United States
Environmental Protection
1=1 m m Agency
EPA/690/R-07/014F
Final
9-27-2007
Provisional Peer Reviewed Toxicity Values for
Dimethylformamide
(CASRN 68-12-2)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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Acronyms and Abbreviations
bw body weight
cc cubic centimeters
CD Caesarean Delivered
CERCLA Comprehensive Environmental Response, Compensation and
Liability Act of 1980
CNS central nervous system
cu.m cubic meter
DWEL Drinking Water Equivalent Level
FEL frank-effect level
FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
g grams
GI gastrointestinal
HEC human equivalent concentration
Hgb hemoglobin
i.m. intramuscular
i.p. intraperitoneal
IRIS Integrated Risk Information System
IUR inhalation unit risk
i.v. intravenous
kg kilogram
L liter
LEL lowest-effect level
LOAEL lowest-observed-adverse-effect level
LOAEL(ADJ) LOAEL adjusted to continuous exposure duration
LOAEL(HEC) LOAEL adjusted for dosimetric differences across species to a human
m meter
MCL maximum contaminant level
MCLG maximum contaminant level goal
MF modifying factor
mg milligram
mg/kg milligrams per kilogram
mg/L milligrams per liter
MRL minimal risk level
MTD maximum tolerated dose
MTL median threshold limit
NAAQS National Ambient Air Quality Standards
NOAEL no-ob served-adverse-effect level
NOAEL(ADJ) NOAEL adjusted to continuous exposure duration
NOAEL(HEC) NOAEL adjusted for dosimetric differences across species to a human
NOEL no-ob served-effect level
OSF oral slope factor
p-IUR provisional inhalation unit risk
p-OSF provisional oral slope factor
p-RfC provisional inhalation reference concentration
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p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
l^g
microgram
[j,mol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
DIMETHYLFORMAMIDE (CASRN 68-12-2)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1. EPA's Integrated Risk Information System (IRIS).
2. Provisional Peer-Reviewed Toxicity Values (PPRTV) used in EPA's Superfund
Program.
3. Other (peer-reviewed) toxicity values, including:
~ Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~ California Environmental Protection Agency (CalEPA) values, and
~ EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are
developed according to a Standard Operating Procedure (SOP) and are derived after a review of
the relevant scientific literature using the same methods, sources of data and Agency guidance for
value derivation generally used by the EPA IRIS Program. All provisional toxicity values receive
internal review by two EPA scientists and external peer review by three independently selected
scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all EPA programs, while PPRTVs are developed specifically for
the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a five-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV manuscripts conclude
that a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and RCRA program offices are advised to carefully review the information provided in
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this document to ensure that the PPRTVs used are appropriate for the types of exposures and
circumstances at the Superfund site or RCRA facility in question. PPRTVs are periodically
updated; therefore, users should ensure that the values contained in the PPRTV are current at the
time of use.
It is important to remember that a provisional value alone tells very little about the adverse
effects of a chemical or the quality of evidence on which the value is based. Therefore, users are
strongly encouraged to read the entire PPRTV manuscript and understand the strengths and
limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
No RfD for dimethylformamide is listed on IRIS (U.S. EPA, 2007) or the Drinking Water
and Health Advisories list (U.S. EPA, 2004). The HEAST (U.S. EPA, 1997) lists a subchronic
oral RfD of 1 mg/kg-day and a chronic oral RfD of 0.1 mg/kg-day based on the derivation of an
Allowable Daily Intake (ADI) for 'rats' [sic] in a Health and Environmental Effects Profile
(HEEP) on dimethylformamide (U.S. EPA, 1986). The HEEP based the provisional acceptable
daily intake (ADI) on a no-observed-adverse-effect level (NOAEL) of 96 mg/kg-day for hepatic
effects in [male] CD-I mice exposed to dimethylformamide in the diet for 119 days (Becci et al.,
1983). In the chronic ADI derivation, an uncertainty factor of 1000 (10 to extrapolate from
animals to humans, 10 to protect sensitive individuals and 10 for the use of a subchronic study)
was applied to the NOAEL. The HEAST dropped the last uncertainty factor of 10 to derive the
subchronic RfD. No additional relevant documents for dimethylformamide are included in the
chemical assessments and related activities (CARA) list (U.S. EPA, 1991a, 1994a).
IRIS (U.S. EPA, 2007) lists a chronic reference concentration (RfC) of 0.03 mg/m3 for
dimethylformamide. The HEAST (U.S. EPA, 1997) lists a subchronic RfC of 0.03 mg/m3,
adopted from the chronic RfC of the same value on IRIS (U.S. EPA, 2007). The chronic RfC was
based on co-principal studies that evaluated hepatic effects in humans following occupational
exposure to dimethylformamide: Cirla et al. (1984), which evaluated 100 exposed/referent pairs
and Catenacci et al. (1984), which evaluated 54 exposed/referent pairs. Catenacci et al. (1984)
found no hepatic enzyme increases in workers exposed to 18 mg/m3 (8-hour TWA), but the power
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of the study was considered insufficient to use this NOAEL as the basis for the risk assessment
(U.S. EPA, 2007). Instead, the RfC was based on a lowest-observed-adverse-effect level
(LOAEL) of 22 mg/m3 (mean concentration) in the study of Cirla et al. (1984) for digestive
disturbances (disulfiram-like alcohol intolerance and dyspepsia) and minimal (i.e., not
statistically significant) hepatic changes suggestive of liver abnormalities (liver enlargement,
elevated serum aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) in workers
exposed for an average of 5 years (range 1-15 years). The LOAEL was adjusted for duration by
multiplying by the ratio of the reference breathing volume for light activity during 8-hour
occupational exposure to the reference daily breathing volume (10/20 rnVday) and by the ratio of
workdays to weekdays (5/7). An uncertainty factor of 300 (10 to protect sensitive individuals, 10
to account for use of a LOAEL and 3 combined for the lack of reproductive toxicity data and the
less than chronic duration of exposure) was applied to the duration-adjusted LOAEL of 7.9
mg/m3 to arrive at the RfC of 0.03 mg/m3. CalEPA (2000) derived a chronic reference exposure
level (REL) of 0.08 mg/m3 for dimethylformamide based on the same adjusted LOAEL of 7.9
mg/m3, but applying a cumulative uncertainty factor of 100 (3 for the use of a LOAEL, 3 for the
subchronic duration and 10 for intraspecies variation). ACGIH (2005a, 2005b), NIOSH (2005),
and OSHA (2006) have all established occupational exposure limits of 30 mg/m3 (10 ppm, 8-hour
time-weighted average [TWA]) for dimethylformamide to protect against toxicity to the liver,
central nervous system, kidney and heart, and irritation of the eyes and respiratory tract.
No cancer assessment for dimethylformamide is available in IRIS (U.S. EPA, 2007), the
Drinking Water Standards and Health Advisories list (U.S. EPA, 2004), or the HEAST (U.S.
EPA, 1997). The HEEP (U.S. EPA, 1986) assigned dimethylformamide to Group D, not
classifiable as to human carcinogenicity, based on inadequate evidence in humans and laboratory
animals and overwhelming evidence that the compound is not mutagenic or genotoxic. The most
recent assessment by IARC (1999, 2002) concluded that dimethylformamide is not classifiable as
to its carcinogenicity to humans (Group 3) based on inadequate evidence in humans, evidence
suggesting a lack of carcinogenicity in experimental animals and consistently negative results for
genotoxicity in well-controlled in vitro and in vivo assays. ACGIH (2005a, 2005b) added an A4
notation to the threshold limit value [TLV]-TWA for dimethylformamide to indicate that the
compound is not classifiable as a human carcinogen.
ATSDR (2007) has not reviewed the toxicology of dimethylformamide. An
Environmental Health Criteria document (WHO, 1991), a Concise International Chemical
Assessment Document (CICAD) (WHO, 2001), a Priority Substances List Assessment Report
(Health Canada, 2001) and IARC (1989, 1999) monographs on dimethylformamide, a toxicity
risk assessment of dimethylformamide (Long and Meek, 2001), a toxicity review on
miscellaneous organic nitrogen compounds (Trochimowicz et al., 2001) and the NTP (2006a,
2006b) management status report and health and safety report for dimethylformamide were
consulted for relevant information. Literature searches were conducted for the period from 1985
to October 2002 to identify data relevant for the derivation of a provisional RfD, RfC and cancer
assessment for dimethylformamide. The following databases were searched: TOXLINE,
MEDLINE, CANCERLIT, NTIS/BIOSIS, RTECS, HSDB, GENETOX, CCRIS, TSCATS,
EMIC/EMICBACK and DART/ETICBACK. An update search of the following databases was
conducted in November 2005: TOXLINE Special (NTIS subfile), TOXCENTER (BIOSIS
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subfile), MEDLINE (plus PubMed cancer subset), TSCATS, CCRIS, DART/EMIC, GENETOX,
HSDB, RETCS and Current Contents (June to November, 2005). An update search of the
PubMed database was conducted in September 2007.
REVIEW OF PERTINENT DATA
Human Studies
Several epidemiological studies have investigated effects on the liver in workers exposed
to dimethylformamide.
Cirla et al. (1984) studied 100 workers exposed to a mean concentration of 22 mg/m3 of
dimethylformamide (range of 8-58 mg/m3, determined with personal air sampler) for an average
of 5 years (range of 1-15 years), and compared the results with those obtained from 100
pair-matched referent controls. The authors accounted for alcohol ingestion, cigarette smoking
and caffeine in selecting the matching characteristics. The population studied was male, with a
mean age of 36 years (range of 21-56 years of age). Their work history was carefully verified,
and the possibility of peak exposures to dimethylformamide was ruled out. The workers were
evaluated by means of a questionnaire of subjective complaints, medical examination and
laboratory studies, including aminotransferase and gamma-glutamyl transpeptidase (GGT) levels.
Dimethylformamide-exposed workers complained more often of headache, dyspepsia,
nonspecific cardiac distress and digestive impairment indicative of hepatic functional impairment.
Symptoms of respiratory irritation that were significantly increased in the dimethylformamide
exposed group included watery eyes, cough and dry throat. Several of the exposed workers
complained of a disulfiram-type reaction upon alcohol ingestion. Among exposed workers, the
prevalence of abnormally high GGT levels (25/100) was statistically significantly increased
versus controls (10/100). Prevalence was also higher in the exposed group for other indicators of
hepatotoxicity, including liver enlargement (20/100 vs 16/100), elevated AST (9/100 vs 3/100),
and elevated ALT (12/100 vs 8/100), although none of these differences achieved statistical
significance. Within the dimethylformamide exposed group, there were no differences between a
subgroup with no dermal exposure and a subgroup with potential dermal exposure (data not
presented), indicating that exposure to dimethylformamide via the dermal route was not a
significant confounder. The average exposure level of 22 mg/m3 in this study is a LOAEL for
digestive disturbances and mild liver abnormalities resulting from occupational exposure to
dimethylformamide.
Catenacci et al. (1984) examined hepatic function in workers exposed to low levels of
dimethylformamide. They studied AST, ALT, GGT and alkaline phosphatase (AP) levels in 54
workers who had been employed in an acrylic fiber plant for more than 5 years. The workers
were divided into two groups; the first group of 28 subjects was exposed to an 8-hour TWA
concentration of 18 mg/m3 (range of 12-25 mg/m3) of dimethylformamide, and the second group
of 26 subjects was exposed to an 8-hour TWA concentration of 3 mg/m3 (range of 1-5 mg/m3) of
dimethylformamide. The control group consisted of 54 workers who were never exposed to
solvents. No significant difference was observed between either of the exposed groups and the
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controls for any of the parameters tested. Thus, the 8-hour TWA concentration of 18 mg/m3 was
a NOAEL for hepatotoxicity in this study. However, the power of this study to detect a
difference in enzyme levels was limited because only 54 matched pairs were used.
Fiorito et al. (1997) evaluated liver function in 75 workers exposed to dimethylformamide
at an 8-hour TWA of 20 mg/m3 (range 2-40 mg/m3) in a synthetic leather factory for up to 3.8
years. From the original pool of 86 workers, 11 were excluded from the study based on
nonoccupational liver complaints, recent drug therapy or high alcohol consumption. The control
group included 75 unexposed workers similar in age, sex, social status and residence. All
subjects underwent a complete physical examination with liver function tests for serum enzymes,
bile acids, bilirubin, serum cholesterol and triglycerides and hepatitis markers. Solvent
absorption was estimated from N-methylformamide (NMF) concentrations in pre- and post-shift
urine samples from a subset of 22 subjects in the same work shift. Fifty percent of exposed
workers reported gastrointestinal symptoms (stomach pain, nausea, loss of appetite). Alcohol
consumption was significantly lower in the exposed group, because alcohol use was reported to
cause symptoms in the workplace. Forty percent of exposed workers reported symptoms of
alcohol intolerance: face flushing (38%), palpitation (30%), headache (22%), dizziness (22%),
body flushing (15%) and tremors (14%). Mean values of liver enzymes (AST, ALT, GGTP, AP)
were statistically significantly increased in exposed workers; there were no effects on serum
cholesterol or other tested indices. In addition, the prevalence of workers with abnormally high
values was significantly increased for ALT (>10%), AST (>19%) and GGT (>12%) in
comparison to controls (all <4%). Multivariate analysis showed that ALT, AST and GGT levels
were not significantly correlated with age or alcohol consumption, and that cumulative exposure
to dimethylformamide (approximated as work seniority) was a stronger predictor of serum
enzyme levels than serum cholesterol levels or body mass index, which were also correlated with
enzyme levels. In this study, the average exposure level of 20 mg/m3 was a LOAEL for hepatic
effects in exposed workers.
Cai et al. (1992) evaluated health effects of dimethylformamide in workers in a
polyurethane plant. A total of 207 workers (114 men and 93 women) in five different
departments were exposed to mean 8-hour TWA concentrations between 0.2 and 9.1 ppm (0.6 to
28 mg/m3) with an overall group average of 4.5 ppm (13.7 mg/m3) and compared to 143 non-
exposed workers (67 men and 76 women) in the same plant. The duration of exposure was
"several years." Subjects were questioned as to the incidence of subjective symptoms during the
preceding 3-months and were evaluated for hematology and serum biochemistry (including AP,
ALT, AST, GGT and bilirubin). Hematology and serum biochemistry values in exposed workers
were not different from those of the controls. However, the prevalences of subjective symptoms
relating to the digestive system (nausea, vomiting, dry mouth and abdominal pain) and other
symptoms (eye irritation, dimmed vision, nasal irritation, sore throat, dizziness, tightness in chest,
breath shortage and unusual taste sensation) were significantly higher in the exposed group than
in the controls. Analysis by department (exposure level) found that the prevalence of self-
reported reduced alcohol tolerance was significantly elevated in subjects exposed to levels of 2
ppm (6 mg/m3) or above, but not average exposures of 0.1-0.6 ppm (0.3-1.8 mg/m3). This study
identified a LOAEL of 6 mg/m3 and a NOAEL of 1.8 mg/m3 based on subjective symptoms of
hepatic effects in workers exposed to dimethylformamide.
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Wrbitzky (1999) evaluated liver function in 126 male employees exposed to
dimethylformamide and in 53 unexposed males in a factory producing synthetic fibers. Workers
were exposed to dimethylformamide at a median concentration of 1.2 ppm (3.6 mg/m3); the range
was <0.1 ppm (the detection limit) to 37.9 ppm (<0.3-113.7 mg/m3). Subjects were evaluated by
a medical questionnaire, liver function parameters (ALT, AST, GGT) and hepatitis antibody
screening. The prevalence of subjective symptoms of alcohol intolerance (flush symptoms,
voluntary reduction in alcohol consumption) was significantly higher in the exposed group. The
exposed group had significantly elevated levels of AST and GGT. However, when the effects of
alcohol consumption and levels of exposure to dimethylformamide on the liver index [(ranking
indices for GGT + AST + ALT) divided by 3 (number of parameters)] were evaluated
statistically, the effect of alcohol (p<0.001) was found to be more pronounced than the influence
of dimethylformamide (p=0.043). For the subset of workers who did not consume alcohol, there
was no difference in the liver index between nonexposed workers and exposed workers,
irrespective of exposure level. In this study, alcohol consumption is a confounding factor.
Therefore, a reliable NOAEL or LOAEL for dimethylformamide is not provided by this study.
Lauwerys et al. (1980) studied 22 workers who were exposed to 1-46.3 mg/m3
dimethylformamide for an average of 5.3 years, and compared the results with those obtained
from 28 control workers. They found no significant difference in liver function tests (ALT, AST,
OCT, GGT, AP, bilirubin) when the exposed workers were compared to the control workers.
However, signs of alcohol intolerance were observed in the workers at dimethylformamide levels
below 30 mg/m3. Similarly, Yonemoto and Suzuki (1980) found no effects on AST, ALT, AP or
GGT in 11 workers exposed to 3-15 mg/m3of dimethylformamide, but the workers reported
episodes of alcohol intolerance.
