Genotoxicity of safrole-related chemicals in microbial test systems

Mutation Research, l01 (1982) 127-140 Elsevier Biomedical Press

127

Genotoxicity of safrole-related chemicals in microbial test systems J u n S e k i z a w a ~ a n d T a k a y u k i S h i b a m o t o 2,, I Ogawa attd Co. Ltd., 1-12-3 Niitaka, Yodogawa-Ku, Osaka, Japan, and 2 Department o/Ent#ronmental Toxicology, Unicersi O' of California, Dacis, Dat~is, CA 95616 (U.S.A.) (Received 18 February. 1981) (Revision received 21 September 1981 ) (Accepted 26 October 1981)

Summary

The genotoxicity of safrole, 9 compounds that are structurally similar to safrole (anethole, cinnamaldehyde, cinnamyl alcohol, estragole, methyl eugenol, eugenol, isoeugenol, isosafrole, piperonal), 5 essential oils (anise oil, cassia oil, cinnamon bark oil, clove oil, fennel oil) which contain the chemicals tested, and 1 oleoresin was studied in 3 microbial test systems. Only anethole showed mutagenicity in the Ames Salmonella reversion assay. All chemicals except anethole, estragole and isosafrole were positive in the Bacillus subtilis DNA-repair test (Rec assay) without $9. All samples tested were negative in the Escherichia coli WP2 uvrA reversion test. The essential oils and pimenta oleoresin were positive in the DNA-repair test. The results obtained are discussed in relation to the nature of the problems encountered with each test method.

Safrole is a major component of the oil of sassafras and has been used as a flavoring agent in soft drinks and in some pharmaceutical preparations. In the early 1960s, safrole was found to induce liver tumors in rats (Long et al., 1961, 1963), and was prohibited for use in foods. Animal studies showed that safrole is metabolized into hydroxy or epoxide derivatives, which are possible carcinogenic metabolites (Borchert et al., 1973; Wislocki et al., 1976, 1977). Miller et al. (1979) reported the metabolic activation pathway from safrole to its ultimate carcinogens. Recently, safrole and structurally related chemicals (i.e. anethole, estragol, eugenol, piperonal and isoeugenol) have been studied for mutagenicity in the Ames Salmonella

* Present adress: Ogawa and Co. Ltd., 6-32-9 Akabanenishi, Kita-Ku, Tokyo, Japan. 0165-1218/82/0000-0000/$02.75 t ' Elsevier Biomedical Press

12~

reversion assay (Swanson et al., 1979; White et al., 1979; Hsia et al., 1979). Dorange et al. (1977) reported that safrole was mutagenic in strain TA 1535 in the presence of liver enzymes. On the other hand, Wislocki et al. (1977) reported that safrole was not mutagenic in strain TA100 or in TA1535. They observed, however, that the safrole metabolite (safrole-2',3'-epoxide) was mutagenic in both strains TA 100 and TA 1535 even in the absence of $9.

Materials and methods Chemicals

All test compounds were obtained from Tokyo Kasei Kogyo Co., Tokyo, except methyl eugenol, which was purchased from Inoue Koryo Seizosho, Osaka. Their chemical structures and degrees of purity (estimated by gas-chromatographic analysis) are shown in Fig. 1 and Table 1, respectively. Reagents were obtained from the following sources: polychlorinated biphenyl (PCB), dimethyl sulfoxide (DMSO) and 2-aminoanthracene (2-AA) from Wako Pure Chemical Industries Ltd., Osaka; sodium azide (NAN3), 2-nitrofluorene (2-NF) and 9-aminoacridine (9-AAc) from Nakarai Chemicals Ltd., Kyoto; chloramphenicol and D-glucose 6-phosphate (G-6-P)

CH=CHCH3

ocl-~

,,+

CH-----CHCHO

,,,+

CH=CHCH20H

CH2CH= CH2

CH2CH~ CH2

CH2CH= CH2

OCH3

OCH3

OH

CH=CHCH.,j

CH=CHCH3

CH0

OCH3

OCH3

VII[~OCH~ VIII[~1 /° IXo~c/O OH

X~ , ~

O--CH 2

--

HE

=cH2

Fig. I. Structures of safrole and related compounds. I, anctholc: IL cinnamaldehyde; IIl, cinnamvl alcohol: IV, estragole: V, methyl cugenol: VI, eugenol: VII, isoeugenol: VIII, isosafrole: IX, piperonal: X, safrole.

