DRUG AND CHEMICAL TOXICOLOGY, 1 4 ( 1 & 2 ) , 173-184 ( 1 9 9 1 )
A NON-INVASIVEMONITORING OF EXPOSURE TO AN INDUSTRIAL ORGANIC SOLVENT, DIMETHYLFORMAMIDE Ming J. W. Chang, Ph.D. Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
Department of Public Health, Chang Gung Medical College, Tao-Y uan, Taiwan and Ruey S. Lin, M.D., Dr P.H. Institute of Public Health, National Taiwan University, Taipei, Taiwan
ABSTRACT A urinary metabolite, N- hydroxymethyl- N- methyl
formamide (DMF-OH) was measured by a colorimetric method in Wistar and Sprague-Dawley rat urine after a single exposure to N, N- dimethyl formamide (DMF).
A linear relationship was
found between the total excretion of DMF-OH in 2 days versus the exposure ranging from 1 to 20% of the LD50 of DMF i.e. 47.2 to 944 mg DMF per kg b. wt. This is proposed to be a non-
invasive biological method for monitoring exposure to DMF. 173 Copyright @ 1991 by Marcel Dekker, Inc.
CHANG AND LIN
174
INTRODUCTION N, N- dimethylformamide (DMF) is a widely used industrial organic solvent for organic and inorganic chemicals. highly volatile and penetrates skin easily.
It is
The vapor
irritates
skin, eyes, and mucous membranes. An accidental dermal and Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
respiratory exposure to DMF was reported to produce severe abdominal pain, hypertension, leucocytosis, and hepatic damage.’
Liver injury was produced in experimental animals
by prolonged inhalation of DMF.zI3
Epidemiological data also
associated liver injury found in synthetic leather and textile workers with exposure to DMF.4,5
The textile and synthetic
leather industries are playing a significant role in the economy of developing countries such as Taiwan, Korea, Indonesia, and Thailand.
In order to protect workers and minimize
unnecessary economical loss, a rapid, sensitive, and noninvasive method for early detection of an exposure to DMF is highly desirable.
The current threshold limit value for a time
weighted average (TLV-TWA) is 30 mg DMF per cubic meter air for both Taiwan and the United States. Assuming 20 cubic meter air is taken daily by an adult man, this would transform the TLV of DMF to 600 rnglday. DMF is rapidly metabolized jn vivo to N- hydroxymethyl-
N-
methyl formamide (DMF-OH) by rat and man.6 Previously, DMF-
OH was not detected by gas chromatography in metabolism studies of DMF.798 This was due to the thermal decomposition
175
EXPOSURE TO DIMETHYLFORMAMIDL
of DMF-OH to N- methyl formamide (NMF) at the injection port.9 Nevertheless, a measurement of NMF in urine was utilized by a few studies as a way to assess workers’ exposure to D M F . ~ J O J ~ A simple analytical method was designed by Scailteur et
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
al.9 to measure DMF-OH in rat urine.
Under alkaline conditions,
0.1 N sodium hydroxide, DMF-OH was stoichiometrically
hydrolyzed to formaldehyde which was then determined by a classical colorimetric method developed by Nash.12 By using this simple analytical method, a linear relationship between the exposure of DMF, ranging from 47.2 to 944 mg/kg, and the total urine excretion of DMF-OH in rat was found in this study. Therefore, determination of urinary DMF-OH by the colorimetric assay of the alkaline hydrolyzed urine is proposed to be an alternative sensitive and non-invasive biological monitoring of exposure to DMF. MATERIALS AND METHODS Female Wistar and Sprague-Dawley rats were used. The animals were housed five per cage and provided with tap water and rat chow ad libitum.
The light cycle of the animal room
was kept from 6 am to 6 pm. Rats were trained one per cage in the metabolic cages prior to the intraperitoneal (ip) administration of DMF which was purchased from Merck Go. (Darmstadt, F.R.G.).
Three rats per strain per dose group were
176
CHANG AND LIN
used.
Each rat was injected once with 1.0-, 0.5,and 0.1-mL
DMF per kilogram body weight or 944-, 472-, and 94.4 mg/kg b.wt., respectively.