Redlich and coworkers investigated an outbreak of liver disease in workers employed in a
fabric coating factory where dimethylformamide, along with other solvents, was mixed with
polyurethane and used to coat fabrics (Redlich et al., 1987a,b; 1988; Riely et al., 1988). Exposure
levels were not quantified, but workers were exposed to large quantities (approximately 15-20
55-gallon drums per week) of dimethylformamide in poorly ventilated areas without appropriate
skin protection. Average duration of employment was 40 months, but 15 workers were employed
for 3 months or less. Fifty-eight of the 66 workers participated in the study. Elevations of either
AST or ALT levels were observed in 36 of the 58 employees, with 19 of these workers showing
increases of twice the normal values, which the researchers considered to be indicative of liver
disease. The liver enzyme levels correlated with job classification; 11 of 12 nonproduction
workers had normal liver enzymes while 35 of 46 production workers had some elevation in liver
enzyme levels. These changes in liver enzyme levels were verified by pathological evidence of
liver disease revealed in liver biopsies taken from seven workers. The changes seen were
variable, but consisted mainly of microvesicular and macrovesicular steatosis, spotty necrosis and
evidence of diffuse regeneration. None of the biopsies showed changes indicative of alcoholism
(i.e., fibrosis or cirrhosis). Furthermore, questionnaire results from 46 workers revealed the
following symptoms: gastrointestinal discomfort (anorexia, abdominal pain or nausea) in 31
respondents, central nervous system solvent intoxication (headaches, dizziness) in 18 respondents
and alcohol intolerance characterized by a disulfiram type reaction (facial flushing and
palpitations after alcohol intake) in 11 respondents. The results demonstrate that occupational
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exposure to high levels of dimethylformamide can result in liver disease. The data are not
sufficient to determine whether the effects were reversible or whether chronic liver disease can
result from long-term exposure. Also, concurrent exposure to other solvents, including
methylethylketone, toluene, 1,1,1-trichloroethane and dichlorobenzene may have potentiated the
hepatotoxicity of dimethylformamide.
A number of additional occupational and case studies report the occurrence of hepatic
effects in exposed workers. These studies are limited by small sample size and uncharacterized
exposure levels. For many, translations were not available, so summaries from secondary sources
or abstracts are discussed below. Massmann (1956a) reported that all liver function tests were
normal in 24 workers exposed to dimethylformamide at levels that were generally below 10 ppm
for 3-24 months. Reinl and Urban (1965) studied 12 workers exposed to dimethylformamide
levels that were generally <20 ppm for 1-32 weeks and reported the occurrence of alcohol
intolerance, vomiting, hepatomegaly, jaundice, urobilinuria and elevated serum aminotransferases
in several of these workers. No increase in serum aminotransferases was observed in workers
exposed to an unknown concentration of dimethylformamide for 6 months (Di Lorenzo and
Graziolo, 1972). Tolot et al. (1968) reported that a liver biopsy performed 3 months after an
acute accidental exposure to an unspecified level of dimethylformamide showed vacuolization of
the parenchymal cells. Abdominal pain, nausea and vomiting, and ethanol intolerance were
observed in four workers exposed to an unknown concentration of dimethylformamide (Chary,
1974). Qian et al. (2007) reported changes in ALT and GGT in workers exposed to
dimethylformamide (concentrations not reported) in a synthetic leather factory. Luo et al. (2005)
examined liver function parameters and incidence of chronic liver disease (as determined by
ultrasonography) in workers (industry not reported) in a "high" dimethylformamide exposure
group (average workplace concentration - 23.9 ppm; range - 5.2-86.6 ppm) to workers in a "low"
exposure group (average - 2.41 ppm; range - 0.9-4.3 ppm); the incidence of abnormal liver
function tests was 13/44 (29.6%) in the high-exposure group and 2/22 (9.1%) in the low-exposure
group; chronic liver disease was reported in 7/44 (15.9%) of the high-exposure workers and 0/22
of the low-exposure workers.
Taken together, these studies demonstrate that dimethylformamide exposure is associated
with hepatic toxicity in humans. Subjective evidence of liver toxicity (e.g., digestive impairment,
alcohol intolerance) is often seen at exposure concentrations below those that cause elevations in
serum enzymes of hepatic function, and thus may serve as a more sensitive indicator of
dimethylformamide-induced hepatic toxicity. The reports indicate that dimethylformamide
concentrations as low as 6-20 mg/m3 may produce these effects. A reliable NOAEL has not been
identified.
One report was located on the reproductive effects of dimethylformamide in humans
(Farquharson et al., 1983). The researchers described the occurrence of three unexplained cases
of small-for-date third trimester intrauterine deaths in female quality control analysts working in
the pharmaceutical industry. This represents a 30% stillbirth rate as compared with the average
rate of 0.26% for the general population of that area. The authors concluded that this occurrence
was not likely due to chance, but cannot be attributed solely to dimethylformamide because these
women were exposed to other agents in addition to dimethylformamide.
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IARC (1999) reviewed carcinogenicity studies in humans exposed to dimethylformamide.
Case reports of testicular cancer among 7 out of 680 F4 Phantom jet aircraft repair technicians
exposed to a solvent containing 80% dimethylformamide (Ducatman et al., 1986) and 3 out of 51
workers in a leather tannery (Levin et al., 1987; Calvert et al., 1990) led to several
epidemiological studies evaluating the carcinogenicity of dimethylformamide. Chen et al. (1988)
studied cancer incidence among 2530 active employees of the DuPont company with potential
exposure to dimethylformamide during 1950-1970 in Virginia and 1329 employees with exposure
to dimethylformamide and acrylonitrile in a plant in South Carolina. For all workers exposed
(alone or with acrylonitrile), the standardized incidence ratio for all cancers combined was 1.1
(95% confidence interval [CI]: 0.9-1.4). Only one case of testicular cancer was identified among
3859 workers (1.7 expected). Walrath et al. (1989) conducted a case-control study for several
cancer types among workers from four DuPont plants. Geometric means for air measurements of
dimethylformamide ranged from a low of <1 ppm (3 mg/m3) to a moderate level around 10 ppm
(30 mg/m3). Mantel-Haenszel odds ratios for exposed individuals were 1 (n=3) (90% CI: 0.4-2.3)
for testicular cancer, indicating no increase. Odds ratios for testicular cancer were 0.9 (90% CI:
0.1-8.6) for the low-exposure group and 3.1 (90% CI: 0.8-11.9) for the moderate-exposure group.
IARC (1999) concluded that these studies do not provide convincing evidence for the
carcinogenicity of dimethylformamide in humans.
No data were located regarding the toxicity or carcinogenicity of dimethylformamide in
humans exposed orally.
Animal Studies
Oral Exposure
The toxicity of dimethylformamide has been analyzed extensively in animals exposed
orally or by inhalation. Oral toxicity data include subchronic dietary studies in rats and mice
(Becci et al., 1983; Kennedy and Sherman, 1986), subchronic drinking water studies in rats and
gerbils (Savolainen, 1981; Elovaara et al., 1983; Llewellyn et al., 1974), a reproductive drinking
water study in mice (Fail et al., 1998; NTP, 1992a) and developmental gavage studies in rabbits,
rats and mice (Hellwig et al., 1991; Saillenfait et al., 1997; Fritz and Giese, 1990; Merkle and
Zeller, 1980). No chronic duration oral studies were located.
Becci et al. (1983) exposed groups of 25 male and 25 female Wistar rats to
dimethylformamide in the diet at concentrations of 0, 215, 750 or 2500 ppm for 104 days.
Reported mean compound consumption was 0, 18, 61 or 210 mg/kg-day for male rats and 0, 20,
69 or 235 mg/kg-day for female rats. Endpoints that were examined throughout the study
included behavior and clinical signs (daily), body weight and food consumption (weekly), and
hematology, clinical chemistry and urine indices (five animals/sex/group after 30 and 90 days).
Hematology indices included hematocrit, erythrocyte count, total and differential leukocyte
counts, platelet count, reticulocyte count, erythrocyte sedimentation rate and hemoglobin level.
Clinical chemistry indices included levels of glucose, urea nitrogen, total protein, bilirubin,
sodium, potassium, SGOT (serum AST), SGPT (serum ALT) and alkaline phosphatase; serum
cholesterol and phospholipids were not assessed. Urine indices included pH, specific gravity,
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color, gross and microscopic appearance, ketones, protein, bilirubin and occult blood. Endpoints
examined at the end of the exposure period included ocular condition, organ weights (10 organs)
and histopathology (25 organs and tissues in all high-dose and control animals and five
animals/sex in low- and mid-dose groups).
Body weight gain was significantly (p<0.05) decreased in the high-dose male and female
rats over the course of the study (8.8 and 12.1% less than controls, respectively). Mean food
consumption per week was significantly decreased in high-dose males (5.3% less than controls)
and mid- and high-dose females (2.5 and 3.4%, respectively). There was no significant effect on
food conversion (gram weight gain/100 grams food consumed), leading the authors to suggest
that the reduced food consumption (and consequently body weight gain) might have been due to
lower palatability of the feed rather than toxicity per se. Clinical chemistry, urinalysis and
hematological parameters were within normal ranges for rats, although a few sporadic changes
achieved statistical significance (decreased hematocrit in low-dose males and increased sodium in
high-dose males). High-dose (210 mg/kg-day) males had significantly increased relative, but not
absolute, weights of brain and testes (11.1 and 16.9% higher than controls, respectively).
Absolute liver weight was increased in females at 235 mg/kg-day (22.3% higher than controls)
and relative liver weight was increased in females at 69 and 235 mg/kg-day (9.8 and 29.5%,
respectively) and males at 210 mg/kg-day (16.0%). No histological changes were observed in the
liver, brain, testes or other organs or tissues. The authors concluded that the increases in relative
brain and testes weights are most likely physiological responses to the decreased feed intake. In
the absence of any hepatic histopathology or abnormal serum transaminase and alkaline
phosphatase levels, the observed increases in liver weight are considered to be an adaptive, rather
than adverse effect. The decreases in body weight are not considered to be adverse because the
reduction was small (8.8-12.1%) and possibly due to palatability-related decreased food
consumption. Due to the lack of clear evidence of an adverse effect on the liver and other organs,
the high dose of 210-235 mg/kg-day is classified as a NOAEL.
Becci et al. (1983) similarly exposed groups of 30 male and 30 female CD-I mice to 0,
160, 540 or 1850 ppm dimethylformamide in the diet for 119 days. Reported mean compound
consumption was 0, 22, 70 or 246 mg/kg-day for males and 0, 28, 96 or 326 mg/kg-day for
females. Endpoints were the same as in the rat part of the study summarized above (although
clinical chemistry was only evaluated after 90 days). No treatment-related effects were observed
on body weight gain, food consumption, food conversion, clinical signs, behavior, ophthalmic
condition or urinalysis indices. Sporadic hematological changes occurred in females
(significantly increased hemoglobin at low-dose and decreased hematocrit at high-dose) but were
within normal historical ranges for mice and not considered treatment-related. Serum ALT levels
were significantly (p<0.05) increased in the high-dose females {66.1% higher than controls; low-
and mid-dose values not reported), although still within the historical normal range. No
differences in clinical chemistry or hematological parameters were observed in male mice.
Absolute and relative weights of the thyroid (11.9 and 15.8% higher than controls, respectively)
and adrenal glands (20.0 and 23.1%) were significantly (p<0.05) increased in high-dose females.
Absolute liver weight was significantly (p<0.05) increased in high-dose males and females (30.0
and 25.0% higher than controls, respectively), and relative liver weight was significantly
increased in mid- and high-dose females (15.7 and 29.4%, respectively) and high-dose males
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(24.5%). Histological examinations showed slight hepatocytomegaly in a majority of high-dose
males and females (incidences and other details not reported), but no changes in the thyroid,
adrenals or other organs or tissues. The toxicological significance of the increased thyroid and
adrenal weights is unclear due to the lack of histopathological changes in these organs. Based on
the increases in liver weight, hepatocytomegaly and serum ALT in high-dose mice, 246-326
mg/kg-day is a LOAEL for hepatic effects. The NOAEL is 70-96 mg/kg-day, the dose that
caused increased liver weight without histological or serum enzyme changes.
Kennedy and Sherman (1986; Haskell Labs, 1960) fed groups of weanling CD rats
(6/sex/group) diets containing 1% peanut oil plus 0, 200, 1000 or 5000 ppm of
dimethylformamide (purity >99%) for 93-94 days. Based on reported average weekly intakes of
dimethylformamide (Haskell Labs, 1960), the average calculated doses are 0, 16, 77 and 426
mg/kg-day for males and 0, 17, 85 and 422 mg/kg-day for females. Rats were examined daily for
mortality and clinical signs; body weights were recorded before exposure, three times per week
for the first 3 weeks and twice per week thereafter. Food consumption was measured weekly.
Hematology (red cell count, total and differential white cell counts, hemoglobin concentration,
hematocrit, red cell diameter, number of nucleated red cells per 100 white cells) and serum
alkaline phosphatase levels were evaluated at 30, 60 and 90 days. Serum levels of SGOT (AST),
/;-phenylenediamine oxidase, cholesterol and phospholipids, as well as liver fat content, were
determined at 90 days. At termination, all rats were necropsied, major organs were weighed
(adrenal, brain, kidney, liver, lung, spleen and testis), a limited number of tissues were examined
for histopathology (organs that were weighed and ovary, heart, pancreas, stomach and small
intestine) and bone (tibia) length was measured.
Overall body weight gain was 12.2 and 11.4% lower than controls in high-dose males and
females, respectively, mainly due to average weight gain reductions of 20.9% in the males during
weeks 1-5 and 39.1% in the females during weeks 1-2. Food consumption was reduced in the
high-dose males during weeks 1-6 (average 4.1% less than controls) and high-dose females
during weeks 1-5 (average 13.2%). Food efficiency (gram weight gain/gram food consumed) was
reduced in the high-dose males during weeks 1-5 (average 16.0% less than controls) and high
dose females during weeks 1-2 (average 28.3%). Hematological alterations, observed at 90 days,
included significantly reduced erythrocyte counts in males and females at 1000 ppm (13.2 and
4.9% lower than controls, respectively) and 5000 ppm (7.3 and 21.5% lower), and increased
leucocyte counts in males at 1000 and 5000 ppm (10.0 and 42.7% higher than controls,
respectively) and females at 5000 ppm (58.2% higher). Serum cholesterol was increased in males
at 5000 ppm and females at >1000 ppm; levels at 200, 1000 and 5000 ppm were 25.2% lower,
2.8%) lower and 37.4% higher than controls in males, and 2.9% lower, 51.4% higher and 84.8%
higher than controls in females. Serum phospholipids were increased in both sexes at 5000 ppm;
levels at 1000 and 5000 ppm (not tested at 200 ppm) were 4.7% lower and 29.9% higher than
controls in males, and 5.9 and 24.3% higher than controls in females. Liver fat (% dry weight)
was decreased in males at >1000 ppm and females at 5000 ppm; levels at 200, 1000 and 5000
ppm were 10.9% lower, 31.5% lower and 34.8% lower than controls in males, and 5.0%> higher,
10.0%) higher and 17.5% lower than controls in females. Absolute and relative liver weights were
significantly increased in both sexes at >1000 ppm; compared to controls at 200, 1000 and 5000
ppm, absolute liver weights were 6.8, 11.6 and 11.7% higher in males and 11.6, 26.3 and 24.0%)
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higher in females, and relative liver weights were increased 4.0, 12.5 and 24.4% higher in males
and 1.8, 13.9 and 31.5% higher in females. Histological changes were limited to the liver and
consisted of a slight variation in size and staining quality of the nuclei in the hepatocytes, which
were classified as barely perceptible liver injury, in 3/6 males and 5/6 females at 5000 ppm
(Haskell Labs, 1960). The hepatocellular changes were further interpreted as increased mitosis
and cellular enlargement (Kennedy and Sherman, 1986).
The main target organ was the liver, as shown by the increases in liver weight and serum
cholesterol and decrease in liver fat at >1000 ppm, and increases in serum phospholipids and
histological alterations at 5000 ppm (Kennedy and Sherman, 1986; Haskell Labs, 1960).
Assessment of the changes in cholesterol, phospholipids and liver fat is complicated by the small
numbers of animals, wide intergroup variations in mean values, unreported standard deviations
and insufficient information on normal ranges. Although increases in serum cholesterol are
possibly indicative of a disruption in liver function, and were also found in 13-week inhalation
studies in rats and mice (Lynch et al., 2003; NTP, 1992b; Senoh et al., 2003), the findings in the
oral study are inconclusive because the value in low-dose males was much lower than in the
control group, the male and female control values appear to be unusually high for CD rats, the
increases were not pronounced, and ranges of normal values could not be located for CD rats of
similar age 16 weeks old) (CCAC, 2006; Charles River Laboratories, Inc., 1994). The
increases in serum phospholipids and decreases in liver fat are similarly inconclusive due to
variability in mean values, incompletely reported statistics, small responses, and lack of
information on normal ranges. Consequently, based on increases in liver weight and slight
histopathology, the LOAEL is 5000 ppm (422-426 mg/kg-day). The NOAEL is 1000 ppm (77-85
mg/kg-day), the highest dose that caused increased liver weight without histological alterations.
Llewellyn et al. (1974) reported that 4/12 female Mongolian gerbils exposed to
dimethylformamide in drinking water at 10,000 mg/L died within 200 days. Based on drinking
water consumption data, the mean total dose was reported by the study authors as >100,000
mg/kg (>500 mg/kg-day, data not shown). Microscopic examination revealed necrotic lesions in
the livers of all the gerbils that died. At a drinking water concentration of 17,000 mg/L, one-half
of the gerbils died within 80 days, and the cumulative dose was 90,206 mg/kg (1128 mg/kg-day).
At concentrations of 34,000 and 66,000 mg/L, one-half of the gerbils died within 6 and 3 days,
respectively. Nearly all of the animals of these higher exposure groups had liver lesions
including diffuse zones of necrosis, abnormal numbers of mitotic figures and many Kupffer cells.
It should be noted that individual water consumption varied widely within groups.