129 TABLE 1 THE PURITIES OF SAFROLE AND RELATED COMPOUNDS Compound

CAS No. a

Purity (%) b

c;strans composition cis

I II III IV V VI VII VIII IX X

Anethole Cinnamaldehyde Cinnamyl alcohol Estragole Methyl eugenol Eugenol Isoeugenol Isosafrole Piperonal Safrole

104-46-1 104-55-2 104-54- I 140-67-0 93-15-2 97-53-0 97-54-1 120-58-1 120-57-0 94-59-7

98.9 99.6 98.2 99.9 99.2 98.9 97.6 97.9 100.0 99.0

0.0 1.3 2. I 33.3 19.7 -

trans

100.0 98.3 96. I -

64.3 78.2

Chemical abstract registration number. b Obtained by gas chromatography.

f r o m S i g m a C h e m i c a l Co., St. Louis, M O ; r e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o tide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) f r o m O r i e n t a l Y e a s t Co. Ltd., T o k y o ; b e n z o [ a ] p y r e n e (BP) f r o m A l d r i c h C h e m i c a l Co., M i l w a u k e e , W I ; s o d i u m a m p i c i l l i n f r o m T a k e d a C h e m i c a l I n d u s t r i e s Co., O s a k a ; m i t o m y c i n C f r o m K y o w a H a k k o K o g y o Co., T o k y o . 2 - ( 2 - F u r y l ) - 3 - ( 5 - n i t r o 2 - f u r y l ) a c r y l a m i d e ( A F - 2 ) was a gift f r o m Dr. T. M a t s u s h i m a , I n s t i t u t e of M e d i c a l S c i e n c e , U n i v e r s i t y of T o k y o , T o k y o . E s s e n t i a l oils

C l o v e oil, c i n n a m o n b a r k oil, f e n n e l oil a n d p i m e n t a o l e o r e s i n w e r e o b t a i n e d f r o m r e l i a b l e c o m m e r c i a l sources. A n i s e oil a n d cassia oil w e r e o b t a i n e d f r o m the

TABLE 2

CHEMICAL COMPOSITION OF THE TESTED ESSENTIAL OILS AND OLEORESIN Sample

Composition (%) "

Anise oil Cassia oil

Anethole (87.3) Cinnamaldehyde (92.3), cinnamyl acetate (1.8), benzaldehyde (0.3) Cinnamaldehyde (78.3), cinnamyl acetate (4.5), eugenol (3.1), linalool (2.9), benzaldehyde t2. I) Eugenol (75.9), eugenyl acetate (16.5), caryophyllene (4.7) Anethole (86.4) Eugenol (76.1), caryophyllene (4.3), methyl eugenol (3.9)

Cinnamon bark oil Clove oil (bleached) Fennel oil (sweet) Pimcnta (allspice) oleoresin " Obtained by GC analysis.

130 People's Republic of China and purified by vacuum distillation before use. The oils were dissolved in ethanol for the tests. The chemical compositions of these materials are given in Table 2.

