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Increased dominant-lethal effects due to prolonged exposure of mice to inhaled ethylene oxide
Mutation Research, 119 (1983) 377-379
377
Elsevier Biomedical Press
Increased dominant-lethal effects due to prolonged exposure of mice to inhaled ethylene oxide W.M. Generoso, R.B. Cumming, J.A. Bandy and K.T. Cain Biology Division, Oak Ridge National Laboratory, P.O. Box Y, Oak Ridge, TN 37830 (U.S.A.)
(Accepted 1 December 1982)
Currently, there is considerable interest in determining the magnitude of the health hazard posed by inhaled ethylene oxide. One component of the hazard may come f r o m c h r o m o s o m a l aberrations that may be induced in germ cells of exposed males. Experiments with mice and rats provided unequivocal evidence that ethylene oxide is able to induce chromosome damage in certain male germ cells. Short-term exposure via inhalation to high concentrations of ethylene oxide or i.p. injection o f this c o m p o u n d have been found to induce dominant-lethal mutations in male rats and mice [1-3]. More importantly, from a genetic hazard point of view, i.p. injected ethylene oxide has been found to induce heritable reciprocal translocations [3]. The germ-cell stage sensitivity pattern for ethylene oxide-induced dominant-lethal mutations is very similar to that for ethyl methanesulfonate and methyl methanesulfonate [2, 3]. The sensitive stages are midspermatozoa to late spermatids (inclusive). Because most human exposures to ethylene oxide is via prolonged inhalation, the question arises as to whether long-term exposures affect the animals in ways that may increase the sensitivity or susceptibility of the germ cells. Hybrid (101 x C3H)F1 males were subjected either to 2 or to 11 weeks of exposure to ethylene oxide via inhalation. In both cases mice were exposed 5 days a week with rests on weekends. Mice in the short-term exposure group were exposed along with those in the long-term group during the last 2 weeks of the 11 weeks exposure. All mice, including those from the control group, were 7 - 9 weeks old at the beginning o f the long-term exposure. The concentration o f ethylene oxide in air was 255 p p m and mice were exposed for 6 h a day. On the last day of inhalation exposure, each male was caged with 2 (C3H x C57BL)F1 females (10-13 weeks By acceptance of this article, the publisher or recipient acknowledgesthe U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering the article. Research sponsored jointly by the National Toxicology Program under NIEHS Interagency Agreement Y01-ES-20085 and the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. 0165-7992/83/0000-0000/$ 03.00 © Elsevier Science Publishers
378 old). Females were e x a m i n e d for a v a g i n a l plug o n the following 4 successive m o r n ings a n d females were killed 1 2 - 1 5 days after m a t i n g for uterine analysis. D o m i n a n t - l e t h a l d a t a are s h o w n i n T a b l e 1. It c a n be seen that the d o m i n a n t lethal effects are higher with l o n g - t e r m t h a n with s h o r t - t e r m exposure. The analyses o f v a r i a n c e indicate t h a t all c o m p a r i s o n s are highly significant for b o t h dead imp l a n t s a n d living e m b r y o s . A t each d a y o f m a t i n g the c o r r e s p o n d i n g values for dead i m p l a n t s a n d for the calculated frequencies o f d o m i n a n t - l e t h a l m u t a t i o n s are higher i n the l o n g - t e r m exposure g r o u p t h a n in the s h o r t - t e r m exposure g r o u p a n d , of course, the same is true for d a t a pooled f r o m all matings. Results also show that there is progressive r e d u c t i o n i n d o m i n a n t - l e t h a l effects with time o f m a t i n g which TABLE 1 DOMINANT-LETHAL EFFECTS OF INHALED ETHYLENE OXIDE (EO) Treatment a
Controle
EO-2t
EO- 11g
Treatment to mating interval (days)b
Number mated
Number Total Living pregnant implants embryos (Ave)¢ (Ave)¢
1/2
20
17
1 1/2
8
8
2 1/2 3 1/2
17 6
Total
Dead Dominant implants lethals (070) (%)d
10.0 9.9 10.0 8.8
8
15 6
10.3 10.0 10.7 8.8
51
46
10.4
9.8
5 47 40 35 26
49 39 37 30
1
6 0
1/2
13
10
9.4
1 1/2
6
6
10.0
2 1/2 3 1/2
15 9
14 8
9.5 9.3
5.0 6.0 6.2 6.9
Total
43
38
9.5
6.0
37
39
1/2 2 1/2 3 1/2
28 13 28 17
24 10 23 15
9.0 9.1 8.7 8.5
4.3 3.1 4.7 5.2
53 66 46 39
56 68 52 47
Total
86
72
8.8
4.4
50
55
1 1/2
aEthylene oxide (EO) in air at 255 ppm 6 h daily on weekdays. bArter last day of exposure. ePer pregnant female. dCalculated using the formula: DL = [1 -
Ave living embryos (experimental) ]×I00 Ave living embryos (control)
The value used for control is the average for all matings (9.8). c36 males were used. f36 males were exposed on weekdays for 2 weeks. g58 males were exposed on weekdays for 11 weeks.
