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Embryo- and fetotoxicity of inhaled methyl ethyl ketone in rats
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
59,62&622
(1981)
Embryo- and Fetotoxicity of Inhaled Methyl Ethyl Ketone in Rats1
Embryoand Fetotoxicity of Inhaled Methyl Ethyl Ketone in Rats. DEACON, M. M., PILNY, M. D., JOHN, J. A., SCHWETZ, B. A., MURRAY, F. J., YAKEL, H. O., AND KUNA, R. A. (1981). Toxicol. Appl. Pharmacol. 59,62&622. Pregnant Sprague-Dawley rats inhaled 0, 400, 1000, or 3000 ppm methyl ethyl ketone (MEK) for 7 hr/day on Days 6 through 15 of gestation. Maternal toxicity, as evidenced by decreased weight gain and increased water consumption, was observed among rats exposed to 3000 ppm MEK. Slight fetotoxicity was observed among litters of rats exposed to 3000 ppm MEK as evidenced by an increased incidence of two minor skeletal variants. The results of this study duplicate the increased incidence of skeletal variants observed in an earlier study by B. A. Schwetz, B. K. J. Leong, and P. J. Gehring (1974, Toxicol. Appl. Pharmacol. 28, 452464), and do not indicate that inhaled MEK causes either an embryotoxic or teratogenic response in rats at exposure levels up to 3000 ppm.
Methyl ethyl ketone (MEK) is an extensively used, nonchlorinated solvent. The major route of human industrial exposure to MEK is by inhalation of vapors and by skin contact with the liquid. The current threshold limit value (TLV) of MEK is 200 wm. MEK (1000 or 3000 ppm) inhaled by pregnant rats for 7 hr/day on Days 6 through 15 of gestation was shown to be embryotoxic, fetotoxic, and potentially teratogenic (Schwetz et al., 1974). Exposure to 3000 ppm MEK resulted in a low incidence of acaudia, imperforate anus, and brachygnathia and an increased incidence of soft-tissue alterations and sternebral variants. Exposure to 1000 ppm MEK resulted in an increase in the total number of litters containing fetuses with skeletal alterations. No significant maternal toxicity was observed. The incidence of major mal’ Sponsored Ethyl Ketone
by the Inter-Industrial Teratology Committee.
0041-008X/81/090620-03$02.00/0 Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
Ad hoc
formations was not sufficiently high to support a positive teratogenic conclusion. This second teratologic study in rats was conducted to determine the repeatability of the fetotoxic effects observed in rats exposed to 3000 ppm MEK; therefore, additional groups of pregnant rats were exposed to 400, 1000, or 3000 ppm MEK for 7 hr/day on Days 6 through 15 of gestation. METHODS Test material. Reagent grade methyl ethyl ketone.’ 99.6% purity, was used for this study. Animals. Adult Sprague-Dawley rats,” weighing 234-332 g, were used. The day on which sperm were found in the vaginal smear was considered Day 0 of pregnancy. The animals were uniquely identified by a numbered metal ear tag. The rats were individually housed in wire-bottom cages and were fed commercial laboratory chow4 and tap water ad libitum, except dur’ Lot No. 36799. Exxon Chemical Co., Linden, 3 Spartan Research Animals, Haslett, Mich. 4 Ralston Purina Company, St. Louis, MO.
Methyl
620
N.J.
SHORT TABLE INCIDENCE
OF
FETAL
INHALING
1
ALTERATIONS
METHYL
AMONG
ETHYL Methyl 0
RATS
KETONE ethyl ketone 400
(ppmp
loo0
3ooo
No. fetuses examined/ No. litters examined External and skeletal examination Soft-tissue examination Bones of the skull
329126 1 I1126 218126
238119 80119 157119
226119 78119 148119
No. fetuses (litters) External examination Multiple defects Rotated and adducted forepaw Soft-tissue examination lnnommate artery missing Skeletal examination Skull. delayed ossification6 Ribs. extm’ Cervical centra. delayed ossitication” Total
major malformations
231/18 79118 150/18
affected
0
0
IllY
0
l(l)
0
0
0
0
l(1)
l(l)
2% 14)
1%10)
2w
23W
0
0
6t4)’ 7(6)’
8X22)
58115)
44( 16)
l(1)
III)
l(l)
l(lY
621
COMMUNICATIONS
114(18) 2I2)
” Rats inhaled 0, 400, 1000. or 3000 ppm of methyl ethyl ketone from Days 6 to 15 of gestation. b This fetus exhibited: acaudia, impetforate anus, short trunk. ectopic ovary. diaphragmatic hernia. and multiple vertebral defects, including missing ribs. vertebrae, and centra. r This fetus exhibited: acaudia, imp&orate anus, short trunk. ectopic ovary. and multiple skeletal defects including asymmetric pelvis, fused ribs, and missing ribs. vertebrae, and centra. d This alteration was considered to be a skeletal variant and was not included in the calculation of the total malformed fetuses. ’ Significantly different from control value by a modified Wilcoxon test. p < 0 05.