Bacterial strains Salmonella typhimurium his tester strains TAI00, TA1535, TA98, TA1537, TA1538 and Escherichia colt WP2 trp were obtained from Dr. Matsushima, Institute of Medical Science, University of Tokyo, Tokyo. Bacillus subtilis H I 7 ~ Rec + and M45 Rec (Rec assay) were donated by Dr. T. Kada, National Institute of Genetics, Mishima, Shizuoka. Mutagenicity assay The mutagenicity assay with S. thyphimurium was conducted as described by Ames et al. (1975) with slight modifications. All tester strains were examined periodically for the markers indicated by Ames et al. (1975). Especially with strains T A I 0 0 and TA98, nearly 100% of the cells showed ampicillin resistance. $9 was prepared from PCB-treated male Sprague-Dawley rats according to Ames et al. (1975) and was stored at - 8 0 ° C . 100/~1 of overnight culture of bacteria and 500/~1 of sodium phosphate buffer (0.1 M, pH 7.4; for assays without $9) or 500 t21 of $9 mix (for assays with $9) were added to 50/xl of sample solution. $9 mix (500/21) consisted of 100/~1 of $9, 50/~moles sodium phosphate buffer (pH 7.4), 4 /~moles MgC12, 16.5 /~moles KC1, 2.5 /2moles G-6-P, 2 /lmoles N A D H and 2 /~moles NADPH. The assays without $9 were performed by the plate-incorporation method. The assays with $9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. The mutagenicity assay with E. co//WP2 uerA trp (Green and Muriel, 1976) was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 /~mole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 /xmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. colt. All test samples were dissolved in DMSO for mutagenicity assays. Revertants per plate (see Results) represent average values from 3 to 5 replications. DNA-repair test," comparison of the repair-conlpetent (Rec ~) and repair-deficient (Rec ) strains for zones of killing The DNA-repair test with Bacillus subtilis was performed as described by Kada et al. (1980). Spores of H I 7 Rec ~ or M45 Rec were poured onto plates with 10 ml of molten nutrient agar to prepare spore-agar plates. Test samples were dissolved in ethanol immediately before use. Sample solutions (20/21) of various concentrations were pipetted onto sterile filter-paper disks (8 mm in diameter), which were carefully placed on the spore-agar plate. After incubation of the plate for 20--24 h at 37°C~ the zones of killing (diameter of growth inhibition zone - diameter of disk) with both

[31

strains (Rec + and Rec ) were measured and the difference between them was taken as the rec effect. The results with the dose at which each sample showed most difference are shown in Tables 5 and 6.

Results Mutagenicity tests No test compounds induced a significant increase in revertant numbers either with Salmonella tester strains or with E. coli WP2 uvrA in the absence of $9 (Table 3). Anethole was the only compound that showed a significant increase in revertants in the presence of $9 (Table 4). Anethole showed a linear dose response against strain TA100 up to the dose of 120/~g (Fig. 2). The number of revertants did not, however, increase to twice the number of the control; this was because of anethole's killing action to bacteria. Numbers of viable cells after pre-incubation

I0

200"

\ 150 -

o

j/oY

8

o

L

I00 b-

4

'O x v

Q. m o

o

"2

50

Q)

2

o

3'o 6'0 9'o

o

o

O

pg/plate

Fig. 2. Mutation and survival of S. o'phimurium TA100 cells treated with anethole in the presence of $9. Cells (I x 10 s cells), $9 mix and each dose level of anethole were incubated together at 37°C for 20 min as described in Methods. Immediately after incubation, portions (20 p_l) of the mixture were diluted with a phosphate buffer (0.1 M, pH 7.4) to estimate the numbers of viable cells on nutrient agar plates (closed circles). The rest (630 ~1) of the mixtures were plated on minimal agar plates to test mutation of the cells (open circles).

