Mutation Research, 187 (1987) 157-163 157 Elsevier

MTR 01140

Cytogenetic studies on 1,1-dichloroethylene and its two isomers in mammalian cells in vitro and in vivo

M . S a w a d a , T . S o f u n i a n d M . I s h i d a t e J r .

Division of Mutagenesis, Biological Safety Research Center, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan)

(Received 27 November 1985) (Revision received 5 November 1986)

(Accepted 7 November 1986)

Key words: 1,1-Dichloroethylene; Mammalian cells; Chromosomal aberration; Sister-chromatid exchange; Chloroacetyl chloride; Chloroacetic acid.

Summary

Chromosomal aberration and sister-chromatid exchange (SCE) tests in vitro on 1,1-dichloroethylene (1,1-DCE), its two isomers, cis- and trans-l,2-DCE, and two possible metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, were carried out using a Chinese hamster cell line, CHL.

1,1-DCE induced chromosomal aberrations in the presence of $9 mix prepared from the rat liver, but not in the absence of $9 mix. SCEs were also slightly induced by 1,1-DCE only in the presence of $9 mix. On the other hand, two isomers and two metabolites of 1,1-DCE induced neither chromosomal aberrations nor SCEs with and without $9 mix.

1,1-DCE, however, was negative even at a sublethal dose in the micronucleus test using mouse bone marrow, fetal liver and blood.

1,1-Dichloroethylene (1,1-DCE, vinylidene chloride), is widely used in the manufacture of plastics and exists in our environment as a low- level contaminant. A mutagenic effect of 1,1-DCE was detected in Escherichia coli using a mouse microsomal activation system (Greim et al., 1975); in Salmonella typhimurium (TA1530, TA1535 and TA100) in the presence of mouse or rat $9 mix (Bartsch et al., 1975; Simmon et al., 1977; Jones and Hathway, 1978a); and in Saccharomyces

Correspondence: Dr. M. Ishidate Jr., Head, Division of Muta- genesis, Biological Safety Research Center, National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 (Japan).

cerevisiae D7 with mouse liver 10000 × g super- natant (Bronzetti et al., 1981).

On the other hand, 1,1-DCE showed no muta- genic activity in the somatic cell mutation assay (8-azaguanine and ouabain resistance) using Chinese hamster cells (V79), though there was a dose-related toxicity in the presence of the super- natant of rat-liver homogenate (Drevon and Kuroki, 1979). Two isomers of 1,1-DCE, cis- and trans-l,2-DCE were also reported to be negative in the Escherichia coli K12 test system (Greim et al., 1975).

Studies on the metabolic pathway of 1,1-DCE in rodents have indicated that chloroacetyl chlo- ride and chloroacetic acid were formed from 1,1- DCE through 1,1-DCE oxide (Jones and Hath-

0165-1218/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

158

way, 1978b). Bacterial mutation assays indicated that 1,1-DCE oxide may be one of the active metabolites of 1,1-DCE (Bartsch et al., 1975, 1979; Henschler, 1980).

In the present study, the cytogenetic activities of 1,1-DCE, cis- and trans-l,2-DCE, and two commercially available metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, were examined with Chinese hamster cells in culture. In addition, a micronucleus test was carried out with 1,1-DCE using mouse bone marrow, and fetal liver and blood.

Materials and methods

Chemicals. 1,1-DCE [CAS registry number 75-35-4] (purity 99%), cis-l ,2-DCE [156-59-2] (97%), chloroacetic acid [79-11-8] (>99%) and metyrapone [54-36-4] (98%) were purchased from Aldrich Chemical Company, Inc. The 1,1-DCE contained 200 ppm of p-methoxyphenol [150-76-5] as a stabilizer, trans-l,2-DCE [156-60-5] (> 99%) were purchased from Tokyo Kasei Kogyo Co., chloroacetyl chloride [79-04-9] from Nakarai Chemicals, and reduced glutathione (GSH) from Wako Pure Chemical Industries.

