Mutation Research, 241 (1990) 125-132

125

Elsevier MUTGEN 01545

Mutagenicity of

3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites in short-term tests in vitro

A t s u k o M a t s u o k a 1, M i c h i k o M a t s u i 1, N a o k i M i y a t a 2, T o s h i o S o f u n i 1 a n d M o t o i I s h i d a t e Jr. 1 1 Division of Geneticsand Mutagenesis and 2 Division of Organic Chemistry, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan) (Received8 May 1989) (Revisionreceived11 December1989) (Accepted20 December1989)

Keywords: Butylhydroxyanisole,metabolites;Reversemutationassay; Chromosomalaberrationtest in vitro

Sulmnary The mutagenicity of 3-tert-butyl-4-hydroxyanlsole (BHA) and its metabolites was investigated in the reverse mutation assay using S. typhimurium strains and the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line, CHL. BHA, tert-butylhydroquinone (BHQ), tert-butylquinone (BQ) and BHA dimer (diBHA) did not show any mutagenlc potential with and without $9 mix in the reverse mutation assay. In addition to the above 4 chemicals, 3-tert-butyl-4,5-dihydroxyanlsole (BHA-OH), 3-tert-butylanisole-4,5-quinone (BHA-o-Q), and tert-butylquinone oxide (BQO) were tested in the chromosomal aberration test. BHA, BHQ and BQ induced chromosomal aberrations only in the presence of $9 mix, while BHA-OH, BHA-o-Q and BQO induced chromosomal aberrations only without $9 mix. DiBHA, however, showed no clastogenic potential with and without $9 mix. The present findings suggest that BHA-OH, BHA-o-Q or BQO may contribute to the clastogenicity of BHA in the presence of $9 mix.

3-tert-Butyl-4-hydroxyanisole (BHA) has been widely used as an antioxidant not only for foods but also for other industrial materials. The report of a Japanese collaborative study on mutagenicity tests on chemicals indicated that BHA was nonmutagenic in both bacteria and mammalian cells, although only a weakly positive response was found in the rec-assay using Bacillus subtilis without metabolic activation (Kawachi et al.,

Correspondence: Dr. A. Matsuoka, Division of Mutagenesis, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku,Tokyo158 (Japan).

1980). Recently, however, it was reported that BHA could be a promoter (Ito et al., 1986) and induce forestomach tumors in rats (Ito et al., 1983). It has been reported that 4-0 conjugates are the main metabolites of BHA in rat, rabbit, dog and man, and that O-demethylation is a minor pathway in rat and dog (Astill et al., 1960, 1962; Dacre et al., 1956). In vitro, BHA has been reported to be metabolized mainly by hydroxylation or O-demethylation by rat liver microsomes (Armstrong and Wattenberg, 1985). In the present study, the mutagenicity of BHA and its metabolites was re-examined in the reverse

0165-1218//90/$03.50 © 1990 ElsevierSciencePublishersB.V. (BiomedicalDivision)

126 TABLE 1 REVERSE M U T A T I O N ASSAYS a ON BHA A N D ITS METABOLITES U S I N G Salmonella typhimurium STRAINS Compound

BHA

Solvent

Acetone

AF-2 c MMC ICR-191 2AA BHQ

Acetone

AF-2 MMC ICR-191 2AA

BQ

AF-2 MMC ICR-191 2AA

Acetone

Dose

TA100

(/~g/plate)

- $9

0 0.5 1 2.5 5 10 25 50 100 250 0.02 0.05 0.01 0.1 2 0 0.5 1 2.5 5 10 25 50 100 250 500 1000 0.02 0.05 0.01 0.1 2 0 0.05 0.1 0.25 0.5 1 2.5 5 10 25 50 100 250 500 0.02 0.05 0.01 0.1 2