ing chamber exposures. Food and water consumption was measured at intervals of 3 days during the experimental period. Exprrimental design. Groups of 25 bred rats were exposed to 400, 1000, or 3000 ppm MEK by inhalation for 7 hrlday on Days 6 through 15 of gestation. A group of 35 bred rats was exposed to filtered room air in an identical chamber to serve as the control group. The exposures were conducted under dynamic aitlIow conditions in glass and stainless-steel chambers with a volume of 0.16 m3. The chamber atmosphere was generated by metering MEK at a calculated rate with a precision syringe pump into a vaporization flask and then into the airstream being drawn into the exposure chamber. Nominal concentrations were calculated as the ratio of the amount of MEK used to the total chamber airflow. The concentrations of MEK in each chamber were monitored 15 min/hr using a Miran I variable filter infrared analyzer at a wavelength of 8.5 Frn. Ana-
lytical concentrations (time-weighted mean) of MEK in the chambers for the 400-, lOOO-, and 3000-ppm exposures were 412 5 31, 1002 2 16, and 3005 2 49 ppm (mean 2 SD), respectively. Maternal and fetal observations. Animals were observed daily beginning on Day 6 of gestation for indications of toxicity. The body weights of bred rats were recorded on Days 6, 8, 10, and 16 of gestation. In addition, maternal body weights and the weight of the maternal livers were recorded at the time of cesarean section, Day 21 of gestation. The method of cesarean section as well as fetal observations and the statistical evaluations employed are described in a previous paper by Deacon et al. (1980).
RESULTS Muternal observations. Exposure of bred rats to 400, 1000, or 3000 ppm of MEK for 7 hr/day had no significant effect on the dam’s appearance or demeanor. Maternal toxicity, as evidenced by decreased weight gain and increased water consumption was observed among rats exposed to 3000 ppm of MEK. No changes were observed in maternal liver weight, or in the food consumption of pregnant rats exposed by inhalation to MEK. Embryo- and fetotoxicity. Inhalation of 400, 1000, or 3000 ppm MEK had no significant effect on the incidence of pregnancy, or on the average number of implantations and live fetuses per dam. The incidence of resorptions was comparable to controls for all exposure levels. No adverse effects on the fetal body weight or crown-rump length were noted among litters of rats exposed to MEK. No external or soft-tissue alterations were observed among fetuses at any of the exposure levels at an incidence which was statistically significantly different from the control incidence (Table 1). When considered collectively no significant increase in the incidence of major malformations in fetuses exposed to MEK was observed. A significant decrease in the incidence of delayed ossification of interparietal bones of the skull was noted among litters of rats
622
SHORT
COMMUNICATIONS
exposed to 3000 ppm of MEK. A significant increase in the incidence of extra lumbar ribs and in the occurrence of delayed ossification of cervical centra was also noted at this exposure level. These skeletal variants have been seen at low incidences among the historical control population in this laboratory, and are therefore considered indicative of a slight fetotoxic effect.