Chromosomal aberration test in vitro. A Chinese hamster lung fibroblast cell line (CHL) was used (Ishidate and Odashima, 1977). The cells were cultured in glass bottle containing Eagle's MEM (Gibco) supplemented with 10% heat-in- activated calf serum (Gibco), and treated with 3 ml of reaction mixture consisting of $9 mix, test compound and culture medium (including serum). The $9 fraction was prepared from the liver ho- mogenate of male F344 rats pretreated with PCB(KC-400). 10 ml of $9 mix consisted of 3 ml of the $9 fraction, 2 ml of 20 mM HEPES buffer, 1 ml each of 50 mM MgC12,330 mM KCI, 50 mM G-6-P, 40 mM NADP and distilled water (Matsuoka et al., 1979). 1,1-DCE, cis- and trans- 1,2-DCE were diluted in DMSO, and chloroacetyl chloride and chloroacetic acid were dissolved in saline. The bottles were sealed tightly with silicon stoppers, because the boiling point of 1,1-DCE was very low (about 32°C). The dose of each chemical was increased to have a positive result or to show distinct cytotoxic effects which were ob- served under a phase contrast microscopy. When a

test chemical did not show any cytotoxic effects, the maximum dose was limited to around 10 mM (Ishidate et al., 1984). After a 6-h treatment, the cells were rinsed with Dulbecco's PBS and recul- tured with fresh medium for another 18 h. The chromosome preparations were made using the standard mr-drying method, and the frequency of the cells with chromosomal aberrations was scored in 100 well-spread metaphases for each concentra- tion group.

Sister-chromatid exchange (SCE) assay in vitro. CHL cells were treated for 6 h in the same way as described above. After rinsing with PBS, the cells were recultured for another 24 h. BUdR (Sigma) was present at a final concentration of 7 /~M throughout treatment and during the subsequent incubation period. The sister-chromatid differenti- ation was made according to the method of Sakanishi- and Takayama (1977). The number of SCEs was counted in 50 metaphases for each concentration group.

Micronucleus test in vivo (a) Bone marrow. 8-week-old male ddY mice

(body weight: 31-34 g) were purchased from Shizuoka Agricultural Co-op. Assoc. In each dose group 6 mice were used; 1,1-DCE dissolved in olive oil was administered once by gavage. Animals were killed by cervical dislocation 24 h after treat- ment, and femoral marrow cells were smeared, fixed with methanol and stained with 3% Giemsa. The sampling time and the maximum dose were determined by the pilot experiment according to Hayashi et al. (1984) in which preparations were made at 5 different points between 18 h and 72 h after administration. 1000 polychromatic erythro- cytes (PCE) per mouse were examined, and the number of micronucleated PCE (MNPCE) was recorded. For the multiple treatment, mice were given, 1,1-DCE by gavage 4 times at 24 h inter- vals, and preparations were made 24 h after the last administration. The number of micronu- cleated normochromatic erythrocytes (MNNCE) among 1000 NCEs was also recorded in addition to the frequency of MNPCE.

(b) Fetal fiver and blood. A transplacental mi- cronucleus test (Cole et al., 1981) was carried out as follows. Pregnant ICR mice purchased from Clea Japan Inc. were injected with 1,1-DCE in-

traperitoneally on the 18th day of gestation. At 24 h after the injection, the fetal liver and fetal blood cells suspended in fetal calf serum were smeared, and fixed with methanol. The cells were then stained with Acridine Orange (Hayashi et al., 1983) instead of Giemsa to distinguish micronuclei from RNA containing basophilic stippling that occa- sionally appeared in the fetal blood cells. 1000 erythrocytes emitting red fluorescence (which cor- respond to PCE in Giemsa staining) were ob- served per fetus and erythrocytes with micronuclei which emitted yellowish green fluorescence were scored.

Results

(1) Chromosomal aberration test in vitro In a system without metabolic activation, 1,1-

DCE did not significantly induce chromosomal aberrations even at the concentration of 2 mg/ml . In the presence of $9 mix, however, 1,1-DCE induced chromosomal aberrations and their fre- quency increased depending on the increasing concentration of the $9 in the reaction mixture. Since clearly positive results were obtained with 1,1-DCE at the relatively higher concentrations of $9, the final concentration of the $9 was fixed at 15% in the following in vitro experiments.

As shown in Table 1, an obvious dose-effect relationship was observed in the induction of chromosomal aberrations by 1,1-DCE: 14% aber- rant cells at 0.25 m g / m l and 54% (maximum) at 1.5 mg/ml . Almost all aberrations were chro- matid-type, and exchanges were more predomi- nant than gaps and breaks. Although 1,1-DCE used here contained 200 ppm of p-methoxyphenol as a stabilizer, 20 g g / m l of p-methoxyphenol induced no chromosomal aberrations in the pres- ence of $9 mix.

