Mutation Research, 119 (1983) 35-43 35 Elsevier Biomedical Press

Mutagenic acitivity of Kathon, an industrial biocide and cosmetics preservative containing 5-chloro-

2-methyl-4-isothiazolin-3-one and 2-methyl- 4-isothiazolin-3-one

Chris t ine Wr igh t a, El l iot G ingo ld a, S tan ley Venit t b and

Chr i s t ophe r C ro f ton -S l e ig h b

aSchool o f Natural Sciences, The Hatfield Polytechnic, P.O. Box 109, College Lane, Hatfield, Herts. ALIO 9AB and bChester Beatty Research Institute: Institute o f Cancer Research, Pollards Wood Research Station, Nightingales Lane, Chalfont St. Giles, Bucks HP8 4SP (Great Britain)

(Accepted 2 August 1982)

'Kathon' is a proprietary name for a family of microbiocides and cosmetics preservatives containing 2 active constituents (Fig. 1): 5-chloro-2-methyl-4-iso- thiazolin-3-one (I) and 2-methyl-4-isothiazolin-3-one (II).

Kathon (in several formulations) has a variety of uses where microbiological con- tamination is a problem. For example, Kathon is used as a preservative for cosmetics and for cutting-oils, and as a biocide in the manufacture of paper and for swimming-pool water.

The formulation tested in this study (Kathon MW 886 Biocide) is an aqueous solu- tion of 10°70 of compound I and 3.4°7o of compound II, and was a gift to Dr. Gingold from Rohm and Haas Ltd., Croydon (Great Britain). The formulation (Kathon CG) sold for use as a preservative in a very wide range of cosmetics consists of 1.15°70 compound I, 0.35o70 compound II, 25°70 magnesium nitrate and 73.5°70 water.

There are no published accounts of the genetic toxicology of Kathon formulations or of the active constituents, although several unpublished studies have been sum- marized in technical literature supplied by Rohm and Haas Ltd. These studies are summarized below, in paragraphs (a), (b), (c) and (d).

(a) In a study conducted in 1976, Kathon 886 ('all magnesium, approx. 14°70 active ingredient') was reported to give negative results in the Ames test, using S a l m o n e l l a

t y p h i m u r i u m strains TA1535, TA1537, TA1538, TA98 and TA100, with or without a rat-liver metabolic activation system prepared from Aroclor-1254-induced rats, and doses ranging from 0.00005 to 0.1 #g of product per plate.

(b) In a second report of results obtained in standard Ames tests, Kathon 886 ( 'NAR Process, approx. 15.5°70 active ingredient') induced a statistically significant

0165-7992/83/0000-0000/$03.00 © Elsevier Biomedical Press

36

increase in revertants in TA100 at concentrations of 0.0005, 0.00075 and 0.001 /~l per plate, without activation. The same formulation was negative at concentrations ranging from 0.0075 to 0.12 nl active ingredient per ml in a mammalian cell transfor- mation test employing C3H 10T1/2 mouse embryo fibroblasts.

(c) Negative results were also obtained in a bone-marrow cytogenetics study per- formed in vivo in male Sprague-Dawley rats forcibly fed for 5 consecutive days with daily doses of a Kathon formulation (Kathon RH-886T) ranging from 0.28 to 28 mg active ingredient per kg body-weight.

(d) Kathon 886 (formulation and doses not stated) produced a positive result in a mammalian cell point-mutation assay (mouse lymphoma TK locus). No further details of this test were available to us.

Kathon CG, at 30 ppm of active constituents, is recommended as a preservative for a variety of cosmetics (including skin- and body-creams, hair shampoos, bubble- baths, sun-screens and mascara). In view of the widespread exposure of people to this preservative (the manufacturers estimate that in 1980 it was used in 55 000 ton- nes of cosmetics end-products in Europe, and 20000 tonnes of end-products in the USA) and in the light of the conflicting unpublished genotoxicity data, we decided to test the ability of a Kathon formulation to induce mutation in 2 species of bacteria.

M a t e r i a l s and m e t h o d s

Chemicals The formulation tested in this study (Kathon MW 886 Biocide, a gift to Dr.

Gingold from Rohm and Haas Ltd.) contained (by weight) 10o70 of compound I, 3.4°7o of compound II, 9°70 magnesium chloride and 15070 magnesium nitrate, made up in water. In this study, 'ng active constituents' refers to the total weight of com- pound I and compound II as calculated from the percentage values mentioned above.

