DRUG AND CHEMICAL TOXICOLOGY, 15(1),
1-14 (1992)
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DEVELOPMENTAL TOXICITY OF DIMETHYLFORMAMIDE IN THE RAT FOLLOWING INHALATION EXPOSURE Steven C. Lewis,> Raymond E. Schroeder,2 and Gerald L. Kennedy,Jr.’ 1ExxOn Corporation, Linden, NJ 07036 ZBiodynamics, Inc., East Millstone, NJ 08873 ’Haskell Laboratory, E.I. du Pont de Nemours and Co.,Inc., Newark, DE 19714
ABSTRACT Dimethylformamide ( D E F ) is a widely used industrial solvent. DMF has been reported to be a developmental toxin when given to rodents by injection or following dermal administration. In this study, groups of pregnant rats were exposed by inhalation to either 0 (control), 3 0 , or 3 0 0 ppm DMF from gestation day 6 through 15. In the 3 0 0 ppm rats, both maternal weight gain during gestation and fetal weights were lower than those of the controls. Fetal resorptions were not increased in this group. No significant differences among either maternal or fetal rats were seen in the 3 0 pprn group compared to controls. Both fetal and maternal toxicity were noted at 300 ppm and the no observed effect level under these experimental conditions was 30 ppm for both the dams and the conceptuses. DMF did not produce malformations in the rat fetus even at a level that was toxic to the dam.
INTRODUCTION
N,N-dirnethylformamide (DMF; Cas Reg. No. 68-12-2) is a versatile industrial solvent with wide organic and inorganic solubility, water miscibility, high boiling and low freezing
Corresponding Author:
Gerald L. Kennedy, JK. Raskell Laboratory E. I. du Pont de Nemours and Co., Inc. P. 0. Box 50 Elkton Road Newark, DE 19714 1
Copyright 0 1992 by Marcel Dekker, Inc.
2
LEUIS, SCHROEDER, AND KENNEDY
point, and good stability.
It is used in the manufacture of
films and fibers and as a booster solvent in coatings and adhesive formulations.
DMF is useful for dissolving
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polyacrylonitrile, polyvinyl chloride, polyamides, polyimides, cellulose derivatives, styrenes, and linear polyesters. The acute toxicity of DMF is quite low with an oral LD50 in rats of 2.0 to 2.2 g/kgl,Z a dermal LD50 in rabbits from 1 to greater than 5 g/kgl*3.4 and an inhalation 4 or 6 hour LC50 of greater than 2,500 ppm.3.5
DMF fed in the diet to cats (215 to
2500 ppm) and mice (160 to 185 ppm) for over 100 days produced increased liver weights without microscopic pathologic changes.6 Other studies in rodents have produced both liver weight increases and liver pathology.l.5#7.*
Rats and mice were exposed
to atmospheres of DIP 6 hr/day, 5 days/week, for 13 weeks9 with the liver being the first indicator of DMF-induced toxicity. Developmental toxicity studies following various routes of administration to various species have been conducted.
DMF given
as a single oral dose of 2 g/kg to rats on gestation day 7 produced little effect.10
The same investigator found n o increase
in fetal resorption or terata using intra-peritoneal doses of
2 g/kg delivered on gestation day 7 or 11.11 were seen in rats when 0.6g/kg gestation days 10 and 11.
No fetal effects
D M F was applied dermally on
Doses of either 1.2 or 2.4 g/kg on the
same days produced an increase in embryomortality.
Doses from
0.6 to 2.4 g/kg on gestation days 11 and 12 produced an increase
in subcutaneous hemorrhage. g/kg
Rabbits treated dermally with 0.2
from days 8 through 16 showed complete fetal resorption.>*
Rabbits given oral doses of 0 . 0 4 4 g/kg from days 6 through 18 showed no adverse maternal or fetal effects.
Fetal weights were
reduced, 3 hydrocephalic fetures were observed, and no maternal weight effects were seen at 0.064 g/kg.
Maternal toxicity
without mortality was seen at 0.19 g/kg, fetal weights were reduced, and 16 progeny from 7 litters were malformed.13
No
3
DEVELOPMENTAL TOXICITY OF DIMETHYLFORWIDE evidence of either embryomortality or teratogenicity was seen in rats inhaling either 18 or 172 ppm, 6 hrs/day from gestation day 6
through 15 .I4
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Malformations were reported in mice and rats following oral administration of 182 mg/kg and 166 mg/kg
respectively.
administration of from 9 4
-
rabbits produced terata.
Fetal weights and lengths were
944
Dermal
mg/kg to rats and 4 0 0 mg/kg to
decreased and early resorptions were increased in rats following inhalation o f 287 2 5 0 ppm.
Rabbit fetuses were generally
unaffected following inhalation of up to 150 ppm but teratogenic effects were seen at 4 5 0 ppm.15
Body weights and the pregnancy
rates were reduced in rats given dermal doses of 2 ml/kg.
Fetal
numbers and fetal weights were also reduced at this dose. Similar, but less pronounced effects, were seen with a dose of 1 ml/kg and no changes were seen at 0 . 2 5 ml/kg.
No increase in
malformations was seen in the DMF-treated fetuses.16 In view of the inconsistent findings regarding the developmental toxicity of DMF and the importance of determining its potential to be damaging following inhalation exposures, the study reported here was conducted to examine both the maternal and fetal responses to DMF using both a different rat strain and a higher dose than was investigated in a previous negative study . l a
METHODS Test Material The test material was DMF, a colorless liquid with a boiling point of 1 4 9 O C .
The sample tested was obtained from the
Exxon Corporation Research and Environmental Health Division, Linden, New Jersey.