379 appears to be more pronounced in the short-term exposure group. Obviously, one cannot tell for sure the cause for the difference between the 2 exposures. However, it is likely that it is attributable to one of two things - (1) longterm exposure affected the ability o f mice to detoxify ethylene oxide resulting in higher effective concentration to which germ cells were exposed and (2) continuous exposure of germ cells at the stem-cell stage and throughout the entire spermatogenic cycle could have altered these cells in ways that increased their sensitivity. Even though the yields of dominant lethals in the present study are already high, still higher effects can be expected from the same exposures when certain other stocks of females are used to mate with the exposed males. It was found earlier that the yield of dominant lethals in (C3H × C57BL)F1 females was lower than that in T-stock females [3]. Finally it should be noted that the ethylene oxide concentration used in this study is only about 5 times that of the current maximum allowable level in the workplace in the U.S.
References 1 Embree, J.W., J.P. Lyon and C.H. Hine (1977) The mutagenic potential of ethylene oxide using the dominant-lethal assay in rats, Toxicol. Appl. Pharmacol., 40, 261-267. 2 Cumming, R.B., and T.A. Michaud (1979) Mutagenic effects of inhaled ethylene oxide in male mice, Environ. Mutagen., 1, 166-167. 3 Generoso, W.M., K.T. Cain, M. Krishna, C.W. Sheu and R.M. Gryder (1980) Heritable translocation and dominant-lethal mutation induction with ethylene oxide in mice, Mutation Res., 73, 133-142.
377
Elsevier Biomedical Press
Increased dominant-lethal effects due to prolonged exposure of mice to inhaled ethylene oxide W.M. Generoso, R.B. Cumming, J.A. Bandy and K.T. Cain Biology Division, Oak Ridge National Laboratory, P.O. Box Y, Oak Ridge, TN 37830 (U.S.A.)
(Accepted 1 December 1982)
Currently, there is considerable interest in determining the magnitude of the health hazard posed by inhaled ethylene oxide. One component of the hazard may come f r o m c h r o m o s o m a l aberrations that may be induced in germ cells of exposed males. Experiments with mice and rats provided unequivocal evidence that ethylene oxide is able to induce chromosome damage in certain male germ cells. Short-term exposure via inhalation to high concentrations of ethylene oxide or i.p. injection o f this c o m p o u n d have been found to induce dominant-lethal mutations in male rats and mice [1-3]. More importantly, from a genetic hazard point of view, i.p. injected ethylene oxide has been found to induce heritable reciprocal translocations [3]. The germ-cell stage sensitivity pattern for ethylene oxide-induced dominant-lethal mutations is very similar to that for ethyl methanesulfonate and methyl methanesulfonate [2, 3]. The sensitive stages are midspermatozoa to late spermatids (inclusive). Because most human exposures to ethylene oxide is via prolonged inhalation, the question arises as to whether long-term exposures affect the animals in ways that may increase the sensitivity or susceptibility of the germ cells. Hybrid (101 x C3H)F1 males were subjected either to 2 or to 11 weeks of exposure to ethylene oxide via inhalation. In both cases mice were exposed 5 days a week with rests on weekends. Mice in the short-term exposure group were exposed along with those in the long-term group during the last 2 weeks of the 11 weeks exposure. All mice, including those from the control group, were 7 - 9 weeks old at the beginning o f the long-term exposure. The concentration o f ethylene oxide in air was 255 p p m and mice were exposed for 6 h a day. On the last day of inhalation exposure, each male was caged with 2 (C3H x C57BL)F1 females (10-13 weeks By acceptance of this article, the publisher or recipient acknowledgesthe U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering the article. Research sponsored jointly by the National Toxicology Program under NIEHS Interagency Agreement Y01-ES-20085 and the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. 0165-7992/83/0000-0000/$ 03.00 © Elsevier Science Publishers