A fourth group of Wistar rats injected
with DMF at 0.05 mUkg b.wt. (or 47.2 mg/kg b.wt.) was also included at the end of the study. Saline was used to dilute DMF
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
to give a final injection volume of 1 mUkg b.wt.
Each 24-hour
urine sample was collected in a glass vessel at room temperature for 2 days and stored in polypropylene Eppendorf tubes at -20°C or lower. The volume of each urine sample was recorded. DMF-OH was determined by the method of Scailteur et al.9 in the thawed and clarified (by a centrifugation of 10,000 x g for 20 min at 4°C) urine supernatant.
Essentially, an aliquot
(0.1 or 0.2 mL) of rat urine was first diluted 1 to 10 or 1 to 5 (when the total volume of a day's urine was more than 15 mL) with deionized water.
Then, two 0.4 mL diluted rat urine
samples were transferred to separate test tubes, and 1.6 mL of a 0.125 N sodium hydroxide solution was added and mixed into one and 1.6 mL deionized water into the other. Each sample was then reacted with two m l of an aqueous reagent containing 2 M ammonium acetate (Merck), 0.05 M acetic acid (Merck), and 0.02 M acetylacetone (Merck) and incubated at 37°C for 40 min. The optical density (absorbance) at 412 nm was then determined for each sample,
The difference in
optical density between the alkalinized and non-alkalinized
177
EXPOSURE T O DIMETHYLFORMAMIDE
(representing the endogenous formaldehyde) was calculated and used to compute the quantity of formaldehyde derived from DMF-OH. A working standard curve of 2-, 4-, 8-, and 12 pg formaldehyde per mL, which was equivalent to 5.9 to 35.6 pg DMF-OH per mL, was included in every batch of the colorimetric determination.
The percent relative error of each
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
standard was kept at no greater than
10 percent.
RESULTS AND DISCUSSION
In the begining of this study, it was noted that DMF-OH
determined by means of assaying the alkaline released formaldehyde in the thawed rat urine decayed to a significant degree after one day storage in a refrigerator.
An
investigation on this assumed stability of DMF-OH was carried out in six randomly selected frozen rat urine samples. Immediately after thawing and clarification, part of each clear supernatant was analyzed for alkaline released formaldehyde and part of it was sealed and stored at 4°C.
One day later, the
stored samples were again analyzed for alkaline released formaldehyde.
The results are shown in Table 1.
Assuming all
alkaline released formaldehyde was derived from DMF-OH, it was noted that at 4°C about half of the DMF-OH was gone in 28 hours.
No significant increase of the endogenous
formaldehyde measured by the optical density at 412 nm of the
CHANG AND LIN
I 7a
TABLE 1 Percent Loss of Urinary DMF-OH Kept at 4°C for 28 Hours
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
Initial concn (mg/mL)
Final concn (mg/mL)
Percent loss
Yo
........................................................ 0.68 0.29 57 0.14 0.05 64 0.84 0.45 46 0.64 0.34 47 0.15 0.07 53 (Mean L SD) 53 L 7
non-alkalinized aliquot was observed after storage. Thereafter, no extra effort was taken to find means to prevent the decay, except that all measurements of DMF-OH in this study was consistently performed on the newly thawed urine samples. The current sampling time recommended by ACGlH (American Conference of Governmental Industrial Hygienist) for industrial settings is at the end of shift.13 We recommend that the collected samples should be kept frozen till analysis. Table 2 summarizes the total excretions of DMF-OH, measured as alkaline released formaldehyde, in 2 days following an ip administration of various doses of DMF to female Wistar and Sprague-Dawley rats. Although DMF-OH was not detected in control rat urine as well as the urine of rats
179
EXPOSURE TO DIMETHYLFORMAMIDE
TABLE 2 Urinary DMF-OH Measured After a Single Dose of DMF ~
Day of Urine Samples DMF-OH (mg/24 h) Collected ........................................................ I. Wistar Rats
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
Dose (mglkg)
944
1 2 total
12.5 11.7 24.2
14.0 5.4 19.4
13.9 10.9 24.8
472
1 2 total
7.2 0.6 7.8
5.4 3.7 9.1
8.9 2.1 11.0
94.4
1 2 total
2.6 0.3 2.9
3.7 0.1 3.8
2.7 0.1 2.8
47.2
1 2 total
0.5 0.0 0.5
0.4 0.1 0.5
0.7 0.0 0.7
5.5
II. Sprague-Dawley Rats
944
1
2 total
19.3 24.8
5.4 14.7 20.1
7.3 23.6 30.9
472
1 2 total
5.7 6.9 12.6
4.0 4.9 8.9
5.1 5.9 11.0
94.4
1 2 total
5.3 0.1 5.4
1.5 0.0 1.5
3.4 0.0 3.4
180
CHANG AND L I N
treated with other chemical such as trichloroethylene and benzo(a)pyrene (data not shown), occasional background readings of about 3 to 5 pg formaldehyde per mL urine (after a correction for dilution) were observed at a frequency of less than 5% of the total determinations.