Bacterial mutation assays (i) Plate-incorporation assays. Mutation assays using the plate-incorporation

method were performed independently in two laboratories, both using the same batch of Kathon 886 MW.

0 Q

C~CH 3 ~'CH 3 I II

Fig. 1. Chemical structure of active constituents of Kathon: (I) 5-chloro-2-methyl-4-isothiazolin-3-one; (II) 2-methyl-4-isothiazolin-3-one.

37

In laboratory 1, assays were performed using the methods described by Ames et al. (1975). Rat-liver $9 was prepared from an Albino Worcester rat supplied with 0.107o phenobarbital in its drinking water for 1 week prior to its death. Preliminary tests showed that Kathon, in the absence of $9 mix, was mutagenic to TA100, but not to TA1535, TA1537 or TA98. S. typhimurium TA100 was therefore assayed in plate-incorporation tests in order to obtain dose-response curves. 3 separate ex- periments were performed, each using l plate per dose of Kathon, which was diluted in sterile distilled water to achieve the desired concentration. In the first experiment, assays were performed (in the dose range 0 -40 nl Kathon per plate, equivalent to 0-4.36/~g active constituents per plate) in the presence of $9 mix containing 4070 $9, and in its absence: $9 mix was omitted in the other 2 experiments. Positive controls consisted of spot-tests with methyl methanesulphonate and 2-aminofluorene.

In laboratory 2, Kathon 886 MW was assayed for mutagenicity in S. typhimurium TA100 and Escherichia coli WP2uvrA(p) by the method described by Venitt and Crofton-Sleigh (1981). In the first experiment, Kathon 886 MW was diluted l:10000 in deionized water, filter-sterilized and assayed in the dose range equivalent to 1-20 nl per plate (134-2680 ng of the active constituents per plate). The assay was per- formed with and without the addition of an $9 mix containing 1007o (v/v) $9 prepared from the livers of Aroclor-1254-induced CB-Hooded male rats. In the sec- ond experiment the dose range was 0 .1-1 .0 nl per plate (13.4-134 ng active consti- tuents per plate), and $9 was not used. 3 plates per dose were used at each dose level in both experiments. Sodium azide was used as a positive control, giving slopes (mutants per /xg) of 755 and 1109 for E. coli WP2uvrA(p) and S. typhimurium TAI00 respectively. Data from both laboratories were analysed by the methods described by Venitt (1982).

(ii) Fluctuation tests. Kathon 886 MW was assayed for mutagenicity, in the absence of an exogenous activation system, in two separate Microtitre fluctuation tests using the method of Gatehouse (1978), and employing S. typhimurium TA100 and E. coli WP2uvrA(p). Positive controls consisted of 10 #g per ml 4-chloro- methylbiphenyl for TA100, (59, 88), and 1.5/xg per ml potassium dichr~omate for E. coli WP2uvrA(p) (84, 87), the numbers in parentheses being the number of positive wells (per 96) for Expts. 1 and 2 respectively. Fluctuation-test data were analysed by the method described by Venitt (1982).

Results and discussion

The results of the plate-incorporation tests are shown in Table 1 and Fig. 2 (Laboratory 1) and Table 2 (Laboratory 2). Laboratory 1 obtained reproducible linear dose-response curves in 3 separate experiments where Kathon 886 MW was tested in the absence of $9 mix. The mean slope of 2.69 +_ 0.28 revertants per ng of active constituents indicates that one or both of the biocidally-active constituents

38

TABLE 1

PLATE-INCORPORATION TESTS OF KATHON (LABORATORY 1)

One plate per dose was used in each experiment.

886 MW USING S. typhimurium TA100

Dose of Kathon 886 MW Number of revertans per plate

nl per plate ng active consti- Expt. l Expt. 2 Expt. 3

tuents per plate - $9 + $9 - $9 - $9

0 0 158 50 141 126 1 134 228 71 206 297 2 268 358 106 338

4 536 956 130 910 5 670 2512 6 804 2196 174 2032

8 1072 3040 224 2808 10 1340 3296 254 3736 4432 15 2010 4408 456 5728

20 2680 202 484 1076 toxic 25 3350 toxic 668 toxic 30 4020 toxic 784

35 4690 toxic 824 40 5360 toxic 1156

Regression analysis Slope, mutants per ng active constituents 2.35 0.19 2.81 2.91 Correlation coefficient 0.9816 0.9902 0.9865 0.9911 Student's t for slope 12.58 23.6 13.03 12.88 Degrees of freedom 6 11 5 3 P <0.001 <0.001 <0.001 <0.001