Additional subacute (Kennedy and Sherman, 1986) to subchronic (Itoh et al. 1987)
duration gavage studies also provide evidence of more severe liver toxicity at relatively high
bolus doses of dimethylformamide, and relatively mild histopathological changes in a 2-week
gavage study using doses that caused hepatocellular hypertrophy in subchronic dietary and
drinking water studies. A subacute toxicity study in which male CD rats were administered 0 or
450 mg/kg-day of dimethylformamide (as a 10% solution in water) for nine doses over a period of
2 weeks reported that the treated rats showed "discomfort" after dosing, and their weight gain was
depressed relative to controls (Kennedy and Sherman, 1986). Histopathological examination of
the livers of the three treated rats sacrificed after the last dose revealed variation in size and
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staining of liver nuclei, frequent mitotic figures and some hepatic cells containing two nuclei. No
histopathological changes were seen in the three treated rats that were sacrificed after an 11-day
recovery period. Gavage treatment of F344 rats (8/sex/group) with 708 or 945 mg/kg of
undiluted dimethylformamide once weekly for 12 weeks resulted in hepatic fibrosis and severe
hepatic necrosis in both sexes, and decreased body weight gain and depressed activity in males
(Itoh et al., 1987). Serum ALT was elevated less than 2-fold in males and unaffected in females,
but blood was obtained for clinical chemistry 1 week after the last dose, just prior to necropsy.
Fail et al. (1998; NTP, 1992a) evaluated the effects of N,N-dimethylformamide (purity
>99%) on fertility and reproduction in Swiss CD-I mice using the Reproductive Assessment by
Continuous Breeding Protocol. The authors cited previous publications for the study methods
(Lamb, 1985; Chapin and Sloane, 1997) and the protocol is also described online (NTP, 2002).
In a 2-week range-finding study, groups of eight mice/sex were exposed to drinking water
containing dimethylformamide at concentrations of 0, 2500, 5000, 7500, 10,000 or 15,000 ppm.
Animals were examined for mortality and clinical signs, but the frequency of observation was not
reported. Body weights and food and water consumption data were recorded weekly. Increased
mortality was observed in males exposed at >10,000 ppm and females at 15,000 ppm. Body
weight was reduced in surviving mice exposed at 15,000 ppm. Decreased water consumption was
observed in both sexes, but the affected groups were not reported; results for food consumption
were not reported. On the basis of the range-finding study, drinking water concentrations of 0,
1000, 4000 and 7000 ppm were selected for the continuous breeding study.
In the continuous cohabitation study, Fail et al. (1998; NTP, 1992a) gave untreated
drinking water to 40 breeding pairs of Swiss CD-I mice and drinking water containing 1000,
4000 or 7000 ppm of dimethylformamide to groups of 20 breeding pairs for a 1-week pre-
cohabitation period and a 14-week cohabitation period (F0 generation). As estimated from the
reported dose ranges, the mean doses were 0, 185, 610 and 1049 mg/kg-day for F0 males and 0,
225, 788 and 1427 for F0 females. Body weights and food and water consumption were
monitored for F0 mice on weeks 1, 8 and 16. Other endpoints in the cohabitation study included
the litter interval, number of litters/pair, the number, sex and weight of pups per litter, external
malformations of pups, and the dam body weight on postnatal day (PND) 0. After week 16 of
exposure, the breeding pairs were separated and F0 females were allowed to deliver and rear the
final litter until PND 21. On PND 0, 4, 7, 14 and 21 of lactation, randomly selected Fi pups were
weaned and saved for the Fi fertility assessment (described below). Following the cohabitation
period, crossover mating trials were conducted using F0 high-dose males mated with control
females (dosed male group), high-dose females mated with control males (dosed female group)
and a control group. Beginning at week 23, breeding pairs were cohabited (untreated) until the
appearance of a vaginal copulatory plug or up to 1 week. At week 24, mice were housed
separately and dosing was resumed. Upon delivery, each crossover litter was evaluated for
lethality, gestation length, sex, number and weight of pups and weight of dams. Selected litters
were evaluated for skeletal malformations and for visceral malformations of the head. After all
crossover litters had been delivered, vaginal smears were collected from control and 7000 ppm F0
females for 12 days. At week 29, all F0 mice were weighed and necropsied. The following organ
weights were measured: liver and paired kidney/adrenal in both sexes, right testis, right
epididymis, prostate and seminal vesicles with coagulating glands in males and right ovary with
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attached oviducts in females. Histopathologic evaluations were conducted on any gross lesions,
livers with gross lesions, right and left kidney/adrenals, right testis and epididymis, prostate,
seminal vesicles and ovary. [Fail et al. (1998) state that all livers were examined
histopathologically, but NTP (1992a) clearly states that only the livers with gross lesions were
examined histopathologically.] Sperm evaluations included assessments of motility,
concentration and morphology (right testis/epididymis) and number of homogenization-resistant
spermatid heads (left testis/epididymis).
In the cohabitation study, exposure of F0 mice to dimethylformamide had no effect on
mortality in either sex (Fail et al., 1998; NTP, 1992a). Treatment had no effect on body weight in
males, but caused significant reductions in females; effects in females were observed in the latter
half of the study at 4000 and 7000 ppm and only after delivery of the last litter (PND 14-21) at
1000 ppm. Food consumption was decreased mainly during the lactation period for the final litter
in the 4000 and 7000 ppm groups, and only during the last week of lactation for the 1000 ppm
group. Water consumption during this period was highly variable, but tended be decreased in the
4000 and 7000 ppm groups. The body weight effects in treated F0 dams were at least partly
related to reduced fertility and fecundity. Five sets of litters were produced during the
cohabitation period. In the first set of litters, fertility was reduced by 10% in the 7000 ppm group
compared to controls. By the time of the final litter, significant reductions in the following
reproductive parameters were observed in mice dosed at >4000 ppm: fertility index (fertile
pairs/cohabiting pairs), the number of litters per pair, the number of live pups per litter, the
percent of live pups, and live pup weight. Treatment had no effect on the cumulative days to
litter. An increase in malformed pups was observed in treated groups. The proportion of litters
with one or more pups with external abnormalities (domed heads, hematomas) was 7.9% in the
control group and 10.5, 90 and 77.8% for the low- to high-dose groups. The authors attributed
the decline in the proportion of pups with external abnormalities in the high-dose group (relative
to the mid-dose group) to the decreased fertility, increased prenatal mortality and increased
postnatal cannibalism of severely affected pups in that group. For the final Fi litter, survival of
pups during lactation was reduced in the >4000 ppm groups, but body weight was not affected in
pups surviving to weaning. Reduced survival appeared to be related to the higher incidence of
craniofacial abnormalities in mid- and high-dose groups.
In the F0 crossover mating trials, results for the 7000 ppm dosed male group were not
significantly different from those of the control group (Fail et al., 1998; NTP, 1992a). However,
the dosed female group had significant reductions in the number of live pups per litter and the live
pup weight. The offspring of dosed females showed significant increases in external
malformations (90.9% of litters affected, compared to 12.5% for control offspring and 0 for
offspring of dosed males mated to control females) and skeletal malformations (95% of pups
within litters affected, compared to 40% for the control group and 38% for the dosed male group;
percentage of litters affected was 100% compared with 83.3% for controls and 81.1% for the
dosed male group). Skeletal malformations (abnormal ossification of cranial plates, abnormal or
incomplete development of sternebrae) were qualitatively more severe in the offspring of dosed
females, compared to the other two groups, in which developmental delay (delayed ossification)
was the primary effect. The fact that the rate of the "malformations" described above in the
control group (83% of litters; 40% of pups) was so high suggests that these might appropriately
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be classified as variations rather than malformations. Offspring of dosed females, but not the
other groups, had increases in facial and brain abnormalities: cleft palate, agnathia, agenesis of
the cerebrum, abnormally-shaped cerebrum and enlarged cerebral ventricles. The results of the
crossover trials indicate that reproductive effects of dimethylformamide are not likely caused by
genotoxicity in sperm, but rather were mediated through the F0 females. Exposure to 7000 ppm
dimethylformamide had no significant effect on the length or stage frequency distribution of the
estrous cycle, but did reduce the number of animals with normal cycle lengths by 20%.
At terminal necropsy of F0 mice, dimethylformamide exposed mice showed no effect on
body weight in males, but exposure at 7000 ppm caused a significant reduction in females (Fail et
al., 1998; NTP, 1992a). In males, the absolute weights of the liver and right cauda epididymis
were significantly increased at >1000 ppm, and the absolute weight of the prostate was
significantly decreased at 7000 ppm. In females, the absolute and relative weights of the liver
and the relative weight of the kidney/adrenals were significantly increased at >1000 ppm. The
authors judged that histopathological findings (centrilobular hepatocellular hypertrophy) in two
mid-dose females and two high-dose males with gross hepatic lesions were related to treatment.
Dimethylformamide had no effect on reproductive histology or on epididymal sperm
concentration, motility or morphology. The authors considered that a significant trend for a slight
decrease in testicular spermatid concentration (significant at low and high doses) was not
biologically relevant. Results for the first generation in the continuous breeding study indicate
that 1000 ppm (185 mg/kg-day for males and 225 mg/kg-day for females) is considered a
NOAEL and 4000 ppm (610 mg/kg-day for males and 788 for females) is considered a LOAEL
for systemic (liver) effects. In the first generation study, the NOAEL for reproductive and
developmental toxicity is 1000 ppm (225 mg/kg-day for females) and the LOAEL is 4000 ppm
(788 mg/kg-day for females) for reductions in fertility, live pups, and pup birth weight, and
increases in external and skeletal abnormalities, mediated through the F0 females. These effects,
however, were considered by the study authors to be due at least in part to the decreased female
body weight and food and water consumption.
The assessment of fertility in Fi mice was conducted using mice randomly selected at
weaning (PND 21) from the four treatment groups (Fail et al., 1998; NTP, 1992a). Weanlings
were dosed at 0, 1000, 4000 or 7000 ppm in drinking water beginning on PND 22. As above,
doses were estimated as 0, 226, 947 and 1814 mg/kg-day for Fi males and 0, 292, 1098 and 2054
for Fi females. At PND 74, males and female nonsiblings were housed for mating for up to 7
days; 20 nonsibling pairs were established for the 0, 1000 and 4000 ppm groups. Since survival
was affected at the highest dose, the group consisted of 15 pairs, some of which were siblings.
After breeding, Fi mice were housed singly through delivery of F2 pups; the same reproductive
endpoints were monitored as for the F0 crossover trial. In addition, body weight and feed and
water consumption were recorded on PND 74, 84 and 112; maternal body weights were recorded
upon discovery of an F2 litter. Vaginal smears were collected for 12 day following delivery. At
scheduled necropsy at PND 119, Fi adults were necropsied as described for F0 mice, except that
histopathology was not conducted on the prostate or seminal vesicles. Treatment with
dimethylformamide reduced body weights of male and female Fi mice exposed at >4000 ppm, but
had no effect on feed consumption. Water consumption was increased for males at >4000 ppm on
PND 84 and at 7000 ppm on PND 112. Exposure to dimethylformamide at 7000 ppm caused
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reductions in the mating index. At >4000 ppm, there were reductions in fertility, the number of
live F2 pups per litter, pup body weight, adjusted (for litter size) pup body weight and proportion
of live pups/litter and an increase in the average days to litter. At 1000 ppm, live pup weight and
adjusted live pup weight were decreased. The proportion of litters with one or more externally
malformed pups was 0, 27.7, 60 and 75%, respectively, for the control and low- to high-dose
groups. Estrous cycle evaluations in control and 7000 ppm females showed that treated mice had
longer cycles and were in either metestrus or diestrus longer than controls. At necropsy, Fi body
weights were reduced in both sexes at >4000 ppm. At >1000 ppm, absolute and relative liver
weights were significantly increased in both sexes, and relative prostate weight was decreased in
males. At >4000 ppm, absolute prostate weight was decreased in males, and relative
kidney/adrenal weight was increased in females. Evaluations of livers with gross lesions from
low- and high-dose group mice demonstrated dose-related centrilobular hepatocellular
hypertrophy. Epididymal sperm concentrations were decreased at 7000 ppm. Average estrous
cycle length was significantly increased in treated Fi females, but treatment had no effect on
sperm morphology. Examination of the skeleton of Fi adults (5/sex/dose) revealed a
malformation rate of 30, 20, 100 and 100% in the control and low- to high-dose groups. High
incidence (30%) in controls could possibly suggest the increased skeletal malformations might be
variants of normal. Malformations in the >4000 ppm groups included abnormal or incomplete
ossification of cranial plates, abnormal cranial sutures, dysplasia of cranial bones and abnormal
sternebrae. For the second generation study, the lowest drinking water concentration of
dimethylformamide, 1000 ppm (equivalent to a dose of 226 mg/kg-day for males and 292 mg/kg-
day for females) is a LOAEL for increased liver toxicity [increased organ weight; centrilobular
hepatocellular hypertrophy (in two livers with gross lesions)] in Fi adults (maternal toxicity) and
for developmental toxicity (reduced live F2 pup weight).
Gestational exposure studies of dimethylformamide have been conducted in rats, mice and
rabbits. Saillenfait et al. (1997) evaluated developmental toxicity in groups of 22-24 pregnant
Sprague-Dawley rats that were exposed to 0, 50, 100, 200 or 300 mg/kg of dimethylformamide
(purity 99.9%) by gavage in distilled water on gestational days (GD) 6-20. Dams were observed
daily for clinical signs; food consumption was measured every third day beginning on GD 6.
Body weights were recorded on GD 0 and every third day beginning on GD 6. At termination on
GD 21, dams were sacrificed and the number of implantation sites, resorptions (including early
resorptions), and dead and live fetuses were recorded. Live fetuses were weighed, sexed,
examined for external anomalies, and then examined either for visceral or skeletal anomalies.
The study methods did not include observation of the dams for clinical signs or necropsy of the
dams at termination. Treatment had no effect on survival of dams. Maternal body weight gain
and food consumption were statistically significantly reduced in the >100 mg/kg-day groups in a
dose-related manner; at 50, 100, 200 and 300 mg/kg-day for GD 6-21, mean maternal body
weight gain was 3.9, 16.9, 33.8 and 40.3% less than controls, mean corrected maternal body
weight gain (maternal body weight gain minus gravid uterine weight) was not different, 41.9, 79.1
and 90.7%) less than controls and mean food consumption was 0.3, 15.0, 24.0 and 20.9% less than
controls. Dose-related reductions in fetal body weight occurred for all (total) fetuses at >100
mg/kg-day, female fetuses at >100 mg/kg-day and male fetuses at >200 mg/kg-day; at 50, 100,
200 and 300 ppm, mean body weight of all fetuses per litter was 0.3, 4.3, 12.1 and 14.1%> less
than controls. Thus, the strongest effect was on the corrected maternal body weight gain, which
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was affected much more severely than maternal food consumption. Effects on fetal body weight
were less severe and appear to be secondary to effects on maternal body weight gain. Sporadic
individual external and visceral malformations were observed in the litters of treated groups, but
the incidences were not dose-related. Incidences of fetuses and litters with two skeletal
variations, absent or incompletely ossified supraoccipital and sternebrae, were statistically
significantly increased at >200 mg/kg-day. At 0, 50, 100, 200 and 300 ppm, for the
supraoccipital skull variation, the incidences of affected fetuses were 2/122, 2/145, 9/140, 64/139
and 119/140 and of affected litters were 0/16, 2/20, 7/19, 16/19 and 20/20, and for the sternebral
variation, the incidences of affected fetuses were 3/122, 13/145, 13/140, 15/139 and 32/140, and
of affected litters were 2/16, 7/20, 7/19, 11/19, and 13/20. These skeletal variations reflect
delayed ossification, and are consistent with body weight depression in the fetuses, which may be
associated with the depression of food consumption and body weight gain in the dams (e.g.,
Fleeman et al., 2005; Hood, 2005). In this study, the maternal NOAEL was 50 mg/kg-day and the
LOAEL was 100 mg/kg-day for reductions in food consumption and body weight gain. Whether
the decreased food consumption was due to discomfort from the gavaged chemical, an affect on
appetite, or other toxic effects of the chemical cannot be determined from the available data. The
developmental NOAEL was 50 mg/kg-day and the LOAEL was 100 mg/kg-day for reduced fetal
body weight gain. Thus, the apparent maternal NOAEL and LOAEL (for reduced food
consumption and body weight gain) were the same as the developmental NOAEL and LOAEL.
Hellwig et al. (1991; BASF 1976a, b) conducted developmental toxicity studies in rats and
mice exposed to dimethylformamide by gavage in water. The study authors stated that testing
was conducted according to FDA (1966) guidelines. Groups of 19-23 pregnant Sprague-Dawley
rats were given gavage doses of 166, 503 or 1510 mg/kg-day on GD 6-15; parallel "untreated'
control groups (18-23 per group) were included. Dams were evaluated for mortality and clinical
signs until GD 20, at which time Caesarian sections were performed. Other parameters analyzed
at termination included gross pathology of dams, numbers of implantation and resorption sites
(early, medium-term and late), numbers of living and diseased fetuses (per group and per dam),
fetal weight, fetal length, fetal sex ratio and placental weight. Viable fetuses were examined for
skeletal (2/3 total number) or soft tissue anomalies (1/3 total). Maternal effects included the
death of one high-dose dam on GD 10, dose-related reductions in placental weights in all treated
groups and dose-related reductions in body weight gain in mid- and high-dose dams (weight gain
data not shown and food consumption not reported). The total number of live fetuses and the
number of live fetuses per dam were significantly reduced in the high-dose group, primarily
because of an increase in mid-term resorptions (>60%). The elevated incidence of early- and
late-term resorptions was statistically significant in the mid-dose group, but not in the low-dose
group. Dose-related increases in the number of resorptions (total and per implantation), in the
total number of runts and in the percent of live fetuses with anomalies (primarily skeletal) were
observed in the mid- and high-dose groups. Dose-related reductions compared to controls were
observed in fetal weights and lengths in the mid- and high-dose groups. Unless specified
otherwise in this description, the data do not appear to be reported on a per litter or per dam basis,
and, thus, the reporting is not in accord with current guidelines. The 4% reduction of placental
weight in the low-dose group, while not biologically significant by itself, represents the low end
of the trend for reductions in placental, maternal and fetal weights observed at the higher doses.