Two isomers of 1,1-DCE, cis- and trans-l,2- DCE, on the other hand, showed no significant increase in the incidence of chromosomal aberra- tions in both systems with and without $9 mix (Table 1). As shown in Table 2, two metabolites of 1,1-DCE, chloroacetyl chloride and chloroacetic acid, showed cytotoxicity, but did not induce chromosomal aberrations.

Metyrapone, an inhibitor of the P-450 activity, was added to the culture at several concentrations

159

T A B L E 1

F R E Q U E N C Y O F C E L L S 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 I N C H L C E L L S T R E A T E D W I T H 1,1-, cis-l,2- and trans-I,2-DICHLOROETHYLENE (DCE)

C o m p o u n d Dose

( m g / ml)

$9 Cells wi th aber ra t ions a (%)

mix ctg ctb cte dic Tota l

1 ,1 -DCE

cis-l,2-DCE

trans- 1,2-DCE

0 + 1 1 0 0 2

0.125 + 2 0 6 0 8 0.25 + 5 4 6 1 14

0.5 + 7 16 20 0 29

1.0 + 12 20 32 1 41 1.5 + 16 27 35 0 54 2.0 + Toxic

0 - 2 0 0 0 2

0.125 - 1 0 0 0 1

0.25 - 1 0 0 0 1

0.5 - 0 0 0 0 0 1.0 - 0 1 1 0 2

1.5 - 1 0 0 0 1

2.0 - 1 1 0 0 2

0 + 0 0 0 0 0 0.25 + 1 1 0 0 2

0.5 + 0 0 0 0 0

1.0 + 0 0 0 0 0 2.0 + 0 0 0 0 0

0 - 2 0 0 0 2

0.25 - 1 1 0 0 2 0.5 - 1 0 0 0 1

1.0 - 1 0 1 0 2

2.0 - 2 0 0 0 2

0 + 0 0 0 0 0 0.25 + 0 0 0 0 0

0.5 + 0 0 1 1 1 1.0 + 0 1 0 0 1

2.0 + 0 0 0 0 0

0 - 1 0 0 0 1 0.25 - 0 0 0 0 0

0.5 - 0 0 1 0 1 1.0 - 0 0 0 0 0

2.0 - 3 0 0 0 3

a ctg, ch roma t id gaps; ctb, ch romat id breaks; cte, ch romat id exchanges; dic, d icent r ic chromosomes .

together with $9 mix, and then 1,1-DCE was added at a final concentration of 1.5 mg/ml . The frequencies of aberrant cells decreased as the con- centration of metyrapone increased (0.1-1.0 raM), and was only 8% at 1.0 mM of metyrapone (Fig. 1). The GSH which plays an important role in the

160

TABLE 2

FREQUENCY OF CELLS WITH CHROMOSOMAL ABERRATIONS IN CHL CELLS TREATED WITH CHLOROACETYL CHLORIDE AND CHLOROACETIC ACID

Compound Dose $9 mix (mg/ml)

Cells with aberrations a (%)

ctg ctb cte dic Total

Chloroacetyl chloride

Chloroacetic acid

0 + 1 0 0 0 1 0.06 + 0 1 1 0 2 0.125 + 0 0 1 0 1 0.25 + 0 0 0 1 1 0.5 + Toxic

0 - 1 1 0 0 2 0.06 - 1 0 0 0 1 0.125 - 0 0 0 0 0 0.25 - 0 0 0 0 0 0.5 - Toxic

0 + 1 0 0 0 1 0.06 + 0 0 1 0 1 0.125 + 1 0 0 0 1 0.25 + 0 1 0 0 1 0.5 + Toxic

0 - 1 1 0 0 2 0.06 - 1 0 0 0 1 0.125 - 1 1 0 0 2 0.25 - 0 0 0 0 0 0.5 - 0 0 0 0 0

a ctg, chromatid gaps; ctb, chromatid breaks; cte, chromatid exchanges; dic, dicentric chromosomes.

d e t o x i f i c a t i o n o f t he ac t ive me tabo l i t e s , a lso

m a r k e d l y i n h i b i t e d the i n d u c t i o n o f c h r o m o s o m a l

a b e r r a t i o n s b y 1 , 1 - D C E w i t h $9 m i x (Fig . 1).