o f K a t h o n 886 M W ( c o m p o u n d s I a n d / o r I I ) is o r a r e p o t e n t m u t a g e n s . I f c o m -

p o u n d I ( 5 - c h l o r o - 2 - m e t h y l - 4 - i s o t h i a z o l i n - 3 - o n e ) is t he m u t a g e n , this s lope is

e q u i v a l e n t to 533 r eve r t an t s pe r n m o l e , t he c o r r e s p o n d i n g v a l u e fo r c o m p o u n d II

( 2 - m e t h y l - 4 - i s o t h i a z o l i n - 3 - o n e ) be ing 1227 r eve r t an t s pe r n m o l e . A d d i t i o n o f

p h e n o b a r b i t a l - i n d u c e d ra t - l ive r $9 d i m i n i s h e d , b u t d id n o t abo l i sh , t he m u t a g e n i c i t y

o f K a t h o n 886 M W , r e d u c i n g the s lope to 38 and 87 r e v e r t a n s pe r n m o l e f o r c o m -

p o u n d s I a n d II r e spec t ive ly . In the a b s e n c e o f $9, K a t h o n 886 M W p r o v e d to be

tox ic a b o v e a d o s e o f 20 nl pe r p l a t e (2.68 #g per p la te ) , c o n f i r m i n g its e f f ec t i venes s

as a b ioc ide . T h e r e d u c t i o n o f t he m u t a g e n i c e f f e c t o f this b i o c i d e by $9 m i x was

39

6000

Q.

~/.000 t2l

>

I I o I I

°l X

x

o! "6 2OOO ~'

°

Z

o i - , , , , , - I 0 1.3/. 2.68 /..02 5.36 dose, jig (active constituents

per pl(ate

Fig. 2. Plate-incorporation tests of Kathon 886 MW Biocide (Laboratory 1). × ,x ©, Expts. 1, 2 and 3 (no $9). O, tested with rat-liver $9. Lines were fitted by regression analysis of revertants per plate upon dose per plate (see Table 1).

accompanied by a reduction of its toxicity, since a linear dose-response curve for mutagenicity was obtained up to and including a dose of 5.36/zg/plate, double the value obtained in the absence of $9 mix.

The mutagenicity of Kathon 886 MW demonstrated in Laboratory 1 was con- firmed in Laboratory 2. Table 2 shows that in plate-incorporation tests, Kathon 886

MW was mutagenic to S. typhimurium TA100 and to E. coli WP2uvrA(p). In the absence of $9, toxic effects in both species of bacteria were noted at doses of 0.134

#g per plate and above, far lower than the toxic dose observed in Laboratory 1. These differences in the lethal effects of the test-material observed in the two

laboratories are puzzling, and we have at present no well-founded explanation for them. The addition of $9 extended the 'window' of mutagenicity to a dose of 1.34 #g per plate. The sharp cut-off in the dose-response curves obtained in Laboratory

2 precluded calculation of slopes for all the data presented. However, the slopes ob- tained for TA100 (1.0, + $9, 1.4, - $9) were well within an order of magnitude of those obtained in Laboratory 1: this degree of interlaboratory variation falls within

the range seen in a recent multi-laboratory trial conducted in the United Kingdom (Venitt, 1982).

In order to confirm the results of plate-incorporation assays, Kathon 886 MW was tested in fluctuation tests, which provide a means of testing very low, non-lethal concentrations of chemicals for mutagenicity (Green et al., 1976). The results of replicate experiments are shown in Table 3 and Fig. 3. Reproducible linear dose-response curves were obtained for both S. typhimurium TA100 and E. coli WP2uvrA(p), the Salmonella strain being about 1.8 times more sensitive to the

40

TABLE 2

PLATE-INCORPORATION TESTS OF KATHON 886 MW USING E. coli WP2uvrA(p) AND S. typhimurium TA100 (LABORATORY 2)

3 plates were used for each dose.