DISCUSSION The results of an earlier teratology study with MEK conducted in this laboratory (Schwetz et al., 1974) were suggestive of a potential teratogenic effect at 3000 ppm MEK. Skeletal alterations were observed among litters of the exposed rats, but the incidence of major malformations was not sufficiently high to support a positive conclusion. In the present study, a teratogenic effect (as defined by an increase in major malformations) was not observed in rats inhaling 400, 1000, or 3000 ppm of MEK. That is, exposure of pregnant rats to MEK vapor did not induce the occurrence of major external, soft-tissue, or skeletal malformations which would severely affect the survival or performance of the young. The few malformations which were observed among litters of rats exposed to MEK were types that have been noted to occur historically at a low incidence among control rats in this laboratory. Some evidence of slight fetotoxicity, as evidenced by an increased incidence of a minor skeletal variant and a delay in the ossification of fetal bones, was observed among litters of rats exposed to 3000 ppm of MEK. Embryotoxic effects, such as an increased incidences of resorptions or preimplantation loss, were not observed among rats exposed to 400, 1000, or 3000
ppm of MEK. Thus, the results of this study duplicate the increased incidence of skeletal variants observed in the earlier study by Schwetz et al. (1974), but do not indicate either an embryotoxic or teratogenie response in rats. In summary, exposure to 3000 ppm of MEK, a slightly maternally toxic level, resulted in some minor variations in the development of the fetal skeleton in rats, a slight fetotoxic effect. Neither significant embryotoxicity nor a teratogenic effect was discerned in rats inhaling up to 3000 ppm MEK for 7 hrlday during the period of major organogenesis. REFERENCES DEACON, M. M., MURRAY, J. S., PILNY, M. K., RAO. K. S., DITTENBER, D. A., HANLEY, T. R., JR., AND JOHN, J. A. (1980). Embryotoxicity and fetotoxicity of orally administered chlorpyrifos in mice. Toxicol. Appl. Pharmacol. 54, 31-40. SCHWETZ, B. A., LEONG, B. K. J., AND GEHRING, P. J. (1974). Em-bryo- and fetotoxicity of inhaled carbon tetrachloride. I. l-dichloroethane and methyl ethyl ketone. Toxicol. Appl. Pharmacol. 28.452-464.
M.M.DEACON
M. D. PILNY J. A. JOHN B. A. SCHWETZ F. J. MLJRRAY~ H. 0. YAKEL R. A. KUNA” Toxicology Research Laboratory Health and Environmental Sciences Dow Chemical U.S.A. Midland. Michigan 48640 Received October 28, 1980 5 Present address: Syntex Corporation, 3401 Hillview Avenue, AZ-168, Palo Alto, Calif. 94304. 6 Exxon Corporation, Research and Environmental Health Division, P.O. Box 45, Linden, N.J. 07036
AND
APPLIED
PHARMACOLOGY
59,62&622
(1981)
Embryo- and Fetotoxicity of Inhaled Methyl Ethyl Ketone in Rats1
Embryoand Fetotoxicity of Inhaled Methyl Ethyl Ketone in Rats. DEACON, M. M., PILNY, M. D., JOHN, J. A., SCHWETZ, B. A., MURRAY, F. J., YAKEL, H. O., AND KUNA, R. A. (1981). Toxicol. Appl. Pharmacol. 59,62&622. Pregnant Sprague-Dawley rats inhaled 0, 400, 1000, or 3000 ppm methyl ethyl ketone (MEK) for 7 hr/day on Days 6 through 15 of gestation. Maternal toxicity, as evidenced by decreased weight gain and increased water consumption, was observed among rats exposed to 3000 ppm MEK. Slight fetotoxicity was observed among litters of rats exposed to 3000 ppm MEK as evidenced by an increased incidence of two minor skeletal variants. The results of this study duplicate the increased incidence of skeletal variants observed in an earlier study by B. A. Schwetz, B. K. J. Leong, and P. J. Gehring (1974, Toxicol. Appl. Pharmacol. 28, 452464), and do not indicate that inhaled MEK causes either an embryotoxic or teratogenic response in rats at exposure levels up to 3000 ppm.
Methyl ethyl ketone (MEK) is an extensively used, nonchlorinated solvent. The major route of human industrial exposure to MEK is by inhalation of vapors and by skin contact with the liquid. The current threshold limit value (TLV) of MEK is 200 wm. MEK (1000 or 3000 ppm) inhaled by pregnant rats for 7 hr/day on Days 6 through 15 of gestation was shown to be embryotoxic, fetotoxic, and potentially teratogenic (Schwetz et al., 1974). Exposure to 3000 ppm MEK resulted in a low incidence of acaudia, imperforate anus, and brachygnathia and an increased incidence of soft-tissue alterations and sternebral variants. Exposure to 1000 ppm MEK resulted in an increase in the total number of litters containing fetuses with skeletal alterations. No significant maternal toxicity was observed. The incidence of major mal’ Sponsored Ethyl Ketone
by the Inter-Industrial Teratology Committee.