All of the data collected
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
in Table 1 and 2 were determined according to a standard operating procedure which dictated a total dilution of 1 to 25 or 50 depending on the total volume of a 24-hour urine sample.
If the volume of a 24-hour urine sample was less than 15 mL, the dilution factor was 50; otherwise, the dilution factor was 25.
The individual variation in the metabolism of DMF is clearly demonstrated in Table 2.
However, a linear relationship was
obtained between the total excretion of DMF-OH in two days and the dose given for both strains of rat.
The correlation
coefficient of both curves was greater than 0.9.
Also, when
the regression equation was evaluated for all experimental rats (n = 21), the linear relationship remained. relationship is also shown in Figure 1.
This linear
It is estimated that on
an average of 200 gram body weight, a rat excreted about 10 mole percent DMF-OH in 48 hours measured by our method for the dose groups of 944, 472, and 94.4 mg/kg but only about 5 mole percent for the 47.2 mg/kg group.
Previously, Scailteur
et al. reported that when Sprague-Dawley rats were exposed to DMF at 1 mL/kg intraperitoneally, about 40 mole percent
181
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
EXPOSURE TO DIMETHYLFORMAMIDE
0
200
400
600
800
1000
DMF Dose (mg/kg)
FIGURE 1 Linear regression of rat urinary DMF-OH excreted in 2 days versus exposure dose.
urinary DMF-OH was measured in 2 days by both GC and colorimetric methods.9 The basis for using the analysis of urinary NMF by gas chromatography (GC) as the method to assess BE1 of DMF was mainly the thermal decomposition of DMF-OH to NMF at the injection port.
The sensitivity of a GC analysis largely
depends on the type of detector (NPD or FID) and the type of GC column (packed or capillary) used. The current ACGIH guidelines recommend a BE1 value for DMF of 40 mg NMF per gm creatinine.13 That is about 60 pg DMF-OH per mg creatinine or 20 pg formaldehyde per mg creatinine after alkalinization.
The normal value of creatinine in adult human
urine is about 0.5 to 1.0 mg/mL.
In our experience, a packed
CHANG AND LIN
182
Tenax GC column (2m x 2mm) coupled with a FID detection barely have the needed sensitivity to measure NMF at a concentration of approximately 40 to 80 pg/mL.
However, one
can make a one to five dilution in the proposed colorimetric determination of DMF-OH and still get an optical density of
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
about 0.500 to 0.550 at 412 nm for 4 pg formaldehyde per mL dilute urine. The assay protocol used in this investigation has a wide margin for enhancing its sensitivity. factor used was 25.