Dose of Kathon 886 MW

nl per ng active plate constituent

per plate

Mean number of revertants per plate, ± standard deviation

S. typhimurium TAI00 E. coli WP2uvrA(p)

Expt. 1 Expt. 2 Expt. 1 Expt. 2

- $ 9 + $ 9 - $ 9 - $ 9 + $ 9 - $9

0.0 0.0 85±7 143+ 9 80± 1 74+ 7 82+__12 28+5 0.1 13.4 70± 10 43+4 0.2 26.8 80± 3 54±7

0.5 67.0 141 + 5 toxic 1.0 134.0 1122+422 336±54 253+20 84+ 15 138+ 9 toxic 2.0 268.0 toxic 557 + 48 toxic 200 ± 20

5.0 670.0 toxic 928 + 62 toxic 231 + 37 10.0 1340.0 toxic 1536±13 toxic 417+24 20.0 2680.0 toxic toxic toxic toxic

Regression analysis Slope, mutants per ng active constituents - 1.01 1.40 - 0.22 0.95 Correlation coefficient - 0.9921 0.9730 - 0.9744 0.9188 Student's t for slope - 28.57 15.2 - 15.04 6.16 Degrees of freedom - 13 13 - 12 7 P - <0.001 <0.001 - <0.001 <0.001

m u t a g e n i c e f f e c t s o f K a t h o n 886 M W t h a n t h e E s c h e r i c h i a s t r a i n , in c o n c o r d a n c e

w i t h t h e d a t a o b t a i n e d in p l a t e - i n c o r p o r a t i o n t e s t s i n L a b o r a t o r y 2. N e g a t i v e r e s u l t s

w e r e o b t a i n e d in a s ing le e x p e r i m e n t u s i n g T A 9 8 ( d a t a n o t s h o w n ) .

W h e n c o r r e c t e d t o t a k e a c c o u n t o f t h e 2 d i f f e r e n t w a y s o f e x p r e s s i n g t h e a p p l i e d

d o s e ( ' u n i t d o s e p e r m l ' f o r f l u c t u a t i o n t e s t s as o p p o s e d t o ' u n i t d o s e p e r p l a t e ' ) t h e

m e a n s l o p e o f 0 .17 r e v e r t a n t s p e r n g - a c t i v e c o n s t i t u e n t s o b t a i n e d in t h e d u p l i c a t e

f l u c t u a t i o n t e s t s u s i n g T A 1 0 0 is in r e a s o n a b l e a g r e e m e n t w i t h t h e d a t a o b t a i n e d us-

ing p l a t e - i n c o r p o r a t i o n t e s t s , t h e c o r r e c t e d s lope b e i n g 3 .38 c o m p a r e d w i t h t h e m e a n

v a l u e o f 2 .69 o b t a i n e d b y L a b o r a t o r y 1.

C l e a r l y , K a t h o n 886 M W is a p o t e n t , d i r e c t l y - a c t i n g m u t a g e n in 2 spec ies o f

b a c t e r i a . B o t h S. t yph imur ium T A 1 0 0 a n d E. coli W P 2 u v r A ( p ) a re b a s e - p a i r

41

TABLE 3

MICROTITRE FLUCTUATION TESTS OF KATHON 886 MW USING E. coli WP2uvrA(p) AND S. typhimurium TA100

Dose of Kathon 886 MW S. typhimurium TA100 E. coli WP2uvrA(p)

Dose (nl per Equivalent to Expt. 1 Expt. 2 Expt. 1 Expt. 2

microtitration ng active consti- N a M b N M N M N M plate) tuents per ml

medium

0.0 0.0 37 0.49 33 0.42 34 0.44 28 0.34 0.1 0.67 58 0.93 41 0.56 0.2 1.34 62 1.04 43 0.59 53 0.80 49 0.71

0.4 2.68 71 1.34 61 1.01 52 0.78 47 0.67 0.6 4.02 68 1.23 60 0.98 56 0.88 58 0.93 0.8 5.36 74 1.47 62 1.04 59 0.95 67 1.20

1.0 6.70 78 1.67 82 1.93 60 0.98 57 0.91 1.5 10.05 88 2.48 84 2.08 68 1.23 80 1.79 2.0 13.40 toxic 80 1.79 82 1.93

3.0 20.10 toxic 87 2.37 4.0 26.80 toxic 90 2.77 6.0 40.20 toxic toxic

87 2.37

Regression analysis (average number of mutants/well vs. ng active constituents/ml) Expt. No. 1 2 1 2 Dose range, ng/ml 0-10.05 0-10.05 0-26.8 0-20.1 Slope, average number of mutants per ng active constituents 0.165 0.172 0.087 0.103 Correlation coefficient 0.9630 0.9525 0.9908 0.9696 Student's t for slope 8.76 7.67 21.96 11.22 Degrees of freedom 6 6 9 8 P <0.001 < 0.001 <0.001 <0.001

aN, number of positive wells per 96-well plate. bM, average number of mutants per well.