0041-008X/81/090620-03$02.00/0 Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
Ad hoc
formations was not sufficiently high to support a positive teratogenic conclusion. This second teratologic study in rats was conducted to determine the repeatability of the fetotoxic effects observed in rats exposed to 3000 ppm MEK; therefore, additional groups of pregnant rats were exposed to 400, 1000, or 3000 ppm MEK for 7 hr/day on Days 6 through 15 of gestation. METHODS Test material. Reagent grade methyl ethyl ketone.’ 99.6% purity, was used for this study. Animals. Adult Sprague-Dawley rats,” weighing 234-332 g, were used. The day on which sperm were found in the vaginal smear was considered Day 0 of pregnancy. The animals were uniquely identified by a numbered metal ear tag. The rats were individually housed in wire-bottom cages and were fed commercial laboratory chow4 and tap water ad libitum, except dur’ Lot No. 36799. Exxon Chemical Co., Linden, 3 Spartan Research Animals, Haslett, Mich. 4 Ralston Purina Company, St. Louis, MO.
Methyl
620
N.J.
SHORT TABLE INCIDENCE
OF
FETAL
INHALING
1
ALTERATIONS
METHYL
AMONG
ETHYL Methyl 0
RATS
KETONE ethyl ketone 400
(ppmp
loo0
3ooo
No. fetuses examined/ No. litters examined External and skeletal examination Soft-tissue examination Bones of the skull
329126 1 I1126 218126
238119 80119 157119
226119 78119 148119
No. fetuses (litters) External examination Multiple defects Rotated and adducted forepaw Soft-tissue examination lnnommate artery missing Skeletal examination Skull. delayed ossification6 Ribs. extm’ Cervical centra. delayed ossitication” Total
major malformations
231/18 79118 150/18
affected
0
0
IllY
0
l(l)
0
0
0
0
l(1)
l(l)
2% 14)
1%10)
2w
23W
0
0
6t4)’ 7(6)’
8X22)
58115)
44( 16)
l(1)
III)
l(l)
l(lY
621
COMMUNICATIONS
114(18) 2I2)
” Rats inhaled 0, 400, 1000. or 3000 ppm of methyl ethyl ketone from Days 6 to 15 of gestation. b This fetus exhibited: acaudia, impetforate anus, short trunk. ectopic ovary. diaphragmatic hernia. and multiple vertebral defects, including missing ribs. vertebrae, and centra. r This fetus exhibited: acaudia, imp&orate anus, short trunk. ectopic ovary. and multiple skeletal defects including asymmetric pelvis, fused ribs, and missing ribs. vertebrae, and centra. d This alteration was considered to be a skeletal variant and was not included in the calculation of the total malformed fetuses. ’ Significantly different from control value by a modified Wilcoxon test. p < 0 05.
ing chamber exposures. Food and water consumption was measured at intervals of 3 days during the experimental period. Exprrimental design. Groups of 25 bred rats were exposed to 400, 1000, or 3000 ppm MEK by inhalation for 7 hrlday on Days 6 through 15 of gestation. A group of 35 bred rats was exposed to filtered room air in an identical chamber to serve as the control group. The exposures were conducted under dynamic aitlIow conditions in glass and stainless-steel chambers with a volume of 0.16 m3. The chamber atmosphere was generated by metering MEK at a calculated rate with a precision syringe pump into a vaporization flask and then into the airstream being drawn into the exposure chamber. Nominal concentrations were calculated as the ratio of the amount of MEK used to the total chamber airflow. The concentrations of MEK in each chamber were monitored 15 min/hr using a Miran I variable filter infrared analyzer at a wavelength of 8.5 Frn. Ana-
lytical concentrations (time-weighted mean) of MEK in the chambers for the 400-, lOOO-, and 3000-ppm exposures were 412 5 31, 1002 2 16, and 3005 2 49 ppm (mean 2 SD), respectively. Maternal and fetal observations. Animals were observed daily beginning on Day 6 of gestation for indications of toxicity. The body weights of bred rats were recorded on Days 6, 8, 10, and 16 of gestation. In addition, maternal body weights and the weight of the maternal livers were recorded at the time of cesarean section, Day 21 of gestation. The method of cesarean section as well as fetal observations and the statistical evaluations employed are described in a previous paper by Deacon et al. (1980).