The low dose of 166 mg/kg-day is a minimal maternal reproductive LOAEL for reduced placental
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weight. More severe maternal effects (more pronounced reductions in placental weight and
reductions in body weight gain) and also developmental effects (e.g., resorptions and reduced
fetal weight) were observed at 503 and 1510 mg/kg-day. The NOAEL and LOAEL for
developmental effects are tentatively identified as 166 and 503 mg/kg-day, but the reliability is
questionable because most of the statistical analyses did not appear to consider the litter as the
unit for analysis and because controls were not gavage-treated with vehicle.
In their developmental study on mice, Hellwig et al. (1991) employed the same protocol
used for rats with the following exceptions. Groups of 24 pregnant NMRI mice were given
aqueous gavage doses of 182 or 548 mg/kg-day on GD 6-15; two "untreated" control groups (23
each) were included. Mouse pregnancies were terminated on GD 18. Treated dams showed no
obvious effects on survival, body weight gain (not evaluated statistically and data not reported) or
incidence of clinical signs. Treatment had no effect on placental weight, or the numbers of
resorptions, implantations or implantations per dam. Gestational exposure resulted in reductions
in fetal length in both groups and in fetal weight in the high-dose group. The number of
anomalies was elevated in the high-dose group. In this study, the high dose of 548 mg/kg-day
was a NOAEL for maternal toxicity. The low dose of 182 mg/kg-day may be considered a
NOAEL for reduced fetal length in the absence of an effect on fetal weight, and 548 may be
considered a LOAEL for reduced fetal body weight and length, and for increased anomalies. The
reliability of the fetal NOAEL and LOAEL, however, is questionable since the statistical analyses
generally did not appear to consider the litter as the unit for analysis, and because controls were
not gavage-treated with vehicle.
Fritz and Giese (1990; Ciba-Geigy, 1979) evaluated the pre- and postnatal toxicity of
dimethylformamide administered at doses of 0 or 750 mg/kg-day by gavage in water to pregnant
Sprague-Dawley rats on GD 10-14. One set of controls and treated dams (10 per group) were
sacrificed near term (GD 21) and the following endpoints were evaluated: number of litters,
number of fetuses, mean fetal body weight, fetal mortality, gross malformations and skeletal
anomalies. The offspring of another set (10 per group) were examined until postnatal day (PND
46). In addition to body weight, 13 randomly-selected pups from the treated group were
evaluated on PND 5 for skeletal anomalies, and the surviving pups were evaluated on PND 32 for
various neurological and behavioral parameters. Maternal endpoints, such as body weight,
clinical signs, food consumption and gross pathology, were not reported for either set of animals,
and fetal endpoints were not reported on a litter basis. In the prenatal study, exposure to
dimethylformamide had no effect on the incidences of fetal mortality or gross malformations, but
mean fetal weight was reduced by about 20% compared to the controls. In addition, the treated
group exhibited significant increases compared to controls in the incidences of delayed
ossification of certain bones: phalangeal nuclei (56.8 vs 8.5% for proximal phalanx V), calcaneus
(93.7 vs 11.1%), frontoparietals (80.2 vs 0%) and supraoccipitals (100 vs 0%). A sex difference
was noted in the frontoparietal effect; all treated male fetuses were affected, but only -65% of
females were affected. In addition, about 53% of treated fetuses exhibited fused sternebrae 4 and
5, an anomaly that was not observed in controls. In the postnatal study, gestational exposure to
dimethylformamide had no effect on the direct pupil reflex, the startle response to a loud noise, or
behavioral parameters (exploratory locomotion, cliff avoidance and negative geotaxis on a 45°
slope). However, body weight gain was slightly reduced in the treated group and pups exhibited
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a persistence of delayed ossification of the frontoparietal bones on PND 5. In this study, 750
mg/kg-day is a LOAEL for developmental effects (increases in delayed ossification and reduced
fetal body weight gain). The delayed ossification effects occurred in such high percentages of
fetuses that the lack of reporting on a litter basis does not impact the judgment that this dose is a
LOAEL.
In an earlier study in rabbits, Merkle and Zeller (1980) treated groups of female Russian
rabbits by gavage with dimethylformamide in aqueous solution on days 6-18 postinsemination.
The dams were killed on day 28 postinsemination. The doses were 0, 46.4, 68.1 or 200 [xL/kg-
day. Using the density of dimethylformamide at 25 °C of 0.9445 g/mL or 0.9445 mg/[xL, the
doses are equal to 0, 43.8, 64.3 or 188.9 mg/kg-day. The highest dose was toxic to the dams,
resulting in decreased food intake and weight gain. The rate of abortion in pregnant does was
1/22, 0/10, 0/16 and 3/11, in order of controls through highest dose. The number of
implantations/dam and the number of live fetuses/dam in the mid-dose group were significantly
(p<0.01) lower than control; however, no significant differences in these parameters were
observed in the high-dose group. Statistically significant decreased mean fetal weight was
observed at the high dose compared with controls (36.3+3.19, 35.3+3.77, 36.0+4.97 and
29.7+5.8 g in the control, low-, mid-, and high-dose groups, respectively). There were
statistically significant increases in the percentage of malformed live fetuses/litter (33.3%) and
the percentage of litters with malformed fetuses (87.5%, seven of eight litters) in the high-dose
group compared with controls (0% for both effects). These parameters were not significantly
elevated in low- (1.1 and 10%) and mid- (3.6 and 13.3%) dose rabbits, although a few malformed
fetuses were observed at the lower doses: one fetus with hydrocephalus in the low-dose group and
three fetuses with hydrocephalus in 2 of 16 litters in the mid-dose group. At the high dose, there
were six fetuses with hydrocephalus, one with abnormal position of the extremities, two with
exophthalmos, three with simple eventration, seven with umbilical hernia and one with cleft
palate. Litter incidences of specific effects were not reported. Thus, the high dose of 188.9
mg/kg-day was teratogenic, fetotoxic and maternotoxic in rabbits. The mid-dose of 64.3 mg/kg-
day is considered a NOAEL in the absence of statistically significant effects on fetal weight and
malformations at this dose.
Inhalation Exposure
The data set for inhalation toxicity of dimethylformamide includes subchronic toxicity
studies in monkeys, rats, mice and other species (Hurtt et al., 1992; Craig et al., 1984; NTP,
1992b; Tanaka, 1971; Massmann, 1956b; Clayton et al., 1963), chronic toxicity studies in rats and
mice (Malley et al., 1994), and developmental toxicity studies in rabbits and rats (Hellwig et al.,
1991; Lewis et al., 1992; Kimmerle and Machemer, 1975; BASF, 1974).
Hurtt et al. (1992) whole-body exposed groups of cynomolgous monkeys (3/sex/group) to
filtered room air or to dimethylformamide (purity >99%) at inhalation concentrations of 30, 100
or 500 ppm (90, 300 or 1500 mg/m3) for 6 hours/day, 5 days/week for 13 weeks. The test
atmospheres were primarily vapor; the aerosol concentration of the 500 ppm atmosphere was
calculated to be less than 0.01% of the total mass of dimethylformamide. Since the lower
concentrations were generated by diluting the 500 ppm atmosphere with filtered air, the aerosol
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concentrations were likewise reduced. The study included satellite groups of two males per
exposure concentration that were given a 13-week recovery period after the last exposure.
Assignment of males to the exposure groups included pre-exposure assessment of sperm
parameters so that group mean values were similar. Monkeys were observed twice daily for
morbidity or mortality and once per week for clinical signs of toxicity. Body weights were
measured prior to the first exposure, weekly thereafter, and at termination. Hematology and
serum chemistry analyses were conducted on blood samples from all monkeys prior to and at the
end of the first exposure, at the end of weeks 2, 4, 8 and 12, and at scheduled necropsy. Two
monkeys/sex/group were placed in metabolism chairs to collect 6-hour samples for urinalysis on
the same schedule. Sperm concentration, motility and morphology were analyzed in semen
collected from males 3 times prior to the start of the study (baseline) and once a week thereafter.
Menstrual cycles were monitored by daily vaginal smears beginning about 2 weeks after the first
exposure. At scheduled necropsy, organ weights were measured for the liver, thyroid/parathyroid
(paired), pituitary, heart, lungs, individual kidneys, testes and ovaries. More than forty tissues,
including three sections of nasal epithelium, were evaluated for histopathology in all animals.
Inhalation exposure to dimethylformamide had no effect on mortality, the incidence of clinical
signs, body weight changes, hematology, serum chemistry, urinalysis, semen volume and sperm
analyses, menstrual cycles, organ weights, gross necropsy findings or incidence of
histopathological lesions. (Some lesions in lung, liver and lymph nodes were attributed to
parasitic infection.) In this study, the highest exposure level, 500 ppm (1500 mg/m3), was a
NOAEL for cynomolgous monkeys exposed by inhalation to dimethylformamide.
Craig et al. (1984) exposed 10 F344 rats/sex or 10 B6C3F1 mice/sex to 0, 150, 300, 600
or 1200 ppm (0, 448, 897, 1794 or 3587 mg/m3) of dimethylformamide for 6 hours/day, 5
days/week for 12 weeks. Animals were observed for clinical signs of toxicity, body weights were
determined biweekly and complete gross necropsies were performed on all animals. Clinical
chemistry analyses were performed on all animals that survived to terminal sacrifice.
Histopathological evaluation was conducted on the lungs, heart, liver, thymus, spleen, pancreas,
kidneys, urinary bladder, testes and nasal passages. The rats exposed to 1200 ppm showed few
signs of overt toxicity, but their weight gain was significantly less than the control animals. The
female rats exhibited an increase in serum AP, and one high-dose animal had high ALT and AST
levels that may be indicative of acute hepatocellular injury. Gross necropsy findings included
increased discoloration of the lungs in high-dose rats, but no lesions were found in the lungs or
nasal turbinates in histological examination. The only treatment-related changes occurred in the
liver. These changes consisted of pale, enlarged livers with an accentuated lobular pattern and/or
prominent capsular blood vessels. Histopathology revealed that the livers of two rats that died
during the study exhibited wide-spread collapse, necrosis, and accumulation of yellow-brown
pigments in Kupffer cells, macrophages and hepatocytes. The livers of the high-dose females
were found to contain areas of collapse near the central veins with occasional fibrosis and
yellow-brown pigment, and large variations in nuclear size and cytoplasmic characteristics. The
livers of the females exposed to 300 or 600 ppm of dimethylformamide showed variation in
nuclear size and cytoplasmic characteristics that were similar to the higher doses, but to a lesser
extent. No changes were seen in the livers of rats exposed to 150 ppm of dimethylformamide.
Livers from the male rats exhibited the same changes as those described for the females except
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that there was no collapse or fibrosis. A LOAEL of 300 ppm (897 mg/m3) and a NOAEL of 150
ppm (448 mg/m3) is identified for these hepatic cell changes.
The mice exposed to dimethylformamide exhibited no clinical signs of toxicity, no
changes in body weight gain and no hematologic or clinical chemistry changes attributable to
treatment (Craig et al., 1984). However, eight high-dose mice and three mice exposed to 600
ppm dimethylformamide died or were sacrificed moribund during the study. The livers from the
treated mice in all groups revealed collapse, necrosis and yellow-brown pigment. Hepatic
cytomegaly was observed in all exposed groups and the incidence and severity of this lesion were
dose-related. No effects were reported in the respiratory tract. These results indicate a LOAEL
of 150 ppm (448 mg/m3) for dimethylformamide in mice; a NOAEL was not identified.
NTP conducted subchronic inhalation toxicity studies of dimethylformamide in rats and
mice (Lynch et al., 2003; NTP, 1992b). Groups of F344/N rats (30/sex/group) were whole-body
exposed to dimethylformamide (purity >99%) vapor at target concentrations of 0 (chamber
control), 50, 100, 200, 400 or 800 ppm (0, 150, 300, 600, 1200 or 2400 mg/m3) for 6 hours (plus
T90)/day, 5 days/week for 13 weeks. Each group was equally divided into three subgroups, a base
study group, a cardiac physiology group and a urinalysis/renal function group. Rats were
observed twice daily for mortality and moribundity; body weights and clinical signs were
measured weekly and at necropsy. Comprehensive hematology and clinical chemistry studies
were performed on cardiovascular group rats at 4 and 23 days and on base-study rats at 13 weeks.
Reproductive system evaluations were performed on all rats exposed to 0, 50, 200 or 800 ppm;
sperm counts, morphology and motility were assessed at necropsy, and vaginal cytology was
evaluated during the 2 weeks preceding necropsy. At 13 weeks, comprehensive urinalysis was
conducted on five male and five female rats in the 0, 50, 200 and 800 ppm groups, and the
kidneys of these rats were evaluated for histopathology. Within 24 hours after the final exposure,
the cardiovascular groups were evaluated for blood pressure and electrocardiograms, and the
hearts were examined for histopathology. At termination, complete necropsies were performed
on rats in the base-study and renal function groups. Organ weights were recorded for the liver,
thymus, both kidneys, both testicles, heart and lungs. Gross lesions and more than thirty tissues,
including nasal turbinates, were evaluated for histopathology in all control and high-dose rats in
the base study; liver histopathology was assessed in all lower dose groups as well.
Dimethylformamide exposure had no effect on survival in the rats (Lynch et al., 2003;
NTP, 1992b). Concentration-dependent depression in body weight gain occurred over the course
of the study in both sexes at 400 ppm (6-11%) and 800 ppm (20-22%). Hematology evaluation of
the males showed increases in hematocrit and hemoglobin concentration at 800 ppm and
erythrocyte counts at >400 ppm at 24 and 91 days; the same parameters were elevated in the 800
ppm females at 4, 24 and 91 days. These findings were characterized as evidence of mild
hemoconcentration consistent with a mild dehydration, and not attributed to dimethylformamide
exposure. Reasons for the mild dehydration were not presented, and water and food consumption
were not monitored. Platelet counts were elevated in males at >100 ppm at 24 and 91 days; in
females, platelets were elevated in the >200 ppm groups at 24 days, but the values at 91 days
were not significantly different from controls. Serum cholesterol was elevated in both sexes at
>50 ppm at 4, 24 and 91 days; at 50, 100, 200, 400 and 800 ppm on day 91, increases were 13.3,
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22.9, 18.1, 18.1 and 61.4% higher than controls in males, and 11.3, 33.0, 18.6, 41.2 and 40.2%
higher than controls in females. Serum ALT was increased in all males at >400 ppm at all time
points. ALT was elevated in females at 800 ppm at all time points and at >100 ppm on day 24.
Total bile acids were increased in males at >400 ppm at all time points and in females at 800 ppm
on day 4, at >200 ppm on day 24 and at >400 ppm on day 91. Isocitric dehydrogenase activity
was increased in both sexes at 800 ppm at all time points. Sorbitol dehydrogenase activity was
increased in both sexes at >200 ppm at all time points; at 50, 100, 200, 400 and 800 ppm on day
91, increases were 17.1, 17.1, 100, 169 and 549% higher than controls in males, and 0, 11.5, 53.8,
84.6 and 558%) higher than controls in females. Concentrations of total protein and albumin were
decreased in males at >200 ppm on day 4, but were elevated in the >400 ppm groups on day 24
and the 800 ppm group on day 91. In females, total protein and albumin were decreased at 800
ppm on day 4, at 400 ppm on day 24 and at >200 ppm on day 91. Absolute lung weights were
significantly decreased in males and females exposed at >50 ppm; relative lung weights were
significantly lower in all exposed groups except for males at 400 ppm and females at 800 ppm.
Relative liver weights were increased in both sexes at >50 ppm; absolute liver weights were
increased in females at >50 ppm and males at >200 ppm. Relative kidney weights were increased
in males at >100 ppm and females at 800 ppm. There were no treatment-related changes in
urinalysis indices. Exposure had no effect on heart rate or blood pressure. Eight exposed rats had
qualitatively abnormal electocardiographic waveforms, but there was no consistent relationship to
concentration. Absolute heart weights were reduced in males and females at >400 ppm, but this
was related to the reduced body weight. Treatment had no adverse effect on sperm parameters or
testicular/epididymal weight or histology. There were no significant group differences in estrous
cycle length, although alterations (prolonged diestrus) occurred at 800 ppm: undefinable or longer
than 12 days in 7/10 animals and slightly longer than controls in 3/10 animals (5.3 vs 5.0 days).
No gross pathology was observed in any organ or tissue and histopathological changes were
found only in the liver. Liver lesions occurred at >400 ppm in both sexes and included
hepatocyte necrosis in 10/10 and 10/10 males and 8/10 and 10/10 females at 400 and 800 ppm,
and pigment in macrophages in 10/10 males and 10/10 females at 800 ppm; incidences of both
lesions were 0/10 in all lower dose and control groups. As summarized above, the liver was the
most sensitive target as shown by effects that included increased serum cholesterol and relative
liver weight at >50 ppm, increased serum sorbitol dehydrogenase at >200 ppm and increased
serum ALT and liver lesions (hepatocyte necrosis) at >400 ppm. The toxicological significance
of the serum cholesterol changes is unclear because the increases were small (particularly at the
lower concentrations) and not clearly dose-related, and information on normal ranges in F344/N
rats of comparable age (19 weeks) was not located. Based on increased serum sorbitol
dehydrogenase at 200 ppm and supporting histological evidence of liver cell necrosis at 400 ppm,
the LOAEL is judged to be 200 ppm (600 mg/m3). The NOAEL is 100 ppm (300 mg/m3), the
highest concentration causing increased liver weight in the absence of the serum enzyme
indication of hepatocellular damage.