Based on chemical reactivity, the material
was assumed to be stable throughout the experimental period.
LEWIS, SCHROEDER, AND KENNEDY
4
Animal s Female Cr1:CD rats, 12 to 14 weeks of age (nulliparous) were received from Charles River Breeding Laboratories, Newfield, New Jersey.
Male rats of the same strain were used for breeding.
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Rats were paired 1:l
and mating was verified by the finding of
sperm in the vaginal smear following overnight cohabitation: the day sperm was noted designated Day 0 of gestation.
The dams were
distributed into control and test groups in rotation by weight such that the mean initial body weight among groups was similar. Each dam was housed individually in suspended, stainlesssteel wire-mesh cages. maintained.
A 12/12 hour light/dark cycle was
Feed (Purina Unmedicated Pellets@ Ralston Purina
Company, St. Louis,
no)
and water were provided ad libitum except
during the actual exposure periods.
Experimental Design Groups of 21 pregnant (mated) rats each were tested by exposure to either 0 (control), 30, or 300 ppm DMF.
Rats were
exposed from gestation day 6 through 15 (10 exposures) by whole-body inhalation for 6 hours per day.
Control rats were
exposed to air only.
Inhalation Exposure Exposures were conducted in stainless steel and glass chambers (Rochester-type) with an effective volume of 760 L.
DMP
was generated by passing a stream of dry air through bubblers containing DMP.
The air flow rate through the bubbler was
monitored with a spherical-float rotameter and was calibrated to volatilize the appropriate amount of test substance.
The
vapor-air mixture was diluted to the appropriate concentration with room air prior t o entering the exposure chamber.
DMF
chamber air flow rates were adjusted to maintain the intended concentrations.
The air flow rates for the 30 ppm group ranged
5
DEVELOPMENTAL TOXICITY OF DIMETHYLFORMAHIDE
from 127 to 380 L/minute; 447 L/minute.
for the 300 ppm chamber from 123 to
Flow'rates for the control chamber were maintained
at the high end of this range.
Six hours after the initiation of
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each daily exposure, the bubblers were turned off, the chambers were allowed to clear with room air ( 3 0 minutes), and rats were returned to their holding cages. DMF concentrations were measured by I R analysis (Miran I e Variable Filter Infrared Analyzer Model I, Wilkes Scientific Corporation, South Norwalk, Connecticut) at 4 intervals during each exposure day.
Parameters Measured The dams were weighed on days 0, 6 through 15, and 21 of gestation.
Clinical signs were observed daily.
On day 21 of
gestation, dams were sacrificed by ether overdose. pathologic examination was made.
A gross
The ovaries and uterus were
removed, the uterus was opened, and the fetuses were removed and examined.
The number of corpora lutea were counted and the
number and position of all live, dead, and resorbed fetuses were recorded. All fetuses were tagged individually for identification, and then weighed, measured crown-to-rump, malformations.
sexed, and examined for
Approximately 2/3 of the fetuses from each litter
w e r e examined for visceral alterations.17
These fetuses were
then eviscerated and placed immediately into aqueous potassium hydroxide solution.
Following staining with alizarin red, the
fetuses were placed in Mall's
solution to remove excess stain
prior to clearing and storing in a benzyl alcohol/glycerine solution.
The skeletons were examined f o r structural
integrity.10 Bouin's
Approximately 1/3 o f the fetuses were fixed in
solution and were examined under 10-20X magnification for
neural and visceral defects using the serial sectioning technique of Wilson.19
LEVIS, SCHROEDER, AND KENNEDY
6
Statistical Evaluations Comparisons between control and DMF-treated groups, were made, where applicable (incidence data), by the chi-square
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method.
Body weights, body weight gains, numbers of corpora
lutea, implantations of the dams, number of fetuses per sex, fetal and litter weights, crown-rump distances, and the number of ossification variations/fetus/litter the F-test and Student's t-test.20 significantly, Student's Cochran's
were compared to control by When variances differed
t-test was appropriately modified using
approximation (t').
Mean reproduction data were
compared to control by the one-tailed t-test.
The level o f
statistical significance was p < 0.05.
RESULTS Chambers Conditions The mean exposure concentrations for this study were 0, 31.2 5 4.6, and 291 5 22.
Since the actual concentrations were
close to the design, this report will use the design concentrations (30 and 300 ppm) throughout.
Maternal Responses All female rats survived the testing period.
No adverse
clinical signs were noted in the DMF exposed rats and outward behavior of these animals was not different than that of the controls.
There was no evidence of an effect of DMF on the
general physical condition of the dams in either of the test groups. Weight gains for dams exposed to 30 ppm compared favorably to those of the control group (Table 1).
The weight gain for
rats exposed to 300 ppm during the exposure period was significantly lower than that for the control group.
The weight
gain from Day 6 through 21 in this group was not significantly
DEVELOPMENTAL TOXICITY OF DIMETHYLFORMAMIDE TABLE 1
BODY WEIGtI'lS OF PEHALE RATS
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INHALING DHF DURING GESTATION
Test Conc
.
Body Weight (g)*
GD:*
6
9
~~~~
Weight Gain (9)
12 ~
15
21
6-9
6-15
6-21
~
0
267(15)
278(15)
296(17)
318(17)
432(29)
ll(1)
50(7)
165(12)
30
266(16)
274(16)
289(19)
311(21)
412(41)
8(1)
45(9)
148(17)
300
268(15)
273(17)
287(19)
307(20)
419(28)
39b(8)
151(12)
5(1)
'Numbers in parenthesis represent standard deviation bp =
*GD
=