The lowest dilution
One can minimize the dilution in
alkalinizing the urine before its incubation with Nash reagent. It is estimated, with our lowest standard (i.e. 2 pg
formaldehyde per mL), that a 5% dilution of 1.9 mL urine with 0.1 mL 2N NaOH can easily assess about 6.2 pg DMF-OH per mL
which gives an optical density of -0.250 and is equivalent to about 4.1 pg NMF per mL. Although our determination of urinary DMF-OH seemed only measure 1/4 of what was determined by Scailteur et al., the proposed method can still determine a BE1 of 16.4 pg NMF per mL urine easily and accurately . In conclusion, we have demonstrated that a determination of DMF-OH in urine by utilizing a colorimetric assay to determine the alkaline released formaldehyde reflects adequately the exposure of rats to DMF ranging from 47.2 to 944 mg/kg b.wt. The method is sensitive enough to assess a
EXPOSURE TO DIMETHYLFORMAMIDE
183
BE1 of DMF at approximately one-half to one-fifth (depending on the concentration of urinary creatinine) of the current ACGIH recommended value i.e. 40 pg NMF per mg creatinine. WI propose that the method described in this report to be used as an alternative non-invasive method to assess BE1 of DMF, especially where a gas chromatography is not easily Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
accessab le. ACKNOWl FKGEMFNTS This work was partially supported by a grant from Chang Gung Medical College, CMRP 279 and a project from EPA/Taiwan, ROC, EPA 79-005-39-1 59.
The skillful technical
support of Ms. S. C. Yuan, S. F. Weng, and Y. C. Chien are acknowledged.
1. H. P. Potter, Arch. Envir. Health,
27, 340 (1973).
2. J. W. Clayton Jr., J. R. Barnes, D. 8. Hood and G. W. H. Schepers, Amer. Ind. Hyg. Assoc. J., 24, 144 (1963). 3. D. K. Craig, R. J. Weir, W. Wagner and D. Groth, Drug and Chem. Tox., Z, 551 (1984). 4. F. Tolot, M. Droin and M. Genevois, Arch. Mal. Prof.,
19,602
(1958). 5. J. D. Wang, M. Y . Lai, J. S. Chen, J. M. Lin, J. R. Chiang, S. J.
Shiau and W. S. Chang, Arch. Envir. Health (in press).
CHANG AND LIN
184
6. V. Scailteur and R. Lauwerys, Chem.-Biol. Interactions,
32,
327 (1984) 7. G. Kimmerle and A. Eben, Int. Arch. Arbeitsmed.,
a,109
(1975). 8. G. Kimmerle and A> Eben, Int. Arch. Arbeitsmed., 524, 127
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
(1975). 9. V. Scailteur, E. de Hoffmann, J.
P. Buchet and R. Lauwerys,
Toxicol., 29, 221 (1984). 10. R. Lauwerys, A. Kivits, M. Lhoir,
P. Rigolet, D. Houbean, J. P.
Buchet and H. A. Roels, Int. Arch. Occup. Environ. Health, 4.5, 189 (1980). 11. I. Yonemoto, Int. Arch. Occup. Environ. Health,
46,159
(1980). 12. T. Nash, Biochem. J., 55, 416 (1953). 13. American Conference of Governmental Industrial Hygienist
(ACGIH), "Threshold Limits Values and Biological Exposure Indices for 1989-1990," ACGIH, Cincinnati, Ohio, (1 989).
A NON-INVASIVEMONITORING OF EXPOSURE TO AN INDUSTRIAL ORGANIC SOLVENT, DIMETHYLFORMAMIDE Ming J. W. Chang, Ph.D. Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
Department of Public Health, Chang Gung Medical College, Tao-Y uan, Taiwan and Ruey S. Lin, M.D., Dr P.H. Institute of Public Health, National Taiwan University, Taipei, Taiwan
ABSTRACT A urinary metabolite, N- hydroxymethyl- N- methyl
formamide (DMF-OH) was measured by a colorimetric method in Wistar and Sprague-Dawley rat urine after a single exposure to N, N- dimethyl formamide (DMF).
A linear relationship was
found between the total excretion of DMF-OH in 2 days versus the exposure ranging from 1 to 20% of the LD50 of DMF i.e. 47.2 to 944 mg DMF per kg b. wt. This is proposed to be a non-
invasive biological method for monitoring exposure to DMF. 173 Copyright @ 1991 by Marcel Dekker, Inc.