The subchronic inhalation study in B6C3Fi mice (Lynch et al., 2003; NTP, 1992b) used
the same experimental design as for the rats, including exposure to 0, 50, 100, 200, 400 or 800
ppm dimethylformamide for 6 hours/day, 5 days/week for 13 weeks, except for smaller numbers
of animals (10/sex/group), minor differences in the tissues selected for histopathology and lack of
hematology, clinical chemistry, urinalysis and satellite groups for cardiovascular and renal
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studies. There were no exposure-related effects on survival, body weight gain or incidence of
clinical signs in mice. Treatment had no effect on testicular/epididymal weights or sperm
parameters. There were no significant group differences in estrous cycle length, although there
was a significant trend (p=0.035) toward an increase in estrous cycle length (4.15, 4.05, 4.55 and
4.80 days at 0, 50, 200 and 800 ppm; data for 400 ppm were not reported). Relative and/or
absolute kidney and lung weights were increased in all exposed female groups, but not in males.
Relative liver weights were increased in both sexes at >50 ppm; absolute liver weights were
increased in males at >200 ppm and in females at >50 ppm. Necropsy showed a gross liver lesion
that may have been exposure-related; tan foci were noted in the liver of one male each in the 400
and 800 ppm groups. Histological changes were also limited to the liver, consisting of minimal to
mild centrilobular hepatocellular hypertrophy in males at >50 ppm and females at >100 ppm.
Incidences of this effect at 0, 50, 100, 200, 400 and 800 ppm were 0/10, 4/10, 9/10, 10/10, 10/10
and 10/10 in males, and 0/10, 0/10, 10/10, 10/10, 10/10 and 10/10 in females. The lowest
exposure level of 50 ppm (150 mg/m3) is classified as a LOAEL based on increases in liver
weight and histological changes (centrilobular hepatocellular hypertrophy) supported by possible
gross liver pathology at 400 ppm.
Senoh et al. (2003) evaluated the subchronic inhalation toxicity of dimethylformamide in
rats and mice. Groups of 10 male and 10 female F344/DuCrj rats were exposed to
dimethylformamide (>99.8% pure) in target concentrations of 0, 50, 100, 200, 400 or 800 ppm (0,
150, 300, 600, 1200 or 2400 mg/m3) for 6 hours/day, 5 days/week for 13 weeks. Deviations of
mean observed concentrations from the target levels were <3.5%. The study conformed to OECD
guidelines for a 90-day inhalation toxicity study. Animals were assessed daily for clinical signs
and mortality and weekly for body weight and food consumption. Comprehensive hematological,
blood biochemical, urinary, gross pathological, organ weight and histological evaluations were
performed on all surviving animals at the end of the 13-week exposure period. Statistically
significant effects included reduced body weight gain in both sexes at >400 ppm, reduced food
consumption in both sexes at 800 ppm, increased serum total cholesterol in males at >50 ppm
(58.9-142.9%) higher than controls) and females at >200 ppm (64.2-107.4%) higher) and increased
serum phospholipids in males at >50 ppm (48.6-70.1% higher than controls) and females at >100
ppm (34.8-64.5%) higher). Other statistically significant changes included increases in
triglycerides in females at >200 ppm and males at 800 ppm, total bilirubin in females at >400
ppm and males at 800 ppm, serum ALT in females at >400 ppm and males at 800 ppm and y-GTP
and ALP in females at >400 ppm; no effects on serum AST and LDH were observed in either sex.
Relative liver weight was statistically significantly increased in males at >100 ppm and females
at >200 ppm. Absolute liver weights tended to increase in both sexes but were not clearly dose-
related (significantly increased in males at 50-200 ppm and females at 50-100 ppm and 800 ppm).
Histopathological changes were limited to the liver and included significantly increased
incidences of single-cell necrosis of hepatocytes in both sexes at >200 ppm (80-100%) compared
to 0%> at 0-100 ppm) and centrilobular hepatocellular hypertrophy at >400 ppm (80-100%)
compared to 0% at 0-200 ppm). As summarized above, the most sensitive effects were increased
serum cholesterol and phospholipids at 50 ppm, increased relative liver weight at 100 ppm and
liver lesions (single-cell necrosis) at 200 ppm. The toxicological significance of the changes in
serum cholesterol and phospholipids is unclear because the increases were not pronounced
(except for cholesterol in high-dose males), standard deviations were not reported and
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information on normal ranges in F344/DuCrj rats could not be located. The LOAEL is 200 ppm
(600 mg/m3) based on liver histopathology (single-cell necrosis) and the NOAEL is 100 ppm (300
mg/m3), the highest level causing increased liver weight in the absence of histopathology.
Groups of 10 male and 10 female Cij:BDFi mice were exposed to 0, 50, 100, 200, 400 or
800 ppm (0, 150, 300, 600, 1200 or 2400 mg/m3) dimethylformamide for 6 hours/day, 5
days/week for 13 weeks (Senoh et al., 2003). The experimental design is the same as in the rat
part of this study summarized above. Statistically significant effects included decreased body
weight gain in males at >50 ppm and food consumption in males at 800 ppm; there were no
effects on these endpoints in females. Blood biochemical changes occurred in both sexes
including statistically significant increases in total cholesterol in females at >50 ppm (26.4-38.9%
higher than controls) and males at 100 and 400 ppm (18.8 and 27.5% higher; non-significantly
increased at 200 and 800 ppm), serum ALT in females at >200 ppm and males at 800 ppm, serum
ALP in males at 800 ppm, and LDH and blood urea nitrogen in females at 800 ppm. Relative
liver weight was significantly increased in males at >50 ppm, absolute liver weight tended to
increase in males but was not clearly dose-related (significantly increased at 100-400 ppm) and
liver weight was not significantly affected in females. Histopathological changes were limited to
the liver and included significantly increased incidences of centrilobular hepatocellular
hypertrophy in males at >50 ppm (40% at 50 ppm and 100% at 100-800 ppm compared to 0% in
controls) and females at 800 ppm (70% compared to 0% at 0-400 ppm), focal necrosis in females
at 100-400 ppm (50-70% compared to 0% in controls and 10% at 50 and 800 ppm) and single-cell
necrosis in males at 800 ppm (60% compared to 0% at 0-200 ppm and 10% at 400 ppm) and
females at 800 ppm (50% compared to 0% at 0-400 ppm). As summarized above, the most
sensitive effects in this study were increased serum cholesterol, increased relative liver weight
and centrilobular hypertrophy at 50 ppm and focal necrosis at 100 ppm. The toxicological
significance of the serum cholesterol changes is unclear because the increases were small,
standard deviations were not reported and information on normal ranges in Crj :BDFi mice could
not be located. The lowest exposure level, 50 ppm (150 mg/m3), is the LOAEL based on
increases in liver weight and hepatocellular hypertrophy at 50 ppm and supporting evidence of
focal necrosis at 100 ppm.
Malley et al. (1994) evaluated chronic inhalation toxicity and oncogenicity in rats and
mice. Groups of CD rats (78/sex/group) were whole-body exposed to humidified air (control) or
atmospheres containing 25, 100 or 400 ppm (75, 300 or 1200 mg/m3) of dimethylformamide
vapor for 6 hours/day, 5 days/week (excluding holidays) for 2 years. Rats were examined twice
daily to detect mortality, morbidity and abnormal behavior and appearance. Rats were weighed
and examined for clinical signs once per week for the first 3 months and once every other week
thereafter. Ophthalmological examinations were conducted prior to the first exposure and
immediately prior to scheduled sacrifice. Hematology, clinical chemistry and urinalysis were
conducted at 3, 6, 12, 18 and 24 months on rats randomly selected prior to the first exposure: one
set of 10 per sex per group examined at the first three time points and sacrificed at 12 months and
a second set examined at 18 and 24 months. All rats were examined by gross necropsy. At
scheduled necropsies, organ weights were recorded for the lungs, brain, liver, kidneys, adrenals,
ovaries and testes. For all animals, gross lesions and more than 40 tissues were processed for
histopathology. All tissues were examined microscopically for the control and 400 ppm groups
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and for rats found dead or euthanized in extremis. The nose, lungs, liver, kidneys and all gross
lesions were examined microscopically for the 25 and 100 ppm groups; the uterus was also
examined in these groups because of the incidence of endometrial stromal polyps in the 400 ppm
females. Cell proliferation in the liver, as measured by hepatocyte labeling following
intraperitoneal injection with 5-bromo-2'-deoxyuridine (BrdU), was evaluated in rats
(5/sex/group) after 2 weeks, 3 months and 12 months of testing; all livers were examined
microscopically, but the control and 400 ppm livers were also evaluated immunohistochemically.
The estrous cycle was monitored in control and 400 ppm females by vaginal smears taken on days
107-131.
Chronic inhalation exposure to dimethylformamide had no effect on survival,
ophthalmoscopic findings, hematology parameters or estrous cycle in rats (Malley et al., 1994).
Mean body weight gains were significantly reduced in males at >100 ppm and in females at 400
ppm. Serum levels of sorbitol dehydrogenase activity were significantly increased in both sexes
exposed at >100 ppm for 3 or 12 months, and in males exposed at >100 ppm and females exposed
at >25 ppm for 18 months; significant elevations in all treated male groups at 24 months were
attributed to an unusually low mean value for control males. Urinalysis results were not reported.
Relative liver weights were significantly increased in both sexes exposed at >100 ppm for 12
months or at 400 ppm for 24 months; significant elevations were also noted in females euthanized
for the hepatic cell proliferation studies on day 19 (all exposed groups) and on day 95 (400 ppm
group only). The authors did not consider the elevation in 25 ppm females on day 19 to be
toxicologically significant because of its transience and because it was not associated with
histopathology. No significant increase in the incidence of gross lesions was observed in rats
exposed for 12 or 24 months. However, the incidence and severity of liver histopathology
increased during the course of the study. At 12 months, the incidence of centrilobular
hepatocellular hypertrophy was elevated in males at 400 ppm and in females at >100 ppm; in
addition, the incidences of single-cell necrosis, accumulation of lipofuscin/hemosiderin, and clear
cell foci were increased in both sexes exposed at 400 ppm. At 24 months, the incidences of
centrilobular hepatocellular hypertrophy and accumulation of lipofuscin/hemosiderin were
elevated in both sexes at >100 ppm; single cell necrosis was increased in males at 400 ppm and in
females at >100 ppm; clear cell foci were increased at >100 ppm in males, and clear and
eosinophilic foci were increased at 400 ppm in females. Hepatic cell proliferation in males at 400
ppm was slightly elevated, but no statistically significant difference was observed for treated rats
of either sex compared to controls. The incidence of endometrial stromal polyps was elevated in
rats at 400 ppm compared to controls (1.7, 5.1, 3.4 and 14.8% for the control and 25, 100 and 400
ppm groups, respectively). Since the incidence of the lesion was considered highly variable, and
the incidence at 400 ppm was within the historical control range for the laboratory (2.0-15.0%),
the authors considered the lesion to be a chance variation and not an oncogenic effect of
treatment. No treatment-related lesions were observed in any other tissue, including the nose or
respiratory tract. In this study, 25 ppm (75 mg/m3) was a NOAEL and 100 ppm (300 mg/m3) was
a LOAEL for hepatic effects (increases in relative organ weight, histopathology and serum
sorbitol dehydrogenase activity) in male and female rats exposed for 24 months.
The protocol for CD-I mice in the chronic inhalation study of Malley et al. (1994) was
similar to that for rats with the following exceptions. Groups of mice (78/sex/group) were
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exposed to dimethylformamide at the same concentrations (0, 25, 100 and 400 ppm or 0, 75, 300
and 1200 mg/m3) for 6 hours/day, 5 days/week for 18 months (study termination). Clinical
evaluations of mice (10/sex/group) at 3, 6, 12 and 18 months only included hematology and not
clinical chemistry or urinalysis. Adrenals were not weighed at scheduled necropsy. The battery
of tissues processed for histopathology included the gall bladder, but excluded the prostate.
Chronic inhalation exposure to dimethylformamide had no effect on survival, ophthalmoscopic
findings, hematology parameters or estrous cycle in mice. Body weight gain was elevated in
males exposed at 400 ppm and in females exposed at >100 ppm during the first year of the study
(results for the period between 12 and 18 months were not reported). Progressive hepatic effects
were observed during the course of the study. Absolute and relative liver weights were
significantly increased in both sexes exposed to 400 ppm at hepatocyte proliferation terminations
on days 19 and 95; at the hepatocyte proliferation termination on day 363, the same weight
increases at 400 ppm were significant in males but slight in females. At 18 months, absolute and
relative liver weights were significantly increased in males at >100 ppm and females at 400 ppm.
Gross necropsies revealed a higher incidence of enlarged livers and liver deformities (not
specified) in males exposed at 400 ppm (Malley et al., 1994). Dose-related increases in liver
histopathology were observed in all treated groups of males and females. Increases in
centrilobular hepatocellular hypertrophy and single-cell necrosis were observed in both sexes
exposed at >25 ppm. Hepatic Kupffer cell hyperplasia with accumulation of lipofuscin and
hemosiderin and an increase in the incidence of inflammatory cells in the liver were observed in
males at >25 ppm and in females at >100 ppm. Mixed hepatic foci were observed in male mice
exposed at >100 ppm. The authors considered that 'secondary changes' observed in livers from
mice exposed at >100 ppm were adaptive or indicative of repair activity: biliary hyperplasia,
increased mitotic figures and multinucleate hepatocytes. The hepatocyte proliferation assays did
not detect any differences between treated and control mice. No treatment-related lesions were
observed in any other tissue, including the nose or respiratory tract. Treatment with
dimethylformamide did not significantly increase the incidence of any specific tumor type. The
authors reported that the incidence of total primary tumors and total benign tumors was
significantly increased in males exposed at 400 ppm because of higher combined numbers for
lung, liver and harderian gland tumors. The authors discounted these as related to treatment
because the individual increased incidences were not statistically significant and because they are
reported to have a high spontaneous incidence in male mice. However, the authors did not
provide any quantitative data for the tumors in this mouse study or the historical control
incidences. In this study, the low dose of 25 ppm was a LOAEL (75 mg/m3) for hepatic effects in
mice: increases in centrilobular hepatocellular hypertrophy and single-cell necrosis in males and
females and Kupffer cell hyperplasia with accumulation of pigment in males. A NOAEL was not
identified.
Tanaka (1971) studied the effects of inhalation exposure to dimethylformamide on female
Sprague-Dawley rats of varying ages. Measurement of liver enzyme activities in serum and liver
histopathology were conducted on 3-, 4-, 5-, 8-, and 12-week-old rats exposed to 200 ppm of
dimethylformamide (598 mg/m3) 8 hours/day 7 days/week for 4 weeks. Serum ALT and AST
activities were increased in the 3- and 4-week-old rats, but not in the rats that were 5 weeks or
older. Serum AP activity was increased in rats that were up to 5 weeks of age. Histopathological
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changes were observed primarily in the livers of the younger animals, including degeneration,
cloudy swelling of liver cells, and isolated cases of fatty degeneration primarily in the central
zone. This study identifies a LOAEL of 200 ppm (598 mg/m3) for liver damage. Tanaka (1971)
also exposed groups of 3-week-old female rats to 200 ppm of dimethylformamide for either 1 or 8
hours/day every day for 4 weeks. The rats were sacrificed after 1, 2 or 4 weeks and liver function
and pathology were evaluated. Serum AST and ALT were elevated in both groups of animals.
The liver damage observed in the exposed animals was qualitatively similar across both groups
(degeneration with evidence of regeneration after 2 and 4 weeks of exposure), but the animals
exposed to dimethylformamide for 8 hours/day showed more severe lesions.
Massmann (1956b) exposed groups of 2 cats and 16 rats to either 100, 230, or 450 ppm
(299, 688 or 1345 mg/m3) of dimethylformamide 8 hours/day, 6 days/week for 120 days. Clinical
signs of toxicity, body weight, hematological parameters and liver function were measured, as
well as the electrocardiogram (in cats only). No overt signs of toxicity were noted in the rats, but
the cats ate less and lost weight. No hematological changes were noted in either species, and the
liver function tests were normal. Necropsy revealed an "irregular incidence" of liver necrosis in
the rats, but the cats exhibited only fatty degeneration without necrosis. Other changes noted in
the rats included bronchopneumonia, hyperemia of the brain, cloudy swelling of the uriniferous
tubules of the kidney and iron deposits in the spleen. Data on incidence of these effects are not
included in the report, but it is implied that liver changes were noted in the cats at dimethyl-
formamide concentrations of 100 ppm or more, while the rats were not adversely affected at
dimethylformamide concentrations below 450 ppm. Therefore, the NOAEL in rats is 230 ppm
(688 mg/m3).
Clayton et al. (1963) reported that rats exposed to 91 ppm (272 mg/m3)
dimethylformamide 6 hours/day for 10 days had increased relative liver weight. Clayton et al.
(1963) also exposed mice (11 females), rats (10/sex), guinea pigs (10 males), rabbits (2/sex), and
dogs (4 males) to 23 ppm of dimethylformamide for 5.5 hours/day, followed by 426 ppm for 0.5
hours/day for 58 weekdays. This exposure regimen was designed to simulate peak exposures that
occur in plant operations. No adverse clinical signs were seen in any species except dogs, where
one of the four animals had a decrease in systolic blood pressure. This dog also exhibited
degenerative myocardial changes at necropsy. Increased liver weights were seen in all species
except the guinea pig, but the difference was statistically significant only in mice. Plasma
cholesterol levels were increased in the rats, rabbits, and in the single dog that demonstrated
cardiovascular changes. Rat liver fat content was also increased. A LOAEL of 56.6 ppm (172
mg/m3) is calculated as a TWA concentration for a 6-hour exposure.