250 750 1500 3000

Cinnamylalcohol

30 60 120 300

60 120 300 600

Cinnarnaldehyde

Estragole

60 120 300 600

Anethole

b

50~1

DMSO

Positive c o n t r o l

Dose a ( ~. g / p l a t e )

6

104± 5 95± 9 91~- 10 109± 8

98~ 5 81± 6 81+13 24±13

78±18 105±16 81+13 83± 9

101+10 105-+ 3 113 + I 98±13

769--+ 54

99±

2

i 4 3 2

2 4 3 4

7~ 6± [I± 6~r

4 3 4 4

10-+ 4 5= 1 7= 1 2= 1

t0± 8± 6 +7~

6~ 10± 11-+ 9m

266 ~ 34

8~

TA1535 4

7 I 4 2

25+ 23 + 27 ~ 25=

6 4 7 4

28± I 24± 1 22~ 8 20-+ 8

31± 34± 40± 30~

30± 4 3 1 -+- 2 31± 4 28 + 8

660 ± 30

24±

2

54= 4± 5=

3 ~ 3 + 5: ~'

5= 4± 3= 3±

5 -+3± 7~ 2=

2 3 3 1

1 1 O 2

I I 2 2

I 1 2 1

822 ~ 88

4±

3

19 -~ 14-17= 14±

9± I0± 14:~ 6-:

2 1 5 4

I 2 0 I

14+ - I 12± 1 14± 4 8-+ 3

11± 0 13~ 1 9± I 7-~ 2

301 + 33

15+ -

9 6 3 6 68= 9 45-' 3 54-~11 63 . ; 1 3

50~ 57:~: 46-33~.

51-' 6 50-*- 1 51 t 8 48± 8

52± 8 45:'- 2 54= I 45= 6

452 -~ 68

55"- 6

W P 2 uvrA T A 1538

S9

T A 100

T A 1537

IN S. o;ohimurium A N D I N E. coli W I T H O U T

Escherichia coil TA98

COMPOUNDS

Salmonella (vphimurium s t r a i n s

R e v e r t a n t s / p l a t e ( m e a n -+ S.D.)

TESTS ON SAFROLE AND RELATED

Sample

RESULTS OF MUTAGENICITY

TABLE 3

133

e,i ,<

3~2 •~ z,

¢~

q'5

e'.

"~

,;.2.

[..,

.-"Z

el

X~

4

Estragole

Cinnamylalcohol

Cinnamaldehyde

Anethole

T A 1538

I N E. coli W I T H

$9

9

124-+ 107+14 119= 9 1 -+

30

60

120

300

6

9

I

72-+11

3000

3

1

5

3

9

4

123-+12

87-+ 1 3 0 -+-

92=

600

750

104=

300

250

110-+ 127 +

94=16

600

60

1 2 9 -+-

300

120

164+11

120 3

134-+15

60

431 + 92

106-+

3

7~

9=

8+

12 +

12=

9--

10 +

7--

11 +

9+

9+

8+

10-+

I1=

10 +

8+

2

2

0

4

2

2

1

2

1

5

4

3

I

2

3

3

158 -" 45

10-+

T A 1535

20+8

34=3

38-+2

28-+4

21+2

24+3

27+3

31+3

28-+6

29-+3

34-+5

31-+5

39+5

36+3

28+6

36+3

180-+ 7

32-+4

7~

8=

7~

8~

4~

8+

7+

9~

9+

7+

10 +

9~

6~

9~

1 2 -~-

11=

4

0

4

0

2

3

2

2

2

0

2

I

I

2

3

I

125 ~ 37

7-"- 2

4

13 +

21 +

18 +

20 +

I1=

21~

18 +

24~

30 +

19 +

18 +

17 +

2 2 -+-

23 +

1 4 -+

22 +

3

7

1

2

0

3

4

8

3

4

3

4

I

2

5

4

154 + t 7

21±

5

6

8

8

4

3

6

8

4

I

7

I

2

9

4

57 +

9

5 3 : + 12

60 ~

54-+

39 ~

42-

62 +

58-

42 +

45 +

53-+

54-+

48±

41 +

55 +

62 +

753 = 95

59 +

W P 2 uvrA

T A 1537

I N S. (vphimurium A N D

T A 100

TA98

COMPOUNDS

Escherichia coli

1500

b

50p,1

RELATED

(mean + S.D.)