(2) S C E assay in vitro T h e n u m b e r o f S C E s p e r cel l was i n c r e a s e d

s ign i f i can t ly b y 1 , 1 - D C E o n l y in t he p r e s e n c e o f

60

~ 40

.~ 20

~ o

i,~,~,,,_ (A)

0

~'-~0.01 0. i 1,0 Metyrapone (mM)

6O

\ 4 0

\ 0 0.01 0.I i'.0

GSH (mM)

Fig. 1. Effect of metyrapone (A) or GSH (B) on the induction of chromosomal aberrations by 1,1-DCE. Metyrapone or GSH was added to the culture together with S9 mix, and then 1,1-DCE (1.5 mg/ml) was added.

$9 m i x ( T a b l e 3). H o w e v e r , the m a x i m u m n u m b e r

o f S C E s p e r cel l (18.7 + 5.9) a t the h ighes t dose

(0.1 m g / m l ) d id n o t r e a c h twice the c o n t r o l v a l u e

( 1 0 . 0 + 3.5), i n d i c a t i n g tha t the S C E - i n d u c i n g

p o t e n t i a l o f 1 , 1 - D C E was re la t ive ly weak . A t t he

d o s e m o r e t h a n 0.1 m g / m l , t he re w e r e a l m o s t n o

cel ls s h o w i n g a d i f f e ren t i a l s t a in ing p a t t e r n o f

s i s ter c h r o m a t i d . B o t h cis- and trans-l,2-DCE, a n d two m e t a b o l i t e s o f 1 , 1 - D C E w e r e n e g a t i v e in

t h e S C E test w i t h a n d w i t h o u t $9 m i x (Tab l e s 3

a n d 4).

(3) Micronucleus test m vivo Resu l t s o f m i c r o n u c l e u s tests o n 1 , 1 - D C E in

the m o u s e b o n e m a r r o w are s h o w n in T a b l e 5. 3

o u t o f 6 a n i m a l s d i e d a f te r a s ingle t r e a t m e n t w i t h

200 m g / k g , a n d 1 a n i m a l d i e d a f te r 4 t r e a t m e n t s

w i t h 100 m g / k g . T h e f r equenc i e s o f M N P C E a n d

M N N C E at e a c h d o s e in b o t h t r e a t m e n t s were n o t

s ign i f i can t ly d i f f e r e n t f r o m those in t he c o n t r o l

g roups . N o s ign i f i can t changes were o b s e r v e d in

T A B L E 3

N U M B E R O F S I S T E R - C H R O M A T I D E X C H A N G E S (SCEs)

I N C H L C E L L S T R E A T E D W I T H 1,1-, c/s-1,2- A N D trans- 1,2-DICHLOROETHYLENE ( D C E )

Compound Dose $9 mix S C E s / c e l l

(mg/ml) Range Mean4- S.D.

1 ,1 -DCE 0 + 2 - 1 9 10.0 4- 3.5 0.025 + 4 - 3 2 12.4 + 5.3

0.05 + 3 - 2 3 13.3 4- 4.4

0.075 + 5 - 2 6 16.4 + 5.0 0.1 + 9 - 3 7 18 .7+5 .9

0 - 1 - 2 0 10.84-4.1

0.025 - 3 - 2 2 11.24-4.1 0.05 - 6 - 2 0 11.5 + 3.0

0.2 - 5 - 2 0 11.24-3.4 1.0 - 4 - 2 0 11.34-3.6

cis-l ,2-DCE 0 + 3 - 2 0 10.7 4- 3.4 0.25 + 5 - 2 0 11.1 + 3.7

0.5 + 6 - 1 9 1 1 . 6 + 3 . 4

1.0 + 6 - 2 4 12.2 + 3.7

2.0 + 7 - 2 1 12.94-4.1

0 - 6 - 2 1 10.8 + 3.6

0.25 - 2 - 1 9 10.94-3.8

0.5 - 4 - 1 8 11.24-3.3 1.0 - 6 - 2 2 11.5 4- 3.9 2.0 - 5 - 2 2 12.44-3.8

trans-l,2-DCE 0 + 3 - 2 0 10.74-3,4

0.25 + 5 - 2 0 10.8 4- 3.4 0.5 + 5 - 2 0 10.5 4- 2,8

1.0 + 4 - 1 9 11.1 4- 3,6 2.0 + 4 - 2 3 10.84-3,8

0 - 6 - 2 1 10.8 + 3.6 0.25 - 5 - 2 5 11.34-4.2

0.5 - 3 - 2 1 10.64-3.7

1.0 - 5 -21 11.5 4- 3.7 2.0 - 4 - 1 8 11 .0+3 .3

* Significantly different from control (t-test, P < 0.01).