CHANG AND LIN
174
INTRODUCTION N, N- dimethylformamide (DMF) is a widely used industrial organic solvent for organic and inorganic chemicals. highly volatile and penetrates skin easily.
It is
The vapor
irritates
skin, eyes, and mucous membranes. An accidental dermal and Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
respiratory exposure to DMF was reported to produce severe abdominal pain, hypertension, leucocytosis, and hepatic damage.’
Liver injury was produced in experimental animals
by prolonged inhalation of DMF.zI3
Epidemiological data also
associated liver injury found in synthetic leather and textile workers with exposure to DMF.4,5
The textile and synthetic
leather industries are playing a significant role in the economy of developing countries such as Taiwan, Korea, Indonesia, and Thailand.
In order to protect workers and minimize
unnecessary economical loss, a rapid, sensitive, and noninvasive method for early detection of an exposure to DMF is highly desirable.
The current threshold limit value for a time
weighted average (TLV-TWA) is 30 mg DMF per cubic meter air for both Taiwan and the United States. Assuming 20 cubic meter air is taken daily by an adult man, this would transform the TLV of DMF to 600 rnglday. DMF is rapidly metabolized jn vivo to N- hydroxymethyl-
N-
methyl formamide (DMF-OH) by rat and man.6 Previously, DMF-
OH was not detected by gas chromatography in metabolism studies of DMF.798 This was due to the thermal decomposition
175
EXPOSURE TO DIMETHYLFORMAMIDL
of DMF-OH to N- methyl formamide (NMF) at the injection port.9 Nevertheless, a measurement of NMF in urine was utilized by a few studies as a way to assess workers’ exposure to D M F . ~ J O J ~ A simple analytical method was designed by Scailteur et
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
al.9 to measure DMF-OH in rat urine.
Under alkaline conditions,
0.1 N sodium hydroxide, DMF-OH was stoichiometrically
hydrolyzed to formaldehyde which was then determined by a classical colorimetric method developed by Nash.12 By using this simple analytical method, a linear relationship between the exposure of DMF, ranging from 47.2 to 944 mg/kg, and the total urine excretion of DMF-OH in rat was found in this study. Therefore, determination of urinary DMF-OH by the colorimetric assay of the alkaline hydrolyzed urine is proposed to be an alternative sensitive and non-invasive biological monitoring of exposure to DMF. MATERIALS AND METHODS Female Wistar and Sprague-Dawley rats were used. The animals were housed five per cage and provided with tap water and rat chow ad libitum.
The light cycle of the animal room
was kept from 6 am to 6 pm. Rats were trained one per cage in the metabolic cages prior to the intraperitoneal (ip) administration of DMF which was purchased from Merck Go. (Darmstadt, F.R.G.).
Three rats per strain per dose group were
176
CHANG AND LIN
used.
Each rat was injected once with 1.0-, 0.5,and 0.1-mL
DMF per kilogram body weight or 944-, 472-, and 94.4 mg/kg b.wt., respectively.
A fourth group of Wistar rats injected
with DMF at 0.05 mUkg b.wt. (or 47.2 mg/kg b.wt.) was also included at the end of the study. Saline was used to dilute DMF
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
to give a final injection volume of 1 mUkg b.wt.
Each 24-hour
urine sample was collected in a glass vessel at room temperature for 2 days and stored in polypropylene Eppendorf tubes at -20°C or lower. The volume of each urine sample was recorded. DMF-OH was determined by the method of Scailteur et al.9 in the thawed and clarified (by a centrifugation of 10,000 x g for 20 min at 4°C) urine supernatant.
Essentially, an aliquot
(0.1 or 0.2 mL) of rat urine was first diluted 1 to 10 or 1 to 5 (when the total volume of a day's urine was more than 15 mL) with deionized water.