161 147 145 149 135 151 158 130 LE b

TA98 + $9 183 190 192 202 184 192 160 167 LE

- $9 21 21 14 19 22 20 20 17 LE

TA102

TA97

+ $9

- $9

+ $9

25

235 248 199 186 215 202 189 170 LE

308

29 29 29 25 22 28 23 LE

323 333 304 304 260 252 237 LE

-$9 291 294 269 295 310 314 266 265 LE

+$9 381 337 451 400 422 419 419 256 LE

2161 235 1691 2 036 8423 161 124 186 149 127 129 LE LE LE

183

194 193 177 149 156 185 LE LE

7741 21 23 18 15 17 10 LE LE LE

25

21 24 26 29 27 18 LE LE

1174 235 210 230 195 163 177 LE LE LE

308

315 293 324 309 259 204 LE LE

1014 187 155 158 119 111 118 LE LE LE

282

254 229 278 217 222 209 LE LE

2161 235 1691 2622 8423 128 109 122 150 148 173 141 21 LE

152

179 188 160 155 183 171 132 LE

7741 19 19 20 18 26 27 22 LE LE

32

21 30 33 22 35 24 23 LE

1174 304 296 274 312 296 257 217 150 LE

383

434 402 395 363 393 359 247 LE

1964 163 115 124 115 149 182 152 70 LE

296

303 308 305 259 247 247 89 LE

1998 198 2187 1743 10299

7671

1112

1 489

127 TABLE 1 (continued) Compound

Solvent

diBHA

Acetone

AF-2 MMC ICR-191 2AA

Dose (/tg/plate)

TA100

0 25 50 100 250 500 1000 2500 5000 0.02 0.05 0.01 0.1 2

134 125 139 166 152 164 173 180 171 1701

- $9

TA98 + $9

169 162 169 190 177 181 183 170 147

- $9

13 11 16 16 11 13 10 11 13

TA102 + $9

29 26 32 48 55 54 21 39 50

- $9

338 349 326 287 323 310 307 268 291

+

$9 372 477 469 502 515 468 498 470 461

TA97 -$9 187 154 182 202 137 113 154 156 221

+$9 282 223 234 219 236 248 284 249 275

201 1633 2 622 9 929

7 706

1210

1964

a Preincubatedfor 20 rain at 37°C. The averagenumber of revertant colonies in 2 plates is shown. b Lethal effect. c Positivecontrol. AF-2, 2-(2-furyD-3-(5-nitro-2-furyl)-acrylamide;MMC, mitomycin C; ICR-191, 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine dihydrochloride;2AA, 2-aminoanthracene.

mutation assay using S. typhimurium strains and the chromosomal aberration test in vitro using a Chinese hamster lung fibroblast cell line, CHL, in the presence and absence of metabolic activation. Materials

and

methods

Chemicals The chemical structures of BHA (CAS No. 121-00-6) (purity 99.4%) and its 6 metabolites, tert-butylhydroquinone (CAS No. 1948-33-0) (BHQ), tert-butylquinone (BQ), BHA dimer (CAS No. 14078-41-2) (diBHA), 3-tert-butyll4,5-dihydroxyanisole (CAS No. 80284-15-7) (BHA-OI-I), 3-tert-butylanisole-4,5-quinone (CAS No. 2940-638) (BHA-o-Q) and tert-butylquinone oxide (BQO), are shown in Fig. 1. BHA and BHQ were purchased from Wako Pure Chemical Industries, Ltd., Osaka and Tokyo Kasei Kogyo Co., Ltd., Tokyo, respectively. BQ, diBHA, BHA-OH and BHA-o-Q were prepared according to the reported procedure (DeStafney et al., 1986; Guarna et al., 1983). BQO was synthesized by the oxidation of BQ using m-ehloroperbenzoic-acid. All chemicals except BHA and BHQ were purified by recrystallization and their purifies measured by highperformance liquid chromatography (HPLC) were

more than 99.0%. For the bacterial mutation assay, only 4 chemicals, BHA, BHQ, BQ and diBHA, w e r e tested because of scarcity of synthesized chemicals and acetone was used as solvent. For the mammalian cytogenetic assay, dimethyl sulfoxide (DMSO) was used as solvent for BHA, BHQ, BQ and diBHA, and physiological saline for BHA-OH, BHA-o-Q and BQO.