s u b s t i t u t i o n m u t a n t s , a n d it is l i ke ly t h e r e f o r e t h a t t h e m u t a g e n i c c o n s t i t u e n t ( s ) o f

t h e t e s t - m a t e r i a l i n d u c e m u t a t i o n b y b a s e - p a i r s u b s t i t u t i o n r a t h e r t h a n b y c h a n g e s

in t h e r e a d i n g f r a m e , b e a r i n g in m i n d t h e n e g a t i v e r e s u l t s o b t a i n e d in p r e l i m i n a r y

p l a t e - t e s t s o f t h e f r a m e - s h i f t m u t a n t s T A 1 5 3 7 a n d T A 9 8 , a n d a n e g a t i v e f l u c t u a t i o n -

t e s t o f T A 9 8 .

T h e n e g a t i v e r e s u l t s o f b a c t e r i a l m u t a t i o n a s s a y s o f K a t h o n 886 M W a n d o t h e r

f o r m u l a t i o n s c o n t a i n i n g , 5 - c h l o r o - 2 - m e t h y l - 4 - i s o t h i a z o l i n - 3 - o n e a n d 2 - m e t h y l -

4 - i s o t h i a z o l i n - 3 - o n e , s u m m a r i z e d in t h e I n t r o d u c t i o n , a r e r a t h e r p u z z l i n g in t h e l i gh t

o f t h e s t r o n g l y p o s i t i v e r e s u l t s o b t a i n e d in t h e p r e s e n t s t u d y . H o w e v e r , t he f a c t t h a t

42

~52 E

E

O

O

~ ~i ~ ~ ~ ~ / ~ (a) (b) /

o

I I I I I I I I 6.7 10.1 6.7 13./, 20.1 26.8

dose, ng active constituents per m[

Fig. 3. Microtitre fluctuation tests of Kathon 886 MW Biocide. Average number of mutants per well as a function of dose (ng active constituents per ml medium). (a) S. typhimurium TA100, (b) E. coil WP2uvrA(p). ---0--- Expt. l, - - - -O--- - , Expt. 2. Lines were fitted by regression analysis of average number of mutants per well upon dose (see Table 3).

Kathon has been developed expressly to kill a wide spectrum of micro-organisms at

very low doses may explain these negative results, since our own data show clearly that the mutagenic effects of this formulation are detectable only within a very small

range of very low doses. It is very important, therefore, to use closely-spaced doses,

and to explore very low concentrations when screening potent biocides for bacterial mutagenicity.

Since Kathon has been shown to be mutagenic to bacteria and to mammalian cells

in vitro, the suspicion must be entertained that it is potentially carcinogenic. There is widespread exposure of the general population to cosmetics which contain Kathon (at concentrations which are mutagenic to bacteria), and its use as an industrial

biocide implies that certain groups of people may be exposed to it at their place of work. There is clearly a need, therefore, for a most thorough evaluation of this

material in long-term carcinogenicity tests using multiple species and routes of administration.

Acknowledgements

S. Venitt gratefully acknowledges financial support from the Cancer Research Campaign and the Medical Research Council of the United Kingdom.

43

References

Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364.

Gatehouse, D.G. (1978) Detection of mutagenic derivatives of cyclophosphamide and a variety of other mutagens in a microtitre (R) fluctuation test, without microsomal activation, Mutation Res., 53, 289-296.

Green, M . H i . , W.J. Muriel and B.A. Bridges (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutation Res., 38, 33-42.

Venitt, S. (1982) UKEMS Collaborative Genotoxicity Trial: Bacterial mutation tests of 4-chloromethylbiphenyl and benzyl chloride: analysis of data from seventeen laboratories, Mutation Res., 100, 91-109.

Venitt, S., and C. Crofton-Sleigh (1981) Mutagenicity of 42 coded compounds in a bacterial assay using Escherichia coli and Salmonella typhimurium, in: F.J. de Serres and J. Ashby (Eds.), Progress in Mutation Research, Vol. 1, Evaluation of Short-Term Tests for Carcinogens, Report of the Interna- tional Collaborative Program, Chapter 32, Elsevier/North Holland, Amsterdam, pp. 351-360.