted. This, therefore, indicates no apparent percutaneous absorption of selenium following a year of use. In conclusion, it is the opinion of the authors that these data not only provide sufficient evidence to clearly demonstrate that selenium sulfide shampoos are as safe or safer than other leading antidandruff shampoos, but in addition it is hoped that these data will be a step forward in removing the fears of “Selenium Toxicity” that are associated with various selenium compounds. ACKNOWLEDGMENTS The invaluable assistance of Mr. D. M. Ebert and Miss Jan Perry are greatly appreciated in obtaining these data. REFERENCES BECKMAN, H. (1961). The Nature, Action and Use of Drugs, 2nd ed., pp. 693. Saunders, Phila-

delphia. CUMMINS, L. M., and MARTIN, J. L. (1967). Are selenocystine and selenomethionine thesized in vivo from sodium selenite in mammals? Biochemistry 6, 3162-3168.

syn-

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DRILL, V. A. (1965).Pharmacology in Medieiw, 3rd ed., pp. 798. McGraw-Hill, New York. GOODMAN, L. S., and GILMAN, A. (1965). The Pharmacological Basis of Therapeutics. 3rd

ed., pp. 984. Macmillan, New York. HENSCHLER, D., and KIRSCHNER, U. (1969). Zur Resorption und Toxicitlt von Selensulhd. Arch. Toxikol. 24, 341-344. LITCHFIELD, J. T., and WILCOXON, F. (1949). A simplifiedmethod of evaluating dose-effect experiments.J. Pharmacol. Exp. Ther. 96,99-101. POTTER, V. R., and ELVEHJEM (1937).The effect of inhibitors on succinoxidase. J. Biol. Chem. 117,341-349.

ROBINSON, W. 0. (1933).Determination of seleniumin wheat and soils.J. ASS. Oflc. Agr. Chemists 16, 423424. ROSENFELD, I., and BEATH, A. 0. (1948).Metabolismofsodiumselenateandselenite by tissues. J.Biol. Chem. 172, 333-341. WATKINSON, J. H. (1966). Fluorometric determination of seleniumin biological material with 2,3-diaminonaphthalene.Anal. Chem. 38,92-97.