Reverse mutation assay Reverse mutation assays using S. typhimurium strains TA100, TA98, TA97 and TA102 were carried out according to the original method (Ames et al., 1975; Maron and Ames, 1983) with a slight modification. The post-mitochondrial supernatant ($9) was prepared from the livers of Fischer rats (Charles River Japan Co.) pretreated with polychlorinated biphenyls (KC-400) 5 days before. 0.5 ml of $9 mix consisted of 50 #1 of $9, 2.5 #mole of glucose 6-phosphate (G-6-P), 2 #mole of N A D P H , 2 #mole of N A D H , 4 #mole of MgC12, 16.5 #mole of KC1 and 50 #mole of phosphate buffer (pH 7.4). Cells cultured overnight were preincubated with both the test compound and $9 mix for 20 min at 3 7 ° C before plating. Duplicate plates were used for each of 8 different concentrations of the compound. The number of revertant

128

OCH 3

, ................................................

OCHa

'~i

OH

OH

O

BHA-OH BHA-o-Q ............................................ . ............. :

BHA ~OH

BHQ

0

:

O

O

O

BQ

BOO

Fig. 1. Chemical structures of B H A and its metabolites. The compounds enclosed in the dotted line were clastogenic to C H L ceils in the absence of $9 mix.

(His + ) colonies was scored after incubation at 37 ° C for 2 days. The result was considered positive if the number of colonies found exceeded twice the number of the solvent control and also if a reasonable dose response was observed.

mix consisted of 3 ml of the $9 fraction, 2 ml of 20 m M Hepes buffer (pH 7.2), 1 ml each of 50 m M MgC12, 330 m M KC1, 50 m M G-6-P, 40 mM N A D P and distilled water (Matsuoka et al., 1979). The final concentration of the $9 fraction in the culture medium was adjusted to 5%. After treatment for 6 h, the reaction mixture was replaced with fresh medium, and then the cells were recultured for 18 h. Both with and without the metabolic activation system, the cells were treated with Colcemid (0.2 /~g/ml) for 2 h, and chromosome preparations were made using a standard air-drying method. The frequency of cells with chromosomal aberrations was scored in 100 well-spread metaphases for each dose. Structural chromosomal aberrations were classified into 6 groups: chromatid gaps (ctg) including chromosome gaps, chromatid breaks (ctb), chromatid exchanges (cte), fragmentation (frg), chromosome breaks (csb), and chromosome exchanges (cse) including dicentric and ring chromosomes. Polyploid ceils were also recorded. Solvent-treated cells served as controls. The experimental groups were judged as negative ( - ) if the total frequency was less than 5.0%, suspicious ( + ) if 5 . 0 - < 10.0% and positive ( + ) if 10% or more. Results and discussion

Chromosomal aberration test in vitro

A Chinese hamster lung fibroblast cell line (CHL) was used (Ishidate and Odashima, 1977). The ceils were cultured with Eagle's minimal essential medium (MEM; Gibco) supplemented with 10% heat-inactivated calf serum. The doubling time of the cells was estimated to be about 15 h, and the modal chromosome number was 25. The cells were treated for 24 and 48 h with a test compound at 3 dose levels at least. The maximum dose of each test compound was estimated from the dose at which distinct cytotoxic effects were observed in a preliminary test on inhibition of cell growth. When the test compound did not show any cytotoxic effects, the maximum dose was limited to around 10 m M (Ishidate et al., 1984). For metabolic activation, the cells were treated with $9 mix together with the test compound. The $9 fraction was prepared from rat liver homogenate as described above. 10 ml of $9

Reverse mutation assay

The results of the bacterial mutation assays are summarized in Table 1. BHA, BHQ and BQ did not show any significant increase in the frequency of revertant colonies with and without $9 mix even at the dose with cytotoxic effects. DiBHA did not show any cytotoxicity or mutagenicity even at the highest dose, 5 mg/plate. These results indicate that BHA, BHQ, BQ and diBHA may not have mutagenic potential in the bacterial system with and without $9 mix. This result on BHA and BHQ is consistent with the study by Hageman et al. (1988). Chromosomal aberration test in vitro

The results of the chromosomal aberration tests are shown in Table 2. In the absence of $9 mix, BHA did not induce chromosomal aberrations at doses up to 0.06 m g / m l at which distinct cyto-

129 TABLE 2 CHROMOSOMAL CULTURE Compound

BHA

BHQ

BQ

ABERRATION

TEST ON BHA AND

ITS METABOLITES

USING

CHINESE

Dose

$9

Time a

Pol b

Cells w i t h c h r o m o s o m a l a b e r r a t i o n s (%) c

(mg/ml)

mix

(h)