the ratio of PCE to total erythrocytes. The micro- nucleus tests using liver and blood of fetuses also showed no significant increase of MNPCE from 25 to 100 mg/kg of 1,1-DCE (Table 6).

Discussion

The present study indicates that 1,1-DCE in- duces chromosomal aberrations in cultured Chi- nese hamster cells (CHL) in the presence of rat $9 mix. 1,1-DCE also significantly increased the

161

T A B L E 4

N U M B E R O F S ISTER C H R O M A T I D E X C H A N G E S (SCEs) I N C H L C E L L S T R E A T E D W I T H C H L O R O A C E T Y L

C H L O R I D E A N D C H L O R O A C E T I C A C I D

Compound Dose S C E s / c e l l

(mg/ml) Range Mean + S.D.

Choroacetyl 0 4 - 1 8 10.6 + 3.2 chloride 0.03 4 - 2 4 10.74- 3.9

0.06 3 - 2 1 10.2 + 3.7

0.125 4 - 2 2 11.6 + 3.7

0.25 3 - 2 3 11.24-4.1

Chloroacetic acid 0 4 - 1 8 10.6 4- 3.2

0.06 2 - 1 6 10.2 + 3.1 0.125 5 - 1 9 10.9 + 3.4 0.25 1 - 2 4 11.7 4- 4.2

Without $9 mix.

number of SCEs in the presence of $9 mix, al- though its induction was relatively weak. It has been reported that 1,1-DCE was mutagenic in the Ames test when bacteria were exposed to gaseous 1,1-DCE in a desiccator (Bartsch et al., 1975; Simmon et al., 1977) or in a gas-tight culture vessel (Jones and Hathway, 1978a). In our experi- ment culture flasks were all sealed, since no chro- mosomal aberrations were observed at any con- centration of 1,1-DCE if the flasks were not sealed. This phenomenon is probably due to the vaporiza- tion of 1,1-DCE because of its lower boiling point (about 32°C).

It is assumed that the induction of chro- mosomal aberrations by 1,1-DCE in the presence of $9 mix may correlate with the activity of cyto- chrome P-450 dependent mixed-function oxidase in the microsomal fraction. The present finding that the induction of chromosomal aberrations was inhibited by the addition of metyrapone (Fig. 1) indicates that cytochrome P-450 in the liver microsome participates in the activation of 1,1- DCE.

It has been known that GSH plays an im- portant role in the detoxification of the active metabolites (Jones and Hathway, 1978b). As shown in Fig. 1, GSH could inhibit the induction of chromosome aberrations by 1,1-DCE, suggest- ing that the active metabolites of 1,1-DCE may be produced in the present metabolic activation sys-

162

TABLE 5

F R E Q U E N C Y OF M I C R O N U C L E A T E D E R YT HR OC YT E S IN BONE M A R R O W OF MICE T R E A T E D WITH 1,1-DICHLO- R O E T H Y L E N E (DCE)

Dose Surviving/ M NP C E a (%) M N N C E b (%) P C E / P C E + NCE (%)

(mg/kg) treated Range Mean -+ S.D. Range Mean -+ S.D. Mean -+ S.D. mice

1,1-DCE 0 × 1 ~ 6 / 6 0.1-0.4 0 .22+0.12 N.D. e 55.4-+4.4 2 5 × 1 6 / 6 0.0-0.4 0.15-+0.16 N.D. 52.7_+3.2 5 0 × 1 6 / 6 0.1-0.3 0.22_+0.10 N.D. 51.3-+6.7

100×1 6/'6 0.0-0.3 0.17-+0.10 N.D. 57.1-+5.2 200 × 1 3/'6 0.2-0.3 0.23 -+ 0.06 N.D. 42.6 _+ 10.9

0 × 4 ~ 6 / 6 0.1-0.4 0.22_+0.10 0.0-0.3 0.10-+ 0.11 50.6-+3.2 25 × 4 6 / 6 0.0-0.1 0.03 + 0.05 0.0-0.2 0.08 -+ 0.08 53.0 -+ 5.6 5 0 × 4 6 / 6 0.0-0.3 0.17-+0.12 0.0-0.3 0.10-+0.13 56.7_+6.9