378 old). Females were e x a m i n e d for a v a g i n a l plug o n the following 4 successive m o r n ings a n d females were killed 1 2 - 1 5 days after m a t i n g for uterine analysis. D o m i n a n t - l e t h a l d a t a are s h o w n i n T a b l e 1. It c a n be seen that the d o m i n a n t lethal effects are higher with l o n g - t e r m t h a n with s h o r t - t e r m exposure. The analyses o f v a r i a n c e indicate t h a t all c o m p a r i s o n s are highly significant for b o t h dead imp l a n t s a n d living e m b r y o s . A t each d a y o f m a t i n g the c o r r e s p o n d i n g values for dead i m p l a n t s a n d for the calculated frequencies o f d o m i n a n t - l e t h a l m u t a t i o n s are higher i n the l o n g - t e r m exposure g r o u p t h a n in the s h o r t - t e r m exposure g r o u p a n d , of course, the same is true for d a t a pooled f r o m all matings. Results also show that there is progressive r e d u c t i o n i n d o m i n a n t - l e t h a l effects with time o f m a t i n g which TABLE 1 DOMINANT-LETHAL EFFECTS OF INHALED ETHYLENE OXIDE (EO) Treatment a
Controle
EO-2t
EO- 11g
Treatment to mating interval (days)b
Number mated
Number Total Living pregnant implants embryos (Ave)¢ (Ave)¢
1/2
20
17
1 1/2
8
8
2 1/2 3 1/2
17 6
Total
Dead Dominant implants lethals (070) (%)d
10.0 9.9 10.0 8.8
8
15 6
10.3 10.0 10.7 8.8
51
46
10.4
9.8
5 47 40 35 26
49 39 37 30
1
6 0
1/2
13
10
9.4
1 1/2
6
6
10.0
2 1/2 3 1/2
15 9
14 8
9.5 9.3
5.0 6.0 6.2 6.9
Total
43
38
9.5
6.0
37
39
1/2 2 1/2 3 1/2
28 13 28 17
24 10 23 15
9.0 9.1 8.7 8.5
4.3 3.1 4.7 5.2
53 66 46 39
56 68 52 47
Total
86
72
8.8
4.4
50
55
1 1/2
aEthylene oxide (EO) in air at 255 ppm 6 h daily on weekdays. bArter last day of exposure. ePer pregnant female. dCalculated using the formula: DL = [1 -
Ave living embryos (experimental) ]×I00 Ave living embryos (control)
The value used for control is the average for all matings (9.8). c36 males were used. f36 males were exposed on weekdays for 2 weeks. g58 males were exposed on weekdays for 11 weeks.
379 appears to be more pronounced in the short-term exposure group. Obviously, one cannot tell for sure the cause for the difference between the 2 exposures. However, it is likely that it is attributable to one of two things - (1) longterm exposure affected the ability o f mice to detoxify ethylene oxide resulting in higher effective concentration to which germ cells were exposed and (2) continuous exposure of germ cells at the stem-cell stage and throughout the entire spermatogenic cycle could have altered these cells in ways that increased their sensitivity. Even though the yields of dominant lethals in the present study are already high, still higher effects can be expected from the same exposures when certain other stocks of females are used to mate with the exposed males. It was found earlier that the yield of dominant lethals in (C3H × C57BL)F1 females was lower than that in T-stock females [3]. Finally it should be noted that the ethylene oxide concentration used in this study is only about 5 times that of the current maximum allowable level in the workplace in the U.S.
References 1 Embree, J.W., J.P. Lyon and C.H. Hine (1977) The mutagenic potential of ethylene oxide using the dominant-lethal assay in rats, Toxicol. Appl. Pharmacol., 40, 261-267. 2 Cumming, R.B., and T.A. Michaud (1979) Mutagenic effects of inhaled ethylene oxide in male mice, Environ. Mutagen., 1, 166-167. 3 Generoso, W.M., K.T. Cain, M. Krishna, C.W. Sheu and R.M. Gryder (1980) Heritable translocation and dominant-lethal mutation induction with ethylene oxide in mice, Mutation Res., 73, 133-142.