(%)

ctg

ctb

0 (DMSO) ~ 0.02 0.04 0.06 0.08 0 (DMSO) 0.02 0.04 0.06 0.08 0 (DMSO) 0.05 0.075 0.1 0.125 0 (DMSO) 0.05 0.075 0.1 0.125 0.15

+ + + + + +

24- 0 24- 0 24- 0 24- 0 24- 0 48- 0 48- 0 48- 0 48- 0 48- 0 6-18 6-18 6-18 6-18 6-18 6-18 6-18 6-18 6-18 6-18 6-18

0 0 0 0 Tox f 1 0 0 0 Tox 0 0 3 1 Tox 0 1 3 2 0 Tox

0 0 3 3

0 0 1 0

0 1 1 1

0 (DMSO) 0.0125 0.025 0.05 0 (DMSO) 0.0125 0.025 0.05 0 (DMSO) 0.02 0.03 0.04 0 (DMSO) 0.02 0.03 0.04

+ + + +

24- 0 24- 0 24- 0 24- 0 48- 0 48- 0 48- 0 48- 0 6-18 6-18 6-18 6-18 6-18 6-18 6-18 6-18

0 1 3 Tox 1 0 1 Tox 0 1 0 Tox .,0 0 1 2

0 (DMSO) 0.0015 0.002 0.0025 0.003 0 (DMSO) 0.002 0.0025 0.003 0 (DMSO) 0.001 0.002 0.003 0 (DMSO) 0.005 0.01 0.02

+ + + +

24- 0 24- 0 24- 0 24- 0 24- 0 48- 0 48- 0 48- 0 48- 0 6-18 6-18 6-18 6-18 6-18 6-18 6-18 6-18

1 5 2 5 7 1 2 3 2 0 1 2 Tox 0 0 2 1

cte

HAMSTER

CELLS IN

Judgement d

frg

csb

cse

total

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 1 1

1 1 0 1

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 2

0 0 0 0

0 0 0 1

0 0 0 0

0 0 0 0

0 0 0 0

0 0 2 0 2

0 0 2 2 5

0 0 0 2 8

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

0 0 4 3 Tox 1 2 2 3 Tox 0 0 0 2 Tox 0 0 4 4 11 Tox

2 2 3

2 0 0

0 0 0

0 0 0

1 0 0

0 0 0

1 2 1

0 0 0

0 0 1

0 0 0

0 0 0

0 0 0

0 2 0

0 0 1

0 0 0

0 0 0

0 0 0

0 0 0

2 1 3 6

0 3 4 7

0 2 9 12

0 0 0 0

0 0 0 0

0 0 0 1

0 3 0 0 3 0 1 1 0 0 0 2

0 2 0 2 3 0 0 1 0 0 1 3

0 1 0 2 1 0 0 0 0 0 0 1

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 1 0 0 0 0

0 0 0 0 1 0 0 0 0 0 0 0

2 1 0 1

0 0 1 9

0 0 0 2

0 0 0 0

0 0 0 0

0 0 0 0

4 2 3 Tox 1 2 2 Tox 0 2 I Tox 2 5 12 19 0 6 0 3 7 0 1 3 0 0 1 5 Tox 2 1 1 11

-

-

-

+

-

-

-

+ + +

+ + +

+

I

I

I

o

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I

I

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I'~ 0

I

p.-b ~.-~ 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

~,-~ 0

0 0 0 ~ 0 0 0 0 0 ~ 0 0 0 0 0 0

o

o 0 0 ~ 0 0 0 0 0 0 0 0 0 0 ~

t'J ~,-~ 0

0 0 0 ~ 0 0 0 0 ~ 0 0 0 0 0 0 ~

o

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0

0 0 0 0 0 0 0 o 0 0 0 0 0 0 0 0

~,-~ ',,o ~-.L o

+~

I

~,1 tJ

t~tJl-~tJ

I

0 0

I

0 0 0

.lu,.lu,,.~.iu,

I

o o o o o o o o o o G ~ o o o o o o 0 0 0 0 0 0

I

~ o ~ ¢ ~ / o ~ ) o ~ o ~ o ¢ ~ o 0 0 0 o 0 0 0 0

I

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++

~

I

,~ 0

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-~1 ~,-L 0

tJl~.)t-~t~)