100×4 5 / 6 0.0-0.2 0.12-+0.08 0.0-0.2 0.12-+0.08 46.4_+7.9

Mitomycin C d 2 × 1 6 / 6 5.5-8.9 6.88 + 1.35 N.D. 34.0 _+ 6.7

a MNPCE, micronucleated polychromatic erythrocytes. b MNNCE, micronucleated normochromatic erythrocytes.

Vehicle control (olive oil, 10 ml /kg) . d Positive control (dissolved in saline, intraperitoneal injection). ¢ N.D., not determined.

TABLE 6

F R E Q U E N C Y OF M I C R O N U C L E A T E D E R YT HR OC YT E S IN MOUSE FETUS AFTER T R E A T M E N T WITH 1,1-DICHLO- R O E T H Y L E N E (DCE)

Dose Number of Tissue a M NPCE b (%) P C E / P C E + N C E (%)

(mg/kg) fetuses used Range Mean _+ S.D. Mean -+ S.D.

0 ~ 4 L 0.1-0.4 0.28 + 0.15 74.3 _+ 3.9 B 0.0-0.3 0.13 +0.13 69.9-+ 7.1

25 4 L 0.2-0.5 0.35 _+ 0.13 75.0 + 6.2 B 0.1-0.3 0.23 + 0.10 68.3 -+ 6.4

50 6 L 0.1-0.4 0.27 + 0.12 75.7 _+ 6.3 B 0.1-0.3 0.22-+0.10 72.2_+3.9

100 6 L 0.1-0.4 0.25 + 0.12 78.7 + 2.8 B 0.0-0.4 0.22 + 0.15 74.2 -+ 5.5

a L, liver; B, blood. b MNPCE, micronucleated polychromatic c Vehicle control (olive oil, 10 ml /kg) .

erythrocytes.

tem, although two metabolites of 1,1-DCE, chlo- roacetyl chloride and chloroacetic acid, were nega- tive in the present tests.

It has been proposed that 1,1-DCE was metabolized to chloroacetyl chloride and chloro- acetic acid via 1,1-DCE oxide in the mouse and rat (Jones and Hathway, 1978b), and that 1,1-DCE

oxide was assumed to be a mutagenic metabolite of 1,1-DCE in the bacterial mutation assays (Bartsch et al., 1975, 1979; Henschler, 1980). The present findings are compatible with the hypothe- sis that 1,1-DCE oxide may be the active metabo- lite of 1,1-DCE. However, the mutagenicity of 1,1-DCE oxide has not been reported so far, be-

cause stabilized 1,1-DCE oxide is very hard to obtain.

I t has been reported that the $9 prepared from mice rather than rats is more effective as an activator of 1,1-DCE in the Ames test (Bartsch et al., 1975; Jones and Hathway, 1978a). Drevon and Kuroki (1979) reported that gaseous 1,1-DCE was negative in mammalian cell mutation assays in the presence of liver S15 fraction derived from either mice or rats pretreated with phenobarbitone, al- though a dose-dependent cytotoxicity was ob- served in the presence of the rat S15 fraction. In our preliminary experiments, the rat $9 was more effective than the mouse $9 in both chromosomal aberration and SCE tests (data are not shown). Mechanisms in such species-dependent variations among $9 fractions are still unclear.

Two isomers of 1,1-DCE, cis- and trans-l,2- DCE, were negative in both cytogenetic tests even at 2 m g / m i . No mutagenic potential was observed in Escherichia coli K12 in the presence of the microsomal fraction from livers of phenobarbital pretreated mice (Greim et al., 1975).

In contrast to the in vitro tests, no significant increase of micronucleated erythrocytes was ob- served in the micronucleus tests with 1,1-DCE. It can be speculated that the life time of the active metabolites may be very short, and a sufficient amount may not have reached the target cells. More detailed information on the pharmaco- kinetic fate of 1,1-DCE is required for the well understanding of the negative results in micro- nucleus test.

Acknowledgement

We are grateful to Dr. M.C. Harnois for re- viewing the manuscript.

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