Then, two 0.4 mL diluted rat urine
samples were transferred to separate test tubes, and 1.6 mL of a 0.125 N sodium hydroxide solution was added and mixed into one and 1.6 mL deionized water into the other. Each sample was then reacted with two m l of an aqueous reagent containing 2 M ammonium acetate (Merck), 0.05 M acetic acid (Merck), and 0.02 M acetylacetone (Merck) and incubated at 37°C for 40 min. The optical density (absorbance) at 412 nm was then determined for each sample,
The difference in
optical density between the alkalinized and non-alkalinized
177
EXPOSURE T O DIMETHYLFORMAMIDE
(representing the endogenous formaldehyde) was calculated and used to compute the quantity of formaldehyde derived from DMF-OH. A working standard curve of 2-, 4-, 8-, and 12 pg formaldehyde per mL, which was equivalent to 5.9 to 35.6 pg DMF-OH per mL, was included in every batch of the colorimetric determination.
The percent relative error of each
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
standard was kept at no greater than
10 percent.
RESULTS AND DISCUSSION
In the begining of this study, it was noted that DMF-OH
determined by means of assaying the alkaline released formaldehyde in the thawed rat urine decayed to a significant degree after one day storage in a refrigerator.
An
investigation on this assumed stability of DMF-OH was carried out in six randomly selected frozen rat urine samples. Immediately after thawing and clarification, part of each clear supernatant was analyzed for alkaline released formaldehyde and part of it was sealed and stored at 4°C.
One day later, the
stored samples were again analyzed for alkaline released formaldehyde.
The results are shown in Table 1.
Assuming all
alkaline released formaldehyde was derived from DMF-OH, it was noted that at 4°C about half of the DMF-OH was gone in 28 hours.
No significant increase of the endogenous
formaldehyde measured by the optical density at 412 nm of the
CHANG AND LIN
I 7a
TABLE 1 Percent Loss of Urinary DMF-OH Kept at 4°C for 28 Hours
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
Initial concn (mg/mL)
Final concn (mg/mL)
Percent loss
Yo
........................................................ 0.68 0.29 57 0.14 0.05 64 0.84 0.45 46 0.64 0.34 47 0.15 0.07 53 (Mean L SD) 53 L 7
non-alkalinized aliquot was observed after storage. Thereafter, no extra effort was taken to find means to prevent the decay, except that all measurements of DMF-OH in this study was consistently performed on the newly thawed urine samples. The current sampling time recommended by ACGlH (American Conference of Governmental Industrial Hygienist) for industrial settings is at the end of shift.13 We recommend that the collected samples should be kept frozen till analysis. Table 2 summarizes the total excretions of DMF-OH, measured as alkaline released formaldehyde, in 2 days following an ip administration of various doses of DMF to female Wistar and Sprague-Dawley rats. Although DMF-OH was not detected in control rat urine as well as the urine of rats
179
EXPOSURE TO DIMETHYLFORMAMIDE
TABLE 2 Urinary DMF-OH Measured After a Single Dose of DMF ~
Day of Urine Samples DMF-OH (mg/24 h) Collected ........................................................ I. Wistar Rats
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
Dose (mglkg)
944
1 2 total
12.5 11.7 24.2
14.0 5.4 19.4
13.9 10.9 24.8
472
1 2 total
7.2 0.6 7.8
5.4 3.7 9.1
8.9 2.1 11.0
94.4
1 2 total
2.6 0.3 2.9
3.7 0.1 3.8
2.7 0.1 2.8
47.2
1 2 total
0.5 0.0 0.5
0.4 0.1 0.5
0.7 0.0 0.7
5.5
II. Sprague-Dawley Rats
944
1
2 total
19.3 24.8
5.4 14.7 20.1
7.3 23.6 30.9
472
1 2 total
5.7 6.9 12.6
4.0 4.9 8.9
5.1 5.9 11.0
94.4
1 2 total
5.3 0.1 5.4
1.5 0.0 1.5
3.4 0.0 3.4
180
CHANG AND L I N
treated with other chemical such as trichloroethylene and benzo(a)pyrene (data not shown), occasional background readings of about 3 to 5 pg formaldehyde per mL urine (after a correction for dilution) were observed at a frequency of less than 5% of the total determinations.
All of the data collected
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
in Table 1 and 2 were determined according to a standard operating procedure which dictated a total dilution of 1 to 25 or 50 depending on the total volume of a 24-hour urine sample.