RESULTS Muternal observations. Exposure of bred rats to 400, 1000, or 3000 ppm of MEK for 7 hr/day had no significant effect on the dam’s appearance or demeanor. Maternal toxicity, as evidenced by decreased weight gain and increased water consumption was observed among rats exposed to 3000 ppm of MEK. No changes were observed in maternal liver weight, or in the food consumption of pregnant rats exposed by inhalation to MEK. Embryo- and fetotoxicity. Inhalation of 400, 1000, or 3000 ppm MEK had no significant effect on the incidence of pregnancy, or on the average number of implantations and live fetuses per dam. The incidence of resorptions was comparable to controls for all exposure levels. No adverse effects on the fetal body weight or crown-rump length were noted among litters of rats exposed to MEK. No external or soft-tissue alterations were observed among fetuses at any of the exposure levels at an incidence which was statistically significantly different from the control incidence (Table 1). When considered collectively no significant increase in the incidence of major malformations in fetuses exposed to MEK was observed. A significant decrease in the incidence of delayed ossification of interparietal bones of the skull was noted among litters of rats
622
SHORT
COMMUNICATIONS
exposed to 3000 ppm of MEK. A significant increase in the incidence of extra lumbar ribs and in the occurrence of delayed ossification of cervical centra was also noted at this exposure level. These skeletal variants have been seen at low incidences among the historical control population in this laboratory, and are therefore considered indicative of a slight fetotoxic effect.
DISCUSSION The results of an earlier teratology study with MEK conducted in this laboratory (Schwetz et al., 1974) were suggestive of a potential teratogenic effect at 3000 ppm MEK. Skeletal alterations were observed among litters of the exposed rats, but the incidence of major malformations was not sufficiently high to support a positive conclusion. In the present study, a teratogenic effect (as defined by an increase in major malformations) was not observed in rats inhaling 400, 1000, or 3000 ppm of MEK. That is, exposure of pregnant rats to MEK vapor did not induce the occurrence of major external, soft-tissue, or skeletal malformations which would severely affect the survival or performance of the young. The few malformations which were observed among litters of rats exposed to MEK were types that have been noted to occur historically at a low incidence among control rats in this laboratory. Some evidence of slight fetotoxicity, as evidenced by an increased incidence of a minor skeletal variant and a delay in the ossification of fetal bones, was observed among litters of rats exposed to 3000 ppm of MEK. Embryotoxic effects, such as an increased incidences of resorptions or preimplantation loss, were not observed among rats exposed to 400, 1000, or 3000
ppm of MEK. Thus, the results of this study duplicate the increased incidence of skeletal variants observed in the earlier study by Schwetz et al. (1974), but do not indicate either an embryotoxic or teratogenie response in rats. In summary, exposure to 3000 ppm of MEK, a slightly maternally toxic level, resulted in some minor variations in the development of the fetal skeleton in rats, a slight fetotoxic effect. Neither significant embryotoxicity nor a teratogenic effect was discerned in rats inhaling up to 3000 ppm MEK for 7 hrlday during the period of major organogenesis. REFERENCES DEACON, M. M., MURRAY, J. S., PILNY, M. K., RAO. K. S., DITTENBER, D. A., HANLEY, T. R., JR., AND JOHN, J. A. (1980). Embryotoxicity and fetotoxicity of orally administered chlorpyrifos in mice. Toxicol. Appl. Pharmacol. 54, 31-40. SCHWETZ, B. A., LEONG, B. K. J., AND GEHRING, P. J. (1974). Em-bryo- and fetotoxicity of inhaled carbon tetrachloride. I. l-dichloroethane and methyl ethyl ketone. Toxicol. Appl. Pharmacol. 28.452-464.
M.M.DEACON
M. D. PILNY J. A. JOHN B. A. SCHWETZ F. J. MLJRRAY~ H. 0. YAKEL R. A. KUNA” Toxicology Research Laboratory Health and Environmental Sciences Dow Chemical U.S.A. Midland. Michigan 48640 Received October 28, 1980 5 Present address: Syntex Corporation, 3401 Hillview Avenue, AZ-168, Palo Alto, Calif. 94304. 6 Exxon Corporation, Research and Environmental Health Division, P.O. Box 45, Linden, N.J. 07036