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+ + + +

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~ o 0 ~ 0 0 0 0 0 0 0 ~ 0 0 0 0

0 0 0 0 0 0 0 o 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

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o o o o o o o o o o o o o o o o 0 0 0 0 o o o 0

+ + + +

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131 TABLE 2 (continued) Compound

BQO

Dose (mg/ml)

$9 mix

Time a (h)

Pol b (%)

0 (Sad) 0.0005 0.001 0.002 0 (Sad) 0.0005 0.001 0.002 0 (Sad) 0.002 0.004 0.006 0 (Sad) 0.002 0.004 0.006

+ + + +

24- 0 24- 0 24- 0 24- 0 48- 0 48- 0 48- 0 48- 0 6-18 6-18 6-18 6-18 6-18 6-18 6-18 6-18

0 1 0 1 0 0 0 0 0 1 1 3 1 0 0 0

Judgement d

Cells with chromosomal aberrations (%) ¢ ctg

ctb

cte

frg

csb

cse

total

1 1 0 2 0 0 1 0 1 4 1 8 0 1 1 2

0 0 0 5 0 0 0 0 0 0 0 8 0 0 1 0

0 0 0 6 0 0 1 0 0 0 1 12 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0

2 1 0 11 0 0 2 0 1 5 2 22 0 1 2 2

+ + + -

a Treatment time - recovery time. b Polyploid. ¢ ctg, chromatid gaps (including chromosome gaps); ctb, chromatid breaks; cte, chromatid exchanges; frg, fragmentation; csb, chromosome breaks; cse, chrolnosome exchanges. d _ if the total frequency was < 5.0~; + if 5.0- < 1 0 ~ ; + if 10.0~ or more. • Solvent: Sal, physiological saline; DMSO, dimethyl sulfoxide. f Almost no metaphases.

toxicity was noted. In the presence of $9 mix, however, BHA induced chromosomal aberrations at the highest dose, 0.125 mg/ml, but the frequency was relatively low (11%). Two metabolites, BHQ and BQ, showed similar results: 19% at 0.04 mg/ml and 11% at 0.02 mg/ml, respectively, in the presence of $9 mix. The order of clastogenic potential in the presence of $9 mix was BQ > BHQ > BHA. These findings suggest that some metabolites other than BQ and BHQ are responsible for the induction of chromosomal aberrations by BHA. On the other hand, BQO, an expected metabolite, converted from BHA via BHQ and BQ (Fig. 1), induced 11% chromosomal aberrations in the absence of $9 mix at a relatively low dose, 0.002 mg/ml, in the 24-h treatment. BQO induced aberrations (22% at 0.006 mg/ml) in the S9 control (treatment for 6 h without S9 mix), while it was negative with $9 mix (Table 2). It is conceivable, therefore, that BQO may be one of the ultimate clastogens converted from BHA and be inactivated by the addition of $9 mix.

It is generally accepted that BQ generates active oxygen species which may contribute to the induction of chromosomal aberrations, and that DMSO used as solvent may be one of the scavengers of active oxygen species. To exclude such effects, BQ and BHQ suspended in physiological saline were also tested, but no significant alteration in the induction of chromosomal aberrations was observed (data not shown). Two metabolites, BHA-OH and BHA-o-Q, which are derived from oxidation of BHA (Fig. 1), also induced chromosomal aberrations without $9 mix: 25% and 24%, respectively, at 0.02 mg/ml in the 24-h treatment. With $9 mix, however, no significant increases were observed. These findings suggest that these 2 metabolites may also be ultimate clastogens. Another BHA derivative, diBHA, showed neither cytotoxicity nor significant increases in the frequency of chromosomal aberrations in both systems with and without $9 mix, at doses up to 0.2 mg/rnl, indicating that diBHA may not be involved in the clastogenicity of BHA.