AND

Salmonella typhimurium s t r a i n s

Revertants/plate

TESTS ON SAFROLE

(/~g/plate)

Dose ~

OF MUTAGENICITY

Positive control

DMSO

Sample

RESULTS

TABLE

118± 6 123±12 126-- 6 124-+ 4

30 90 150

300

98±13 111± 3 123-+19 120-'-16

133 + 6 106+14

120 300

300 600 1200 2400

98-+10 114-+ 5

95± 8 98±22

30 60

113±15

120

300 600

5

94-+ 0 102±

600

60

98+12 100 + 6 119 + 4

105-+ 8 91-+12

120 300

60 120 300

98 + - 6 111+10

30 60

2 3

I

I

I 4 1 5

11 +

0

8 -+ 2 12-+ 5 13 + 3

1 3 +16± 14-+ 9+

9-+ 2 12 + 3

12-+ 2 9-+ 1

16 + 2 12-+ 0

9-+

l0 +

12± 0

8-+ 3 10± 2 10± 1

8-'- 3 7+ 2

8+ 9+

34-+1

30-+2 39-+9 39-+0

35+1 36-+1 36-+4 34-+1

32-+4 26--+5

30-+2 35-+5

31+5 37+6

25-+2

25+7

33+3

28±5 24+1 30-+3

30+3 29+2

32±3 33±5

1

0

4

3 1 3

I 2

2 2

6 -+-

5

[[-+ I I1-+ 5 8-+ 4

8+ I 7-+ 3 10 + 3 6+ 2

8-+ 3 4+ 2

I I ~- 3 7+ 1

7-+ I 9+ 3

7-+

8 -+

7+

9+ 9+ 9+

5+ 5+

4 -+ 5 -+ 6 7

5 7

5 7

1

6

8

18-+ 0

32 + 8 28-+ 2 21-+ 5

23 + 0 23-+ 3 23+10 21-+ 1

20-+ 4 18 + 1

19 + 2 22 + I

28 + 20 +

23-+

29 +

18±

1 9 +- 5 2 1 -+ 5 18-+ 8

20 + 18 +

25 + 24 +

a All test c o m p o u n d s s h o w e d killing on b a c t e r i a at the highest doses used ( e x a m i n e d with a dissecting microscope). b T A I 0 0 (BP, 5); T A 1 5 3 5 ( 2 - A A , 5); T A 9 8 (BP, 5); T A 1 5 3 7 ( 2 - A A , 5); T A I 5 3 8 ( 2 - A A , 2); W P 2 uvrA ( 2 - A A , 80).

Safrole

Piperonal

Isosafrole

Isoeugenol

Eugenol

Methyl eugenol 8 0

1

I

6 I

4 2 4 8

49 +

8

55-+ 8 72 + I 55 + 2

60-+ 58-+ 54-56 +

59 + 7 48-+ 8

60 + 67±

54-+ 4 49 + 1

57-+

63-+ 4

56-+

57+10 53-+ 6 56-+ 4

69 + 63 +

65-+ 9 6 3 -+ 5

t~

136

with anethole in each dose are also shown in Fig. 2. Induced mutation frequencies [(number of revertants with test p l a t e ) - (number of revertants with control p l a t e ) / n u m b e r of viable cells with test plate] were calculated as described by Green and Muriel (1976). By this calculation, the number of induced revertants is normalized for the number of viable cells and thus the effect of bactericidal activity on the revertant number can be eliminated. Induced mutation frequencies of T A I 0 0 cells with anethole at each dose were as follows (doses shown in parentheses): 1.58 × 10 v (30 /~g), 3.74 × 10 7 (60 /~g), 6.59 × 10 v (90 >g), 1.01 × 10 6 (120/~g), 1.31 × 10 6 (150 t~g). These calculations s h o w a clear dose-dependent increase of induced mutation frequencies of T A I 0 0 cells with anethole.