Hellwig et al. (1991) evaluated the developmental toxicity of dimethylformamide in
rabbits and rats exposed by inhalation. Groups of 15 artificially inseminated Himalayan rabbits
were exposed (whole-body) to air or dimethylformamide vapor at concentrations of 50, 150 or
450 ppm (150, 450 or 1350 mg/m3) for 6 hours/day on GD 7-19. Dams were observed until GD
29 and then subjected to caesarean section and necropsied. The following endpoints were
evaluated: maternal body weight, clinical signs, food consumption, gross pathology, corpora
lutea, uterine and placental weight, conception rate, living and resorbed implantations, pre- and
postimplantation loss, live fetuses, fetal weight and fetal length. Live fetuses were evaluated for
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external, soft-tissue or skeletal findings (retardations, variations or anomalies). Exposure to
dimethylformamide vapor had no effect on mortality, the incidence of clinical signs, necropsy
findings, uterine weights, or conception rate in dams. Maternal body weight gain was reduced in
dams at 150 ppm, and body weight loss occurred in the 450 ppm dams during the period of
exposure (GD 7-19); maternal body weight gain for the entire study (GD 0-29) was lower in
treated groups than in controls, but the difference was not statistically significant and did not
show a clear concentration-response. Exposure at 450 ppm resulted in significant reductions in
fetal weights, and significantly increased total incidences of external malformations (mostly
hernia umbilicalis) and skeletal abnormalities (sternal anomalies and variations). In the 150 ppm
group, one hernia umbilicalis and a slight increase, of borderline significance, in the incidence of
fused sternebrae was observed (p>0.053 in Fisher Exact test conducted for this review). In this
study, the maternal NOAEL was 50 ppm (150 mg/m3) and the LOAEL was 150 ppm (450 mg/m3)
for reduced body weight gain (during exposure). The developmental NOAEL was 50 ppm (150
mg/m3) and the LOAEL was 150 ppm (450 mg/m3) for minimal increases in the incidences of
certain anomalies (hernia umbilicalis and fused sternebrae) that were statistically significant at the
highest concentration.
Hellwig et al. (1991) evaluated the inhalation developmental toxicity in rats in two
separate experiments using different discontinuous exposure protocols. In each experiment,
groups of 30 pregnant Sprague-Dawley rats were whole-body exposed to air or 287 ppm (860
mg/m3) of dimethylformamide vapor for 6 hours/day. In experiment I, exposures were on GD
0-1, 4-8, 11-15 and 18-19; in experiment II, rats were exposed on GD 0-3, 6-10 and 11-18.
Randomly selected dams from each group were permitted to litter and raise offspring (satellite
group). The following parameters were recorded: clinical signs, mortality, gross pathology,
uterine weights, conception rates, implantations (total, live and dead), resorptions (early, medium-
term and late), pre- and postimplantation loss, placental weight, and sex, length and weight of live
fetuses. Live fetuses were examined for external anomalies and then for soft tissue (1/3) or
skeletal anomalies (2/3). Satellite groups were evaluated for conception rate, implantations and
resorptions, litter size, pup weight gain, pup mortality, viability and lactation index; on postnatal
day 20, pups were subjected to a gross necropsy, including assessments of organ weights and
head structure. Diseased pups were evaluated for skeletal anomalies. Results were similar in the
two experiments. Under either protocol, exposure at 287 ppm for 6 hours/day reduced maternal
weight gain by about half. The number of early resorptions and dead implants was elevated in
both experiments; the increase in dead implants was statistically significant in experiment I. In
both experiments, there were significant reductions in fetal weight, fetal length and placental
weights in the groups exposed at 287 ppm. Exposed groups showed increases in the incidence of
skeletal variations (sternal aplasia and displacement) and retardations (not specified) compared to
the controls. Exposure had no effect on pups in the satellite group. The single exposure level of
287 ppm (860 mg/m3) is a LOAEL for maternal toxicity (reduced maternal weight gain and
reduced placental weight) and developmental toxicity (reduced fetal weight and increases in
skeletal variations).
Lewis et al. (1992) evaluated developmental toxicity in groups of 21 pregnant CD rats
exposed to air or to 30 or 300 ppm (90 or 900 mg/m3) of dimethylformamide vapor by whole-
body inhalation for 6 hours/day on GD 6-15. Dams were evaluated daily for clinical signs,
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weighed on GD 0, 6-15 and 21, and given a gross pathological examination on GD 21. The
following endpoints were examined: number of corpora lutea, number and position of live, dead
and resorbed fetuses, and fetal weight, length, sex, external malformations (all), visceral
alterations (2/3) and skeletal changes (1/3). Exposure had no effect on survival or the incidence
of clinical signs or necropsy findings in dams. Body weight gain was reduced during GD 6-15 in
the dams exposed at 300 ppm. Fetal weight was also reduced in the 300-ppm group compared to
controls. There were no dose-related malformations. In this study, 30 ppm (90 mg/m3) was a
NOAEL for both maternal and developmental toxicity and 300 ppm (900 mg/m3) was a LOAEL
for reduced body weight gain in dams and reduced fetal body weight.
Kimmerle and Machemer (1975) reported that exposure of 22 or 23 female rats to 18 or
172 ppm (54 or 514 mg/m3) of dimethylformamide 6 hours/day on gestation days 6-15 produced
no evidence of maternal toxicity or fetal malformations. However, fetal body weight was
significantly reduced in the high-dose group as compared with the controls, suggesting a LOAEL
at 172 ppm (514 mg/m3) and a NOAEL at 18 ppm (54 mg/m3).
In an unpublished experiment conducted by BASF (1974), pregnant rats were exposed to
either 220 or 520 ppm (660 or 1554 mg/m3) of dimethylformamide on gestation days 4-8. A
significant reduction in maternal weight gain was observed at 520 ppm, and a reduction in fetal
weight and length was observed in both treated groups. The mean number of live fetuses was
reduced in animals exposed to 520 ppm. The authors report that there was "an increased number
of retardations and variations" found in the offspring of animals exposed to 520 ppm of
dimethylformamide, but these effects were not evident in the data presented. This study identifies
a LOAEL for a reduction in fetal weight and length at 220 ppm (660 mg/m3).
Other Studies
Biochemical Changes
In a study by Savolainen (1981), groups of 10 male Wistar rats were exposed to
dimethylformamide in the drinking water at concentrations of 0, 1.4, 6.8 or 13.7 mmol/L (0,
102.3, 497.0 or 1001 mg/L); five rats/group were killed after 2 and 7 weeks, and determinations
of brain enzyme activities were made. Based on body weight and water consumption data, the
rats consumed total doses of dimethylformamide of 0, 1.5, 7.5 or 14.4 mmol/kg (0, 7.8, 47.0 or
75.2 mg/kg-day) after 2 weeks and 0, 4.7, 24.0 or 43.9 mmol/kg (0, 7.0, 35.8 or 65.5 mg/kg-day)
after 7 weeks. No effects were observed on body weight gain or behavior. High-dose rats had
significantly (p<0.05) decreased levels of cerebral glutathione at 2 and 7 weeks. A dose-related
decrease in the activity of cerebral succinate dehydrogenase occurred that was significantly lower
than control in the mid- and high-dose rats at 2 weeks; the activity remained depressed at 7
weeks. Azoreductase activity was decreased significantly at all dose levels at 7 weeks, while
RNA content of the cerebrum was increased significantly at the high dose after 7 weeks.
Biochemical changes in the glial cells included significantly increased acid proteinase in the high-
dose group at 2 and 7 weeks, significantly dose-related increased activity of 2',3'-cyclic
nucleotide 3'-phosphohydrolase at 2 weeks, and significantly decreased glial cell succinate
dehydrogenase at all levels after 7 weeks. These biochemical changes were discussed in relation
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to the inhibitory action of formic acid, a minor metabolite of dimethylformamide, on
mitochondrial cytochrome oxidase; the investigators concluded that dimethylformamide may
affect glial cells by inhibiting mitochondrial respiration. This study found biochemical changes
in the brain occurring primarily at doses of 36 mg/kg-day or above. Only a few minor changes
were seen at 7 mg/kg-day. None of the endpoints monitored are clearly indicative of an adverse
effect.
In a similar study of the effects of dimethylformamide on the drug-metabolizing enzymes
of the liver and kidney, groups of 10 male Wistar rats received the compound in the drinking
water at 0, 100, 500 or 1000 mg/L (Elovaara et al., 1983). By analogy to the Savolainen (1981)
study conducted in the same laboratory, corresponding doses were approximately 0, 7, 36 or 66
mg/kg-day. Five rats/group were killed after 2 and 7 weeks, and livers and kidneys were
removed, weighed and homogenized. No effect on body weight gain or kidney weight was
observed; however, there was a dose-related significant increase in relative liver weight after 2
weeks (statistically significant in the 36 and 66 mg/kg-day groups), which became more
pronounced at 7 weeks (statistically significant in all treated groups). In the liver, no treatment-
related effects on cytochrome P450 content or NADPH-cytochrome c reductase activity occurred,
but glutathione levels, 7-ethoxycoumarin o-deethylase activity, and most markedly, UDP
glucuronyl transferase activity all increased in a time- and dose-related manner (statistically
significant differences from control primarily at 36 and 66 mg/kg-day). Although incubation of
dimethylformamide with rat liver microsomes did not result in liberation of formaldehyde, there
was a significant increase in endogenous formaldehyde liberation by microsomes from rats
treated at 66 mg/kg-day for 7 weeks, and significantly decreased activities of formaldehyde and
propionaldehyde dehydrogenases at 36 and 66 mg/kg-day. In the kidneys, glutathione content
and deethylase activity increased and cytosolic formaldehyde dehydrogenase activity decreased.
This study found biochemical changes in the liver and kidney, and an increase in relative liver
weight, occurring at doses of 7 mg/kg-day or above. However, these changes are considered to
be an adaptive response to dimethylformamide, not evidence of a toxic effect.
Toxicokinetics
Dimethylformamide is readily absorbed by all routes of exposure. Following an
application of 2 ml of liquid dimethylformamide over an area of 100 cm2 on the forearm of
volunteers, percutaneous absorption occurred at a rate of 9 mg/cm2/hour (IARC, 1999). A
significant amount of percutaneous absorption occurred in volunteers exposed to atmospheres
containing 50 mg/m3 of dimethylformamide vapor for 4 hours while breathing fresh air through
masks. Under conditions in which volunteers also inhaled the atmosphere, percutaneous
absorption accounted for 13-36% of a specific urinary metabolite, A-hydroxymethyl-A'-methyl-
form amide (HMMF) (IARC, 1999). Inhalation absorption in human volunteers exposed to
concentrations between 10 and 60 mg/m3 for 8 hours or 30 mg/m3 for 8 hours/day on 5
consecutive days, was approximately 90% (IARC, 1999). Analyses of urine recovered 0.3% as
unmodified dimethylformamide, 22% as HMMF, 13% as A-hydroxy methyl form amide and 13%
as the mercapturic acid conjugate A^-acetyl-,.Y-f7V-m ethyl carbamoyl )cysteine (IARC, 1999).
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The primary metabolic pathway for dimethylformamide in humans and rodents is the
a-hydroxylation of one alkyl moiety, specifically by cytochrome P450 2E1 to form HMMF
(IARC, 1999; Amato et al., 2001). HMMF decomposes to A^-m ethyl form amide, which undergoes
P450-mediated oxidation to form N-acetyl-S-(N-methylcarbamoyl)-cysteine (AMCC) via the
reactive carbamoylating intermediate, methylisocyanate (MIC). Humans and animals differ in the
proportion of specific urinary metabolites. In 10 volunteers exposed to 60 mg/m3 for 8 hours,
9.7-22.8% of the dose was excreted as AMCC in urine over 72 hours, whereas in rats, mice or
hamsters exposed by intraperitoneal injection, only 1.1-5.2% was excreted as AMCC (Mraz et al.,
1989). The higher production of AMCC in humans is thought to reflect the higher potential for
reactive intermediate generation in the human liver. This metabolic difference appears to account
for the greater hepatic sensitivity to dimethylformamide in humans as compared to laboratory
animals.
Dimethylformamide and its metabolites readily passed through the placenta and were
found in embryonic/fetal tissues following gavage administration of 100 mg/kg of the chemical to
16-hour fasted pregnant Sprague-Dawley rats on GD 12 or 18 (Saillenfait et al., 1997). Fasting,
however, is known to affect the pharmacokinetics (and toxicity) of chemicals. No qualitative or
quantitative differences were seen between maternal plasma and placenta, amniotic fluid or
embryo/fetus in the concentrations of parent compound and metabolites. HMMF was the most
prominent metabolite, followed by NMF. In rats dosed on GD 12 and monitored for 8 hours,
peak concentrations of parent compound had been attained by the first sampling time, 1 hour,
remained fairly constant through the second sampling time, 4 hours, and decreased by the third
sampling time, 8 hours. Levels of metabolites increased through the 8 hour sampling period such
that HMMF concentrations were equal to or higher than levels of parent compound by 8 hours.
In rats dosed on GD 18 and monitored for 24 hours, results were similar, but the major decline in
concentrations of parent compound occurred between 8 and 16 hours. In the same study,
concentrations of dimethylformamide, HMMF and NMF in the milk of lactating rats were in
equilibrium with concentrations in maternal plasma following gavage dosing of the non-fasted
dams on day 14 postpartum with 100 mg/kg of dimethylformamide.
A decrease in hepatic cytochrome P450 content has been reported in animals treated with
dimethylformamide following bioactivation of dimethylformamide. In an in vitro experiment,
incubation of 0-20 mM dimethylformamide with hepatic microsomes from pyridine-induced rats
resulted in concentration-dependent reductions of cytochrome P450 as high as 28% (Tolando et
al., 2001). Electron spin resonance analysis indicated that the suicidal inactivation of P450 was
the result of the modification of the prosthetic heme moiety by free radical intermediates of
dimethylformamide.
Hemoglobin adducts have been detected in the blood of workers in the polyacrylic fiber
industry who were exposed to dimethylformamide (Angerer et al., 1998). Blood levels of
hemoglobin with N-methylacarbomoylated valine residues were 100 times higher in
occupationally exposed individuals than in the general population. The formation of hemoglobin
adducts would be expected to result in deformation of erythrocytes, leading to their removal by
the spleen. This process may contribute to the reduced erythrocyte counts observed in the
subchronic dietary studies in rats (Kennedy and Sherman, 1986).
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Genotoxicity
Extensive genotoxicity testing on dimethylformamide yielded generally negative results in
bacterial and other non-mammalian systems. Dimethylformamide did not induce reverse
mutations in Salmonella typhimurium strains TA100 (16 studies), TA1535 (11 studies), TA1537
(12 studies), TA92, TA1530, TA1531, TA1532 and TA1964 with or without metabolic
activation, or in strains TA98 (14 studies) or TA1538 (7 studies) without activation (U.S. EPA,
1986; IARC, 1989, 1999). False positive results with activation were reported in a single study
on TA98 and TA1538, but all other tests with activation on these strains (14 and 8 studies,
respectively) yielded negative results (U.S. EPA, 1986; IARC, 1999). Dimethylformamide did
not induce forward or reverse mutations in Escherichia coli K-122/343/113 without activation, or
reverse mutations in E. coli WP2 uvrA strains with or without activation (IARC, 1999). With or
without activation, dimethylformamide gave negative results in the SOS repair test in S.
typhimurium TA1535/pSK1002 and in assays for differential toxicity in Bacillus subtilis (rec
strain) and E. coli (DNA-repair deficient strains) (IARC, 1999). Dimethylformamide did not
induce forward mutations in Saccharomyces cerevisiae XV185-14C or in Saccharomycespombe
with or without activation (IARC, 1999). It gave positive results with or without activation in one
test for homozygosis by mitotic recombination or gene conversion in S. cerevisiae D7, but
negative results in 'race XII' and strains D4 and JD1 (IARC, 1999). In tests for differential
toxicity in DNA-repair-deficient strains, dimethylformamide gave positive results in S. cerevisiae
rad and negative results in 'race XII' (IARC, 1999). Dimethylformamide did not induce heritable
sex-linked recessive lethal mutations in germ cells of male Drosophila melanogaster when
administered either in feed or by injection (IARC, 1999).
In vitro genotoxicity studies for dimethyformamide in mammalian cells were primarily
negative. With or without S9, dimethylformamide did not increase the mutation frequency in
human fibroblasts and in three studies on murine L5178Y tk+/~ lymphoma cells, but increased the
mutation frequency 2-fold in a single study on the lymphoma cells without S9 (IARC, 1999).
Without activation, dimethylformamide gave weakly positive results for unscheduled DNA
synthesis in cultured primary hepatocytes from Fischer 344 rat, but negative results in other tests
on hepatocytes from rat, mouse and hamster (IARC, 1999). Dimethylformamide did not induce
chromosomal aberrations in Chinese hamster ovary (CHO) cells, rat liver RLi cells, or, in one
study at a concentration of 80 mg/mL, in human lymphocytes; another study reported positive
results for chromosomal aberrations in human lymphocytes at a concentration of 7 ng/mL (IARC,
1999). Dimethylformamide did not induce sister chromatid exchanges (SCE) in human
lymphocytes without activation or CHO cells with or without activation (IARC, 1999).
In vivo genotoxicity studies in mammals provided only weak evidence for genotoxicity.
Studies on occupationally-exposed workers yielded mixed results (IARC, 1999). Berger et al.