132

The present study indicates that 3 metabolites of BHA, BHA-OH, BHA-o-Q and BQO, are clastogenic to the Chinese hamster cells in the absence of $9 mix. By chemical analysis, BHA-OH has been identified in the BHA mixture incubated with the rat rnicrosome fraction (Armstrong and Wattenberg, 1985). As BHA-OH can be readily oxidized to BHA-o-Q, it is possible that BHA-OH and BHA-o-Q are the ultimate clastogens of BHA, which are formed in the presence of $9 mix. The present findings also indicate that some clastogens may be converted from BQ in the presence of $9 mix, and that BQO is possibly one of such clastogens. Further studies are necessary to clarify which metabohte(s) among the 3, BHA-OH, BHA-o-Q and BQO, effectively contribute to the clastogenicity of BHA in the presence of $9 mix. BHA has been reported to be carcinogenic in rats and hamsters (Ito et al., 1986). It has been reported that in man BHA is excreted as a glucuronide mostly within 24 h (Astill et al., 1962). The present study has shown that BHA has clastogenic potential in cultured mammalian cells. The hazard of BHA to man should be reevaluated. References Ames, B.N., J. MeCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364. Armstrong, K.E., and L.W. Wattenberg (1985) Metabolism of 3-tert-butyl-4-hydroxyanisole to 3-tert-butyl-4,5-dihydroxyanisole by rat liver microsomes, Cancer Res., 45, 1507-1510. AstiU, B.D., D.W. Fassett and R.L. Roudabush (1960) The metabolism of phenolic antioxidants 2. The metabolism of butylated hydroxyanisole in the rat, Biochem. J., 75, 543551. Astill, B.D., J. Mills, D.W. Fassett, R.L. Roudabush and C.J.

Terhaar (1962) Fate of butylated hydroxyanisole in man and dog, J. Agric. Food Chem., 10, 315-319. Dacre, J.C., F.A. Denz and T.H. Kennedy (1956) The metabolism of butylated hydroxyanisole in the rabbit, Biochem. J., 64, 777-782. DeStafney, C.M., U.D.G. Prabhu, V.L. Sparnins and L.W. Wattenberg (1986) Studies related to the mechanism of 3-BHA-induced neoplasia of the rat forestomach, Food Chem. Toxicol., 24, 1149-1157. Guarna, A., L.D. Corte, M.G. Giovannini, F.D. Sarlo and G. Sgaragli (1983) 2,2'-Dihydroxy-3,3'-di-t-butyl-5,5'-dimethoxydiphenyl, a new metabolite of 2-t-butyl-4-methoxyphenol in the rat, Drug Metab. Dispos., 11, 581-584. Hageman, G.J., H. Verhagen and J.C.S. Kleinjans (1988) Butylated hydroxyanisole, butylated hydroxytoluene and tert-butylhydroquinone are not mutagenic in the Salmonella/microsome assay using new tester strains, Mutation Res., 208, 207-211. Ishidate, M., Jr., and S. Odashima (1977) Chromosome tests with 134 compounds on Chinese hamster cells in vitro - a screening for chemical carcinogens, Mutation Res., 48, 337-354. Ishidate, M., Jr., T. Sofuni, K. Yoshikawa, M. Hayashi, T. Nohmi, M. Sawada and A. Matsuoka (1984) Primary mutagenicity screening of food additives currently used in Japan, Food Chem. Toxicol., 22, 623-636. Ito, N., S. Fukushima, A. Hagiwara, M. Shibata and T. Ogiso (1983) Carcinogenicity of butylated hydroxyanisole in F344 rats, J. Natl. Cancer Inst., 70, 343-349. Ito, N., M. Hirose, S. Fukushima, H. Tsuda, T. Shirai and M. Tatematsu (1986) Studies on antioxidants: their carcinogenic and modifying effects on chemical carcinogenesis, Food Chem. Toxicol., 24, 1071-1082. Kawachi, T., T. Komatsu, T. Kada, M. Ishidate, Jr., M. Sasaki, T. Sugiyama and Y. Tazima (1980) Results of recent studies on the relevance of various short-term screening tests in Japan, in: G.M. Williams et al. (Eds.), The Predictive Value of Short-term Screening Tests in Carcinogenicity Evaluation, Elsevier, Amsterdam, pp. 253-267. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Matsuoka, A., M. Hayashi and M. Ishidate, Jr. (1979) Chromosomal aberration tests on 29 chemicals combined with $9 mix in vitro, Mutation Res., 66, 277-290.

Mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites in short-term tests in vitro.

The mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites was investigated in the reverse mutation assay using S. typhimurium strain...
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