DNA-repair test D N A - d a m a g i n g activities of test c o m p o u n d s assayed with a pair of B. subtilis strains in the absence of $9 are summarized in Tables 5 and 6. Kada et al. (1980) used 2 × 106 spores per plate for this D N A - r e p a i r test. We used 2 × 10 s spores per plate, as the sensitivity of the test was increased by this reduction. The results obtained from the test with D M S O as a solvent did not show any significant differences (data not shown). D N A - r e p a i r tests with $9 were not successful.

TABLE 5 RESULTS

OF

DNA-REPAIR

TEST ON

SAFROLE

AND

RELATED

COMPOUNDS

WITH

B.

subtilis S T R A I N S

Compound

Dose

Zone of killing ( r a m ) a

(mg/disk)

Controls Ethanol (solvent control) Chloramphenicol (negative control) Mitomycin C (positive control) Test eompounds Anethole Cinnamaldehyde Cinnamyl alcohol Estragole

Methyl eugenol Eugenol Isoeugenol

Isosafrole Piperonal Safrole

20.0 2.5 x 10 2 . 0 × 10

10.0 0.2 1.0 4.0 1.0 0.4 0.8 20.0 5.0 20.0

3 s

M 4 5 Rec

H 17 R e c "

0.0 14.0 16.2

0.0 14.5 3.6

0.0 -0.5 12.6

5, l 22.9 15.5 8.9 16.3 19.7 23.4 7.5 18.6 16.2

2.0 13.9 I 1.0 6.3 9.8 12.8 18.2 5.1 12.0 10.8

3.1 9.0 4.5 2.6 6.5 6.9 5.2 2.4 6.6 5.4

~' Mean values from 3 independent Expts. b Difference greater than 4 mm was taken as evidence of preferential killing of R e c

Difference h (Rec Rec " }

cells.

137 TABLE 6 RESULTS OF DNA-REPAIR TEST ON ESSENTIAL OILS A N D AN OLEORESIN WITH B. subtilis STRAINS Compound

Dose (mg/disk)

Zone of killing (mm) a M45 "Rec

H17 Rec +

Difference b (Rec -- Rec +)

Controls Ethanol (solvent control) Chloramphenicol (negative control) Mitomycin C (positive control)

20.0 2.5× 10 3 2.0)< 10-5

0.0 14.8 18.0

0.0 14.5 4.6

0.0 0.3 13.4

Test compounds Anise oil Cassia oil Cinnamon bark oil Clove oil Sweet fennel oil Pimenta oleoresin

20.0 0.1 0. I 0.2 20.0 0.4

14.2 22.2 21.7 13.7 10.0 18.4

7.2 11.0 10.3 7.9 5.8 11.0

7.0 1 1.2 11.4 5.8 4.2 7.3

Mean values from 3 independent Expts. b Difference greater than 4 mm was taken as evidence of preferential killing of Rec a

cells.

Discussion Among the test c o m p o u n d s in this report, safrole, estragol and isosafrole are known as weak carcinogens in rodents (Long et al., 1963; Drinkwater et al., 1976; Innes et al., 1969). Alkenylbenzene structures are common features of these chemicals and they were found to be metabolized in a similar manner by liver enzymes (Miller et al., 1979). The test compounds used in this study were 6 alkenylbenzenes, 2 alkenylbenzene derivatives (alcohol and aldehyde) and 1 aldehyde with a 3,4methylene dioxybenzene moiety which is present in safrole (Fig. 1). Both the mutagenicity test (S. typhimurium and E. coli) and the DNA-repair test ( B. subtilis) were used to assess genetic toxicity of the test compounds. In the reverse mutation test, anethole showed an increase in the number of revertants, but not twice as many as that of the solvent control. For the chemicals that showed a killing effect at the doses at which they showed mutagenic activity, the 2-fold rule may turn out to be too restrictive a requirement for a positive effect in strain TA100, where the number of spontaneous revertants ranges around 100 or higher (Chu et al., 1981). The fluctuation test, sometimes effective in detecting very weak mutagenicity, was not applied for anethole, because the active metabolite can be formed from anethole only in the presence of $9, which may have too short a half-life to yield meaningful results. Furthermore, the test chemical was toxic to bacteria (Green and Muriel, 1976). When we analyzed the data in Fig. 1 by Student's t test, 3 consecutive values (number of revertants) at the doses 60, 90 and 120 ~g were significantly