(1985) reported an increased frequency of chromosomal gaps and breaks in 20 workers exposed
to mono-, di- and trimethylamines in addition to dimethylformamide (12 mg/m3) compared to 18
unexposed controls (1.4 vs. 0.4%, respectively); however, the authors noted that the control value
was low compared to other studies and the study did not address the effect of smoking (IARC,
1999). In a study that obtained blood samples at 4- or 6-month intervals from 40 workers
exposed to dimethylformamide and trace amounts of other organic compounds (2-butanone, butyl
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acetate, toluene, cyclohexanone and xylene), the frequencies of chromosomal aberrations in
peripheral lymphocytes were 3.82, 2.74, 1.59, 1.58 and 1.49%, respectively, following average
dimethylformamide exposures of 180, 150, 50, 40 and 30 mg/m3 (Koudela and Spazier, 1981);
aberration frequencies in two unexposed control groups were 1.61 and 1.10%. An abstract
reported that there was no evidence for an increased chromosomal aberration frequency in
occupationally-exposed workers, but no details were provided (IARC, 1999). Seiji et al. (1992)
compared the rates of sister chromatid exchanges in peripheral lymphocytes in 22 women
occupationally-exposed to dimethylformamide and 22 matched controls. The exposed group was
subdivided by level of exposure: 17.4 mg/m3 (high group), 2.1 mg/m3 in combination with an
equivalent amount of toluene (medium group) and 0.9 mg/m3 (low group). Compared to the
matched controls, the frequency of SCE was significantly higher in the medium and high
exposure groups but not in the low exposure group. In mice intraperitoneally injected with
dimethylformamide without exogenous activation, micronuclei were induced in the bone marrow
of the Kunming strain injected with a dose of 1 mg/kg, but not other strains injected at higher
doses: CD (1500 mg/kg), ICR (1600 mg/kg), B6C3Fi (2.5 mg/kg) or BALB/c (2000 mg/kg)
(IARC, 1989, 1999). Following intraperitoneal injection of dams with 3 mL/kg of
dimethylformamide, there was no evidence for morphological transformation in cultured Syrian
hamster embryos (IARC, 1999). An abstract reported no dominant-lethal effect in Sprague-
Dawley rats exposed to dimethylformamide by inhalation at a concentration of 900 mg/m3 for 6
hours/day on 5 consecutive days (IARC, 1999). Dimethylformamide gave negative results in a
bone marrow cell cytogenetics assay in rats exposed at 1200 mg/m3 for 7 hours or 7 hours/day for
5 days and in a mouse sperm morphology assay (IARC, 1999).
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC
ORAL RfD VALUES FOR DIMETHYLFORMAMIDE
No information was located regarding the oral toxicity of dimethylformamide in humans.
Oral systemic toxicity data in animals are available from subacute and subchronic studies in rats,
mice and gerbils, as summarized in Table 1; chronic oral testing has not been conducted. These
studies collectively indicate that the liver is the most sensitive systemic target of oral
dimethylformamide, as shown by effects that increased in severity with dose level. The target
organ effects included non-adverse changes in rats at 7-235 mg/kg-day (induction of microsomal
enzymes and increased liver weight, with no accompanying increases in serum ALT, AST or AP
or histological changes) (Becci et al., 1983; Elovaara et al., 1983; Kennedy and Sherman, 1986;
Haskell Labs, 1960); slight histological alterations (hepatocellular hypertrophy, increased mitosis)
and/or increases in serum ALT in mice at 246-326 mg/kg-day and rats at 422-426 mg/kg-day
(Becci et al., 1983; Kennedy and Sherman, 1986; Haskell Labs, 1960), liver necrosis with death in
gerbils at >500 mg/kg-day (Llewellyn et al., 1974) and severe hepatic necrosis and fibrosis in rats
at 708 mg/kg once weekly (Itoh et al., 1987). Additional considerations in the weight of evidence
for liver toxicity are that some subchronic and chronic inhalation studies in rats and mice report
that liver enlargement, hepatocellular hypertrophy, and single-cell or focal necrosis of hepatocytes
co-occurred at the lower exposures, and more severe hepatic necrosis and other more clearly
adverse changes occurred at higher exposures (Malley et al. 1994; Senoh et al. 2003). In addition,
occupational studies have reported elevated indices of liver toxicity and suggestive symptoms of
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Table 1. Summary of Non-Cancer Oral Data for Dimethylformamide in Animals
Study type
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Effects
Subacute exposure
Itohetal., 1987
Rats, F344, 8/sex/group
Gavage: undiluted, once weekly, 12 weeks
0, 708, 945 mg/kg-week
—
708 (once
weekly)
(PEL)
Hepatic fibrosis and severe hepatic necrosis in both
sexes, decreased BW gain and depressed activity in
males.
Subacute exposure
Kennedy and
Sherman, 1986
Rats, CD, 6 M/group
Gavage: 10% in water, nine doses/2 weeks
0, 450 mg/kg-day
450
"Discomfort" after dosing; variation in size and
staining of liver nuclei, frequent mitotic figures,
some hepatic cells contained two nuclei (these
changes seen in three treated rats examined after
the 9th dose but not in three rats following 11-day
recovery).
Subchronic
exposure
Kennedy and
Sherman, 1986;
Haskell Labs, 1960
Rats, CD, 6/sex/group
Dietary: 0, 200, 1000, 5000 ppm; 93-94 days
M: 0, 16, 77, 426 mg/kg-day
F: 0, 17, 85, 422 mg/kg-day
1000 ppm diet
77 (M)
85(F)
5000 ppm diet
426 (M)
422 (F)
Increased absolute and relative liver weight and
increased hepatocellular mitosis and enlargement.
Subchronic
exposure
Becci et al., 1983
Rats, Wistar, 25/sex/group
Dietary: 0, 215, 750, 2500 ppm; 104 days
M: 0, 18, 61, 210 mg/kg-day
F: 0, 20, 69, 235 mg/kg-day
2500 ppm diet
210 (M)
235 (F)
Changes in body and organ weights due to
decreased food consumption, not considered
adverse; no histological changes.
Subchronic
exposure
Becci et al., 1983
Mice, CD-I, 30/sex/group
Dietary: 0, 160, 540, 1850 ppm; 119 days
M: 0, 22, 70, 246 mg/kg-day
F: 0, 28, 96, 326 mg/kg-day
540 ppm diet
70 (M)
96(F)
1850 ppm diet
246 (M)
326 (F)
66% increase in serum ALT in females, increased
absolute and relative liver weight,
hepatocytomegaly (histopathologic examinations
conducted on all high dose and control mice but
only on 5/sex/dose for mid and low dose).
Subchronic
exposure
Llewellyn et al.,
1974
Gerbils, 12 females
DW: 10,000 ppm, 200 days
>500 mg/kg-day; no controls
10,000 ppm
DW
>500 (FEL)
Necrotic lesions in livers of four gerbils that died
{uncertain whether livers of survivors were
examined).
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Table 1. Summary of Non-Cancer Oral Data for Dimethylformamide in Animals
Study type
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Effects
Continuous
breeding/
2-Generation
Fail et al., 1998;
NTP, 1992a
Mice, Swiss CD-I, 40 (control) and 20 (treated
groups) breeding pairs at start
DW: 0, 1000, 4000, 7000 ppm
F0 M: 0, 185, 610, 1049 mg/kg-day
F0 F: 0, 225, 788, 1427 mg/kg-day
Fi M: 0, 226, 947, 1814 mg/kg-day
Fj F: 0, 292, 1098, 2054 mg/kg-day
F0
1000 ppm DW
185 (M)
225 (F)
Fi pups
1000 ppm DW
292
F0
4000 ppm DW
610 (M)
788 (F)
Fi pups
4000 ppm DW
1098
FI adults
1000 ppm DW
226 (M)
292 (F)
F2 pups
1000 ppm DW
292
Liver weight effects at >1000 ppm. Dose-related
effects on body weight primarily at mid and high
dose; centrilobular hepatocellular hypertrophy (in
livers with gross lesions) and decreased fertility at
mid and high dose.
Increased external malformations, particularly of
the head, and decreased pre- and post-natal
survival, decreased pup weight; crossover F0
mating study showed pup effects were mediated
through the dams and included abnormal or
incomplete ossification of cranium and sternebrae
and brain abnormalities.
Increased absolute and relative liver weights,
centrilobular hepatocellular hypertrophy (in livers
with gross lesions). At mid and high doses, also
liver weight effects and decreased body weights
and decreased fertility; malformations involving
abnormal or incomplete ossification of cranium and
sternebrae.
Decreased live F2 pup body weight at low dose.
At mid and high doses, decreased live pups,
increased pup malformations.
Developmental
Hellwig et al., 1991
Rats, SD, 19-23 pregnant rats/group
Gavage: in water
166, 503, 1510 mg/kg-day GD 6-15:
Controls were untreated
Maternal
Developmental
166
Maternal
166
Developmental
503
Reduced placental weight (4%), dose related
through higher doses; dose-related decreases in
maternal BW at mid and high doses but food
consumption not reported.
Increased resorptions (per litter); decreased fetal
BW and increased skeletal anomalies (but data not
analyzed per litter).
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Table 1. Summary of Non-Cancer Oral Data for Dimethylformamide in Animals
Study type
Reference
Description
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Effects
Developmental
Hellwig et al., 1991
Mice, NMRI, 24 pregnant mice/group
Gavage: in water
182, 548 mg/kg-day GD 6-15:
Controls were untreated
Maternal
548
Developmental
182
Developmental
548
No obvious effect on maternal BW and BW gain
but statistical analysis not performed and data not
provided, no clinical signs.
Reduced fetal length at low dose; fetal BW and
length decreased and anomalies increased at high
dose; but data not analyzed per litter.
Developmental, pre-
and post-natal
Fritz and Giese,
1990; Ciba-Geigy,
1979
Rats, SD, 10/group/duration
Gavage: in water
0, 750 mg/kg-day GD 10-14
Maternal
No data
Developmental
Maternal
No data
Developmental
750
Maternal endpoints including BW and food
consumption not reported.
Reduced fetal BW, delayed ossification lasting
through PND 5.
Developmental
Merkle and Zeller,
1980
Rabbits, Russian, 11-18/group
Gavage (in water)
0, 43.8, 64.3, 188.9 mg/kg-day GD 6-18
Maternal and
Developmental
64.3
Maternal and
Developmental
188.9
Decreased maternal food consumption and weight
gain; decreased fetal BW, increased percentage of
fetuses and litters with malformations, but litter
incidences of individual malformations not
reported.
Developmental
Saillenfait et al.,
1997
Rats, SD, 22-24 pregnant rats/group
Gavage: 0, 2.5, 5, 10, 15 % in water
0, 50, 100, 200, 300 mg/kg-day GD 6-20
Maternal and
Developmental
50
Maternal and
Developmental
100
Decreased maternal food consumption and BW
gain; decreased fetal BW.
At higher doses, dose-related depression of
maternal food consumption and maternal and fetal
BW gain and increased fetal skeletal variations
related to delayed ossification.
BW = body weight, DW = drinking water, F = female, M = male
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liver effects in exposed workers. The chronic RfC on IRIS is based on a LOAEL for "digestive
disturbances and minimal hepatic changes suggestive of liver abnormalities" in occupational
studies (U.S. EPA, 2007).
Critical effect levels for hepatic effects were identified in the Becci et al. (1983) and
Kennedy and Sherman (1986; Haskell Labs, 1960) subchronic dietary studies in rats and mice.
The Becci et al. (1983) study used larger numbers of animals (25-30/sex/dose), was
comprehensive in scope and identified hepatic NOAELs of 210-235 mg/kg-day in rats and 70-96
mg/kg-day in mice (for increased liver weight in the absence of serum enzyme and histological
changes), no LOAELs in rats, and LOAELs of 246-326 mg/kg-day in mice (for hepatocytomegaly
and increased serum ALT). The Kennedy and Sherman (1986; Haskell Labs, 1960) study
evaluated a small number of rats (6/sex/dose) and endpoints and identified NOAELs of 77-85
mg/kg-day (for increased liver weight in the absence of serum enzyme and histological changes,
and LOAELs of 422-426 mg/kg-day (for increased hepatocellular mitosis and enlargement). The
histology and serum enzyme data indicate that mice are more sensitive than rats, as shown by the
LOAELs of 246 mg/kg-day (males)-326 mg/kg-day (females) in mice and 422-426 mg/kg day in
rats. The highest systemic NOAELs in mice are 70 mg/kg-day (males) and 96 mg/kg-day
(females) (Becci et al., 1983).
A continuous breeding/2-generation study of dimethylformamide administered in the
drinking water to mice (Fail et al., 1998; NTP, 1992a) found systemic and fetal effects at dose
levels similar to the 246-326 mg/kg-day LOAEL for liver toxicity in the Becci et al. (1983)
subchronic dietary study in mice, as summarized in Table 1. In the Fi mice, absolute and relative
liver weights were increased, and hepatocellular hypertrophy was observed (in livers with gross
lesions) at the lowest dose (226 and 292 mg/kg-day in males and females, respectively); F2 pup
weights were also decreased at this dose level. A crossover mating study with the F0 mice had
determined that effects on the pups were mediated through the dams. A NOAEL for systemic
toxicity was not identified, and the developmental effects occurred at minimally maternally toxic
doses.
Developmental toxicity studies, all of which were conducted by gavage, have identified
LOAELs for maternal and developmental toxicity of dimethylformamide that are lower than those
seen in the subchronic and reproductive studies conducted using dietary or drinking water
exposure (Table 1). Gestational exposure of Sprague-Dawley rats by gavage resulted in maternal
toxicity (reduced food consumption and body weight gain) and developmental toxicity (reduced
fetal body weight) at >100 mg/kg-day and increases in skeletal variations indicative of delayed
ossification at >200 mg/mg-day (Saillenfait et al., 1997); the NOAEL for maternal and
developmental toxicity was 50 mg/kg-day. In a developmental gavage study in Sprague-Dawley
rats, 166 mg/kg-day was a minimal maternal reproductive LOAEL for reduced placental weight
(Hellwig et al., 1991), which was a likely precursor to reduced fetal weight gains at higher doses.
In rabbits, 189 mg/kg-day was teratogenic (increased incidence of total malformations), fetotoxic
(decreased fetal weight) and maternotoxic (reduced body weight gain and food intake), while 64
mg/kg-day was a NOAEL for developmental and maternal effects (Merkle and Zeller, 1980). The
lowest LOAEL is 100 mg/kg-day in rats and the corresponding NOAEL is 50 mg/kg-day
(Saillenfait et al., 1997).
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Effects on fetal weight and ossification in the gavage studies, however, may have been
secondary to maternal effects. Data for maternal food consumption and maternal and fetal body
weight in the study of Saillenfait et al. (1997) are presented in Table 2. The effect on corrected
maternal body weight gain (corrected by subtracting the weight of the gravid uterus) as compared
with the effect on fetal body weight indicates that the effect was primarily on the dams. Effects
on fetal ossification occurred at dose levels >200 mg/kg-day, where the corrected maternal body
weight gain was decreased by 79-90% and the fetal body weight was decreased by 12-14%, and
are consistent with delayed ossification, which is likely related to the depression of maternal and
fetal body weight (e.g., Fleeman et al., 2005; Hood, 2005).
Table 2. Food Consumption, Body Weight, and Ossification Effects in Sprague-Dawley
Rats Treated by Gavage with Dimethylformamide on Days 6 to 20 of Gestation
(Saillenfait et al., 1997)
Dose, mg/kg-day
0
50
100
200
300
Maternal food consumption,
g/dam/day
(% decrease)
28.7±0.7a
28.6±0.6
(0.3)b
24.4±0.6°
(15.0)
21.8±0.6°
(24.0)
22.7±0.6°
(20.9)
Maternal body weight gain, g
(% decrease)
GD 6-21
154±7
148±8
(3.9)
128±5°
(16.9)
102±5°
(33.8)
92±5°
40.3)
GD 6-21 minus
gravid uterus
43±5
48±3
25±4°
(41.9)
9±4°
(79.1)
4±4°
(90.7)
Fetal Body weight/litter, g
(% decrease)
5.54±0.05
5.52±0.04
(0.3)
5.30±0.05°
(4.3)
4.87±0.05°
(12.1)
4.76±0.06°
(14.1)
Supraoccipital: absent or
incomplete ossification
Litter incidence
0
2/20
7/19
16/19°
20/20°
Sternebrae: absent or
incomplete ossification,
Litter incidence
2/16
7/20
7/19
ll/19d
13/20°
aMean ± standard error of the mean
bPercent decrease relative to vehicle control value
Significantly different from vehicle control value, p<0.05
dSignificantly different from vehicle control value, p<0.01
Consideration was given to whether the results seen in the gavage developmental studies
indicate an increased sensitivity of pregnant animals to dimethylformamide, or whether they
indicate that once a day bolus dosing is more toxic than routes of exposure more relevant to
human exposure (i.e., dietary and drinking water exposures). The results from the continuous
breeding/2-generation drinking water study in pregnant mice (Fail et al., 1998; NTP 1992a) were
compared to those from the Becci et al. (1983) subchronic dietary toxicity study in nonpregnant
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mice. This comparison suggests that systemic toxicity (liver effects) in females occurred at
approximately the same exposure level in the reproductive study (292 mg/kg-day, resulting from
1000 ppm in the drinking water) as in the subchronic study (326 mg/kg-day, resulting from 1850
ppm in the diet). Thus, pregnant mice do not seem more sensitive to dimethylformamide than
nonpregnant female mice.
Three studies were considered as the basis for the subchronic RfD - the developmental
toxicity study by Saillenfait et al. (1997), the continuous breeding/2-generation study by Fail et
al. (1998; NTP, 1992a), and the subchronic toxicity study by Becci et al. (1983).
As noted above (see also Table 1), gavage administration of dimethylformamide in studies
by Saillenfait et al. (1997) and others has been shown to cause developmental toxicity in rats,
mice and rabbits, generally at doses associated with maternal toxicity. The presence of maternal
toxicity complicates the interpretation of the developmental toxicity findings. As noted in U.S.
EPA's Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991b), "when
adverse developmental effects are produced only at doses that cause minimal maternal toxicity...,
the developmental effects are still considered to represent developmental toxicity and should not
be discounted as being secondary to maternal toxicity. At doses causing excessive maternal
toxicity..., information on developmental effects may be difficult to interpret and of limited
value." These guidelines also note that where maternal toxicity is seen at dose levels lower than
those producing developmental toxicity or at exposure levels lower than that in other evaluations
of adult toxicity, "this implies that the pregnant female is likely to be more sensitive than the
nonpregnant animal," and that data from such studies should be used in the overall assessment of
risk.