If the volume of a 24-hour urine sample was less than 15 mL, the dilution factor was 50; otherwise, the dilution factor was 25.
The individual variation in the metabolism of DMF is clearly demonstrated in Table 2.
However, a linear relationship was
obtained between the total excretion of DMF-OH in two days and the dose given for both strains of rat.
The correlation
coefficient of both curves was greater than 0.9.
Also, when
the regression equation was evaluated for all experimental rats (n = 21), the linear relationship remained. relationship is also shown in Figure 1.
This linear
It is estimated that on
an average of 200 gram body weight, a rat excreted about 10 mole percent DMF-OH in 48 hours measured by our method for the dose groups of 944, 472, and 94.4 mg/kg but only about 5 mole percent for the 47.2 mg/kg group.
Previously, Scailteur
et al. reported that when Sprague-Dawley rats were exposed to DMF at 1 mL/kg intraperitoneally, about 40 mole percent
181
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
EXPOSURE TO DIMETHYLFORMAMIDE
0
200
400
600
800
1000
DMF Dose (mg/kg)
FIGURE 1 Linear regression of rat urinary DMF-OH excreted in 2 days versus exposure dose.
urinary DMF-OH was measured in 2 days by both GC and colorimetric methods.9 The basis for using the analysis of urinary NMF by gas chromatography (GC) as the method to assess BE1 of DMF was mainly the thermal decomposition of DMF-OH to NMF at the injection port.
The sensitivity of a GC analysis largely
depends on the type of detector (NPD or FID) and the type of GC column (packed or capillary) used. The current ACGIH guidelines recommend a BE1 value for DMF of 40 mg NMF per gm creatinine.13 That is about 60 pg DMF-OH per mg creatinine or 20 pg formaldehyde per mg creatinine after alkalinization.
The normal value of creatinine in adult human
urine is about 0.5 to 1.0 mg/mL.
In our experience, a packed
CHANG AND LIN
182
Tenax GC column (2m x 2mm) coupled with a FID detection barely have the needed sensitivity to measure NMF at a concentration of approximately 40 to 80 pg/mL.
However, one
can make a one to five dilution in the proposed colorimetric determination of DMF-OH and still get an optical density of
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
about 0.500 to 0.550 at 412 nm for 4 pg formaldehyde per mL dilute urine. The assay protocol used in this investigation has a wide margin for enhancing its sensitivity. factor used was 25.
The lowest dilution
One can minimize the dilution in
alkalinizing the urine before its incubation with Nash reagent. It is estimated, with our lowest standard (i.e. 2 pg
formaldehyde per mL), that a 5% dilution of 1.9 mL urine with 0.1 mL 2N NaOH can easily assess about 6.2 pg DMF-OH per mL
which gives an optical density of -0.250 and is equivalent to about 4.1 pg NMF per mL. Although our determination of urinary DMF-OH seemed only measure 1/4 of what was determined by Scailteur et al., the proposed method can still determine a BE1 of 16.4 pg NMF per mL urine easily and accurately . In conclusion, we have demonstrated that a determination of DMF-OH in urine by utilizing a colorimetric assay to determine the alkaline released formaldehyde reflects adequately the exposure of rats to DMF ranging from 47.2 to 944 mg/kg b.wt. The method is sensitive enough to assess a
EXPOSURE TO DIMETHYLFORMAMIDE
183
BE1 of DMF at approximately one-half to one-fifth (depending on the concentration of urinary creatinine) of the current ACGIH recommended value i.e. 40 pg NMF per mg creatinine. WI propose that the method described in this report to be used as an alternative non-invasive method to assess BE1 of DMF, especially where a gas chromatography is not easily Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
accessab le. ACKNOWl FKGEMFNTS This work was partially supported by a grant from Chang Gung Medical College, CMRP 279 and a project from EPA/Taiwan, ROC, EPA 79-005-39-1 59.
The skillful technical
support of Ms. S. C. Yuan, S. F. Weng, and Y. C. Chien are acknowledged.
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