higher than the value with the solvent control by 98% confidence limits. Additionally, they showed a dose-dependent increase. From this statistical analysis and the estimation of induced mutation frequency shown in the Results section, it was concluded that anethole was mutagenic in strain TA100. Low-purity estragole (96.0%) showed weak mutagenicities both in basesubstitution-type strains (TA100 and TA1535) and in frameshift'-type strains (TA98 and TA1537) in the absence of $9 (data not shown). The higher purity estragole (99.9%) did not, however, show any mutagenicity with or without $9 in any tester strains used. Swanson et al. (1979) reported that estragole was very weakly mutagenic in strain TA100 and that the mutagenicity was enhanced by the addition of liver enzymes (13 000 g supernatant from liver homogenate). They used ethanol as a solvent. When our experiments were performed with ethanol as a solvent instead of DMSO, no activity was, however, observed in any tester strains used in this study (data not shown). Swanson et al. (1979) claimed the purity of the estragole used to be more than 99% as measured by either TLC or HPLC. There are several reports on mutagenicity of safrole with positive results. However, some of these reports did not specify the purity of the samples. Even a very low level of impurities may play an important role in the mutagenic activity of the samples. Dorange et al. (1978) used liver enzymes from methylcholanthrene-treated female rats as an activation system in detecting mutagenicity in safrole with strain TA1535 (the purity of the sample was not reported and the results were obtained at only a single dose). Although a highly purified safrole was used as a test sample, mutagenicity was not detected in any tester strain under the conditions used in this study. These differences in results obtained from mutagenicity tests of safrole-related compounds may be derived from differences in metabolic activation methods, from differences in the sample purities, or from difficulties in obtaining clear dose response relationships owing to the killing action of the samples on bacteria. In the DNA-repair test, many test compounds exhibited positive responses even in the absence of $9. The DNA-repair test can, therefore, be applied to samples that are toxic to bacteria. Kada et al. (1980) reported that carcinogens that were not detected by their mutagenicities in the Ames test showed positive responses in the DNA-repair test with B. subtilis. Safrole was included in the list of these compounds (Kada et al., 1980). Rosenkranz and Poirier (1979) also reported that safrole exhibited a positive response in the DNA-repair test using E. coli p o l A - and p o l A . It is important to use a variety of test systems that are based on different principles in order to examine genetic toxicities of objective samples. The test compounds in this study were somewhat oily and thus were not easy to diffuse effectively in an aqueous agar layer. On the other hand, Rec + cells grew faster than Rec cells: the doubling time of each strain in nutrient-broth medium was measured to be 48 min for H17 Rec ÷ and 75 rain for M45 Rec . Consequently, it may be that, for the test compounds in this report, the Rec + cells grew too fast and gave a visible lawn of bacteria before the sample diffused effectively; it would, then, eventually give smaller inhibition zones than the Rec cells. We are currently testing

139

this possibility by applying a liquid-suspension procedure which is a modification of the DNA-repair test (Rosenkranz and Poirier, 1979).

Acknowledgements We thank Dr. T. Matsushima (Institute of Medical Science, University of Tokyo) for his useful advice in the preparation of this manuscript. Thanks are also due to Mr. T. Yoshikawa and Miss M. Yoshimoto for their technical assistance.

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