The possible influence of gavage administration merits some consideration. The analysis
of data for pregnant mice from Fail et al. (1998; NTP 1992a) that involved nongavage (drinking
water) administration with mouse data from Becci et al. (1983) that also involved nongavage
(dietary) administration suggested that pregnant mice were not more sensitive to
dimethylformamide than nonpregnant animals. Therefore, it is possible that the lower LOAEL in
the developmental gavage toxicity studies may been due to enhancement of dimethylformamide
toxicity by bolus dosing. However, overt fetal malformations were observed in multiple
developmental toxicity studies following dimethylformamide exposure, lending uncertainty as to
whether maternal toxicity was a major factor in the observed toxicity in some studies, thus data
from developmental (gavage administration) studies was considered in the derivation of a
subchronic p-RfD.
Of the available developmental toxicity studies, Saillenfait et al. (1997) reported maternal
and developmental toxicity at the lowest doses. In this study, pregnant SD rats received
dimethylformamide by gavage in drinking water at doses ranging from 50 to 300 mg/kg-day on
GD 6-20. Investigators reported dose-related reductions in maternal body weight gain and food
consumption in dams, reductions in fetal body weight, and increased incidences of two skeletal
variations (absent or incompletely ossified supraoccipital and sternebrae) at 100 mg/kg-day
(LOAEL); the NOAEL was 50 mg/kg-day. Although effects on the fetus may have been
secondary to effects on maternal body weight gain, the study nevertheless provides clear evidence
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of treatment-related toxicity at a dose of 100 mg/kg-day. These findings are supported by Merkle
and Zeller (1980), which identified a NOAEL and LOAEL of 64.3 and 189 mg/kg-day,
respectively, for maternal and developmental toxicity in the rabbit.
A provisional subchronic RfD of 0.5 mg/kg-day for dimethylformamide based on the
Saillenfait et al. (1997) study was derived by applying an uncertainty factor of 100 (10 to
extrapolate from rats to humans and 10 to protect sensitive individuals) to the rat NOAEL of 50
mg/kg-day. An additional uncertainty factor for database limitations was not applied, since
reproductive and developmental effects have been adequately studied. The derivation is as
follows:
subchronic p-RfD = NOAEL / UF
= 50 mg/kg-day / 100
= 0.5 mg/kg-day
The continuous breeding/2-generation study identified a LOAEL for developmental
toxicity (decreased F2 pup weight) at the same dose level as liver effects in the dams (increased
liver weight and hepatocellular hypertrophy), and did not identify a NOAEL for either
developmental or systemic toxicity (Fail et al., 1998; NTP, 1992a). The investigators concluded
that maternal toxicity contributed to fetal and neonatal problems; however, reproductive effects
and more severe developmental effects, including skeletal and visceral malformations, that
occurred at the two higher exposure levels in this study did not appear to reflect simple
developmental delays. Thus, the LOAEL of 292 mg/kg-day for developmental toxicity
(decreased F2 pup weight) from the continuous breeding/two generation study of Fail et al. (1998;
NTP, 1992a) was considered for subchronic p-RfD derivation.
A provisional subchronic RfD of 0.3 mg/kg-day for dimethylformamide based on the Fail
et al. (1998; NTP, 1992a) study was derived by applying an uncertainty factor of 1000 (10 to
extrapolate from a LOAEL to a NOAEL, 10 to extrapolate from mice to humans and 10 to protect
sensitive individuals) to the mouse LOAEL of 292 mg/kg-day for reduced F2 pup weight. As
noted above, an additional uncertainty factor for database limitations was not considered
necessary because reproductive and developmental effects have been adequately studied. The
derivation is as follows:
subchronic p-RfD = LOAEL / UF
= 292 mg/kg-day / 1000
= 0.3 mg/kg-day
Finally, consideration was given to liver toxicity as the basis for the subchronic p-RfD.
The most appropriate study was considered the Becci et al. (1983) study, which was a robust
study of systemic toxicity that identified liver toxicity at relatively low doses. The highest
NOAEL (96 mg/kg-day) for hepatotoxicity in female mice from Becci et al. (1983) was used to
derive a subchronic p-RfD. The NOAEL for the female mouse was the higher of the NOAELs
for the male (70 mg/kg-day) and female (96 mg/kg-day) mouse in this study.
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A provisional subchronic RfD of 1 mg/kg-day for dimethylformamide based on the Becci
et al. (1983) study was derived by applying an uncertainty factor of 100 (10 to extrapolate from
rats to humans and 10 to protect sensitive individuals) to the rat NOAEL of 96 mg/kg-day. As
noted above, an additional uncertainty factor for database limitations was not considered
necessary. The derivation is as follows:
subchronic p-RfD = NOAEL / UF
= 96 mg/kg-day / 100
= 0.96 mg/kg-day or 1 mg/kg-day
Candidate subchronic p-RfDs based on study data from Saillenfait et al. (1997), Fail et al.
(1998; NTP, 1992a), and Becci et al. (1983) yielded values ranging from 0.3 to 1 mg/kg-day - a
range of only 3.3-fold. The subchronic p-RfD of 0.3 mg/kg-day based on developmental effects
as reported by Fail et al. (1998; NTP, 1992a), which represents the most sensitive effect, is
selected as the subchronic p-RfD.
Confidence in the critical study is medium. The study examined a suitable number of
endpoints and was adequately documented. Confidence in the database is high. Supporting data
are available from subchronic studies, as well as developmental studies in multiple species. The
oral database is also supported by numerous studies conducted for the inhalation route. Medium
confidence in the subchronic p-RfD for dimethylformamide results.
No chronic oral systemic toxicity studies are available for derivation of the chronic p-RfD.
The same three studies considered for derivation of the subchronic p-RfD were therefore
considered as the basis for chronic p-RfD derivation. The p-RfD of 0.3 mg/kg-day based on
reduced pup weight in the mouse in the continuous breeding/2-generation study (Fail et al., 1998;
NTP, 1992a) and the p-RfD of 0.5 mg/kg-day based on developmental effects in the rat
(Saillenfait et al., 1997) are considered appropriate for the assessment of chronic exposure
without addition of an uncertainty factor for extrapolation to chronic exposure. The
developmental period is recognized as a susceptible lifestage where exposures during certain time
windows are more relevant to the induction of developmental effects than lifetime exposure (U.S.
EPA, 1991b).
A provisional chronic RfD of 0.1 mg/kg-day for dimethylformamide based on the Becci et
al. (1983) study is derived by applying an uncertainty factor of 1000 (10 to extrapolate from mice
to humans, 10 to protect sensitive individuals and 10 to extrapolate from subchronic to chronic
duration) to the NOAEL as follows:
Chronic p-RfD = NOAEL / UF
= 96 mg/kg-day / 1000
= 0.096 or 0.1 mg/kg-day
Candidate chronic p-RfDs based on study data from Fail et al. (1998; NTP, 1992a),
Saillenfait et al. (1997), and Becci et al. (1983) yielded values ranging from 0.1 to 0.5 mg/kg-day
- a range of only five-fold. The chronic p-RfD of 0.1 mg/kg-day based on liver toxicity as
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reported by Becci et al. (1983), which represents the most sensitive effect, is selected as the
chronic p-RfD.
Confidence in the critical study is medium, as it was well-conducted and reported, and
examined a suitable number of endpoints. Confidence in the database is medium because
although it includes oral subchronic, reproductive and developmental studies in multiple species,
and is supported by an extensive inhalation database, it does not include a chronic oral study.
Medium confidence in the chronic p-RfD for dimethylformamide results.
DERIVATION OF PROVISIONAL SUBCHRONIC INHALATION RfC VALUE
FOR DIMETHYLFORMAMIDE
A chronic RfC of 0.03 or 3E-2 mg/m3 is available for dimethylformamide on IRIS (U.S.
EPA, 2007). The RfC, which was verified 8/23/90, was derived based on a LOAEL of 22 mg/m3
(duration adjusted to 7.9 mg/m3) for digestive disturbances and minimal hepatic changes
suggestive of liver abnormalities in workers exposed for an average of 5 years (Cirla et al., 1984).
Epidemiological studies demonstrate that dimethylformamide exposure is associated with
hepatic toxicity in humans (Cirla et al., 1984; Fiorito et al., 1997; Cai et al., 1992; Wrbitzky,
1999; Lauwerys et al., 1980; Yonemoto and Suzuki, 1980; Redlich et al., 1987a, b, 1988; Riely et
al., 1988; Reinl and Urban, 1965; Tolot et al., 1968; Chary, 1974). Several of these studies found
hepatotoxicity indicated by effects on serum biomarkers, such as GGT, ALT, AST, AP and
bilirubin (Cirla et al., 1984; Fiorito et al., 1997; Wrbitzky, 1999; Redlich et al., 1987a, b, 1988;
Riely et al., 1988), and liver biopsy (Redlich et al., 1987a, b, 1988; Riely et al., 1988; Tolot et al.,
1968). Although other studies found no effect on serum biomarkers (Catenacci et al., 1984; Cai
et al., 1992; Lauwerys et al., 1980; Yonemoto and Suzuki, 1980; Massmann, 1956a; Di Lorenzo
and Graziolo, 1972), many of these did find subjective evidence of liver toxicity (e.g., digestive
impairment, alcohol intolerance) (Cai et al., 1992; Lauwerys et al., 1980; Yonemoto and Suzuki,
1980). Subjective symptoms indicative of hepatotoxicity were also reported in the studies that
found serum biomarker changes (Cirla et al., 1984; Fiorito et al., 1997; Wrbitzky, 1999; Redlich
et al., 1987a, b, 1988; Riely et al., 1988) and in other studies (Chary, 1974). The reports indicate
that dimethylformamide concentrations as low as 6-20 mg/m3 may produce these effects. Cirla et
al. (1984) found significantly increased prevalence of abnormally high GGT levels (and also non-
significant increases for abnormally elevated ALT, AST and enlarged liver) and increased
subjective complaints potentially related to hepatotoxicity in 100 workers exposed for an average
of 5 years to a mean concentration of 22 mg/m3, compared with a pair-matched control group.
Fiorito et al. (1997) found significantly increased prevalence for abnormally elevated ALT, AST
and GGT, as well as significantly increased mean values for these biomarkers, and increased
subjective complaints indicative of hepatotoxicity in a group of 75 workers exposed to an average
of 20 mg/m3 for up to 3.8 years. Increased subjective indicators of liver dysfunction, but no effect
on serum biochemistry, was reported for 207 workers in a polyurethane plant who were exposed
to levels up to 21 mg/m3 for several years, compared with 143 non-exposed workers (67 men and
76 women) in the same plant (Cai et al., 1992). Analysis by department (exposure level) found
that the prevalence of self-reported reduced alcohol tolerance was significantly elevated in
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subjects exposed to levels of 2 ppm (6 mg/m3) or above, but not average exposures of 0.1-0.6
ppm (0.3-1.8 mg/m3). This study identified a LOAEL of 6 mg/m3 and a NOAEL of 1.8 mg/m3
based on subjective symptoms of hepatic effects in workers exposed to dimethylformamide.
The inhalation toxicity database for animals supports the identification of the liver as the
main target organ of dimethylformamide and indicates that humans may be more vulnerable to
dimethylformamide than the other tested species. In CD-I mice exposed for 18 months, 75
mg/m3 was a LOAEL for liver histopathology (centrilobular hepatocellular hypertrophy and
necrosis) (Malley et al., 1994). In the companion study in rats exposed for 2 years, 75 mg/m3 was
a NOAEL and 300 mg/m3 was a LOAEL for increased relative liver weights and serum enzyme
values (Malley et al., 1994). Subchronic toxicity studies on dimethylformamide indicate that
rodents may be more susceptible than cynomolgous monkeys to hepatic effects. Thirteen-week
bioassays identified hepatic LOAELs of 300-600 mg/m3 in rats (liver cell necrosis) and 150
mg/m3 in mice (increases in centrilobular hepatocellular hypertrophy and liver weight) (NTP,
1992b; Lynch et al., 2003; Senoh et al., 2003). Hepatic effects were also seen at higher
concentrations in other subchronic studies in rodents (Craig et al., 1984; Tanaka, 1971;
Massmann, 1956b). No adverse effects were observed in cynomolgous monkeys exposed to
concentrations as high as 1500 mg/m3 for 13 weeks, although the statistical power of the study
was limited due to the small group sizes (3/sex/group) (Hurtt et al., 1992). Gestational inhalation
exposure studies in several species reported that developmental effects occurred at maternally
toxic concentrations. In rabbits, 150 mg/m3 was a LOAEL for maternal toxicity (reduced body
weight gain) and fetal toxicity (minimal increase in developmental anomalies) and the NOAEL
for both was 50 mg/m3 (Hellwig et al., 1991). In CD rats, 90 mg/m3 was the maternal and fetal
NOAEL and 900 mg/m3 was the LOAEL for reduced body weight gain in dams and reduced body
weight in fetuses (Lewis et al., 1992). In Sprague-Dawley rats exposed under nonstandard
protocols (for a total of 13 or 16 gestational days), 860 mg/m3 was a LOAEL for reduced body
weight gain in dams and reduced fetal weight and increased skeletal variations in fetuses (Hellwig
et al., 1991).
The occupational studies of Cirla et al. (1984), Fiorito et al. (1997) and Cai et al. (1992)
are chosen as co-principal studies for the subchronic RfC for dimethylformamide. The Cirla et al.
(1984) and Fiorito et al. (1997) studies both reported elevated liver enzymes and symptoms
indicative of liver abnormalities (alcohol intolerance, gastrointestinal disturbances) at similar
8-hour TWA LOAELs: 22 mg/m3 in Cirla et al. (1984) and 20 mg/m3 in Fiorito et al. (1997). Cai
et al. (1992) found subjective evidence of liver toxicity (e.g., digestive impairment, alcohol
intolerance), without serum enzyme changes, in workers with an average exposure of 13.7 mg/m3
over several years, and also did a dose-response analysis that identified a LOAEL of 6 mg/m3 and
NOAEL of 1.8 mg/m3 for prevalence of self-reported reduced alcohol tolerance. These studies
together define a LOAEL of 6-22 mg/m3 and a NOAEL of 1.8 mg/m3 for hepatotoxicity in
workers subchronically exposed to dimethylformamide. Because of self-reported reduced alcohol
intolerance, the dose-response for hepatotoxicity is questionable. The LOAEL (20 mg/m3)
identified in Fiorito et al. (1979) for elevated liver enzyme changes represents critical liver
function, thus considered as the POD for derivation of the subchronic p-RfC. The subchronic
LOAEL is adjusted for duration by multiplying by the ratio of the reference breathing volume for
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light activity during occupational exposure to the reference daily breathing volume (10 m3/ 20
m3) and by the ratio of workdays to weekdays (5/7) (U.S. EPA, 1994b), as follows:
LOAELadj = (LOAEL)(VOLoccup/VOLdaily)(DAYSexposed /DAYSweek)
= (20 mg/m3) (10 m3/20 m3) (5/7)
= 7.14 mg/m3
The provisional subchronic RfC of 7E-2 mg/m3 for dimethylformamide is derived by
applying an uncertainty factor of 100 (10 for human variability to the duration-adjusted
subchronic LOAEL and 10 for use of a LOAEL). An additional uncertainty factor for database
limitations was not applied, since the database included adequate human studies to define a
NOAEL and LOAEL, and supporting animal data for systemic and developmental effects that
showed humans to be more sensitive to liver effects than effects noted in animals. The derivation
is as follows:
subchronic p-RfC = LOAELadj / (UF)
= (7.14 mg/m3)/ (100)
= 0.07 or 7E-2 mg/m3
Confidence in the key subchronic studies is medium-to-high because they carefully
evaluated specific endpoints relevant to the primary target organ in an adequate number of
exposed workers and controls, were well-designed, well-conducted and adequately-documented,
but exposure concentrations and durations were not thoroughly characterized. Confidence in the
database is medium-to-high. Supporting human and chronic, subchronic and developmental
toxicity studies in multiple species are available for inhalation exposure, but reproductive toxicity
has been studied only by oral exposure. Medium-to-high confidence in the provisional
subchronic RfC for dimethylformamide results.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
DIMETHYLFORMAMIDE
Weight-of-Evidence Descriptor
Although reports of clusters of testicular cancer in workers exposed to dimethylformamide
(combined inhalation and dermal exposure) were a matter of concern (Ducatman et al., 1986;
Levin et al., 1987), subsequent investigations failed to confirm any association between
dimethylformamide exposure and cancer (Calvert et al., 1990; Chen et al., 1988; Walrath et al.,
1989; IARC, 1999). Inhalation exposure to dimethylformamide at concentrations as high as 1200
mg/m3 did not induce tumor formation in rats exposed for 2 years or in mice exposed for 18
months (Malley et al., 1994). An extensive array of in vitro and in vivo genotoxicity assays on
dimethylformamide yielded largely negative results (IARC, 1999). On the basis of inadequate
evidence in humans and negative evidence in adequate inhalation assays in rats and mice (Malley
et al., 1994), and in accordance with U.S. EPA (2005) guidelines, the data are inadequate for an
assessment of human carcinogenic potential for dimethylformamide.
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Quantitative Estimates of Carcinogenic Risk
The absence of positive carcinogenicity data for dimethylformamide precludes the
calculation of quantitative estimates of carcinogenic risk.
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Amato, G., E. Grasso, V. Longo and P.G. Gervasi. 2001. Oxidation of TV,TV,-dimethylformamide
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Angerer, J., T. Goen, A. Kramer and H.U. Kafferlein. 1998. N-methylcarbamoyl adducts at the
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ATSDR (Agency for Toxic Substances and Disease Registry). 2007. Toxicological Profile
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Atlanta, GA. Online, http://www.atsdr.cdc.gov/toxpro2.html
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