Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity oral: BASF AG, 1977, Repeated dose 28-day oral gavage study in rats, 238, 475, 950, 1900 mg/kg in water, comparable to the OECD TG 407. NOAEL = 238 mg/kg bw (both sexes).
Repeated dose toxicity dermal: data can be waived from oral or inhalation studies;
Repeated dose toxicity inhalation: Malley et al., 1994. Chronic toxicity/oncogenicity of dimethylformamide in rats and mice following inhalation exposure; according to the OECD TG 451, NOAEC = 80 mg/m³

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented report which meets basic scientific principles.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
Principles of method if other than guideline:
- Principle of test: Repeated dose 28-day oral gavage study in rats.
- Short description of test conditions: Sprague-Dawley rats received DMF at dose levels of about 238, 475, 950 and 1900 mg/kg bw/day by gavage on 5 days/week during four weeks.
- Parameters analysed / observed: Clinical signs, body weights and findings by necropsy were recorded.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: WIGA, Sulzfeld, Germany
- Age at study initiation: 32 days
- Weight at study initiation: male: 104-120 g; female: 104 - 122 g
- Housing: 3 per cage
- Diet: Altromin-R, Altrogge, Germany ad libitum
- Water: tap water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
no details given
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
5 d/w
Dose / conc.:
238 mg/kg bw/day (nominal)
Remarks:
250 µL/kg
Basis: nominal in water
Dose / conc.:
475 mg/kg bw/day (nominal)
Remarks:
500 µL/kg
Basis: nominal in water
Dose / conc.:
950 mg/kg bw/day (nominal)
Remarks:
1000 µL/kg
Basis: nominal in water
Dose / conc.:
1 900 mg/kg bw/day (nominal)
Remarks:
2000 µL/kg
Basis: nominal in water
No. of animals per sex per dose:
10
Control animals:
other: aqua bidest
Details on study design:
no information given
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 10 days before the start of the study and directly before the last substance administration.
- How many animals: all
- Parameters examined: mean cell haemoglobin concentration, haemoglobin, erythrocytes, haematocrit, thrombocytes, leucocytes, differential count, mean cell volume, mean cell haemoglobin.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 10 days before the start of the study and direclty before the last substance administration.
- How many animals: all
- Parameters examined: urea, chloride, creatinine, calcium, glucose total, bilirubin, albumin, phosphorus (as phosphate), total protein, lipids, sodium, potassium, carbon dioxide, alkaline phosphatase activity, glutamate-pyruvat transfaminase activity.

URINALYSIS: Yes
- Time schedule for collection of urine: day 21 or 22
- Parameters examined: pH, protein, glucose, bilirubin, sediment.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes: heart, lung, thyroid, stomach, duodenum, jejunum, ileum, mesenteric lymph nodes, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, testes and ovaries and brain.
Other examinations:
Body weight and organ weights of heart, liver, kidneys, spleen, thyroid, adrenals, testes, uterus and ovary were determined.
Statistics:
Statistical calculations (t-Test; x2-Test) were done for clinical, pathological and clinical chemistry data as well as for data from haematology and urinalysis.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The animals in the highest dose group showed piloerection, apathy, anaemia, dyspnoea, hyperthermia, lateral position.
Mortality:
mortality observed, treatment-related
Description (incidence):
At the highest dose group all animals died, mostly during the first 5 days of substance application. The animals in the highest dose group showed piloerection, apathy, anaemia, dyspnoea, hyperthermia, lateral position.
At 950 mg DMF/kg the general state of health was reduced (in male animals already beginning in study week 1, in female animals at the end of study week 3). 4 male animals (on study days 7, 8, 14 and 19) and one female animal (after 15 substance applications) died.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The animals in the highest dose group showed reduced body weight gain already after the first treatment. At 950 mg DMF/kg significantly reduced body weight when compared to the controls (at the end of the study for male animals 28 % lower, and for female animals 21 % lower than control). At 475 mg/kg significantly reduced body weight when compared to the control animals (14.6 % lower than controls) were seen.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The animals in the highest dose group showed reduced food consumption already after the first treatment. At 950 mg DMF/kg the animals showed significantly reduced food consumption (up to 36 % reduced in the males and up to 40 % reduced in the females). At 238 and 475 mg/kg reduced food consumption in the male animals were seen.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
950 mg/kg: 1 male and 2 females showed an extreme decrease in thrombocytes and the males an increase in segmented granulocytes and decrease in lymphocytes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 950 mg DMF/kg hepatic damage was represented by changes in clinical chemistry values (increased total bilirubin, increased enzyme values, i.e. GPT, AP) and disturbances in kidney function were represented by elevated urea (in 2 of 9 female animals) and creatinine values (in all animals of the 950 mg/kg dose group).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No abnormalities
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weights were increased in both sexes' and relative kidney weights were increased in the male animals at 950 mg/kg. At 238 and 475 mg/kg in both sexes increased relative liver weights and in the males increased relative kidney weights were observed, however without histopathological correlates.
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histologically an acute to subacute haemorrhagic liver dystrophy with necrosis was found in the animals of 950 mg/kg and the highest dose group.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
238 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant effects
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEL
Effect level:
475 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced body weight
Critical effects observed:
not specified

The repeated application of dimethylformamide in doses from 950 mg/kg onwards leads to distinct acute haemorrhagic liver dystrophy with necrosis. Females were in general more tolerant to dimethylformamide.

Conclusions:
NOAEL of 238 mg/kg bw and LOAEL of 475 mg/kg bw were established for both sexes.
Executive summary:

Study design

This non-GLP in vivo study was conducted similar to OECD TG 407 (Repeated Dose 28-Day Oral Toxicity in Rodents). In the present study, Sprague-Dawley rats received 250, 500, 1000 and 2000 µL N,N-dimethylformamide /kg bw (about 238, 475, 950 and 1900 mg/kg bw/day) by gavage on 5 days/week during four weeks.

Results

In the highest dose group all animals died, mostly at the beginning of the study. At 1000 µL/kg bw/day all animals affected by reduced food consumption and reduced body weight, males already at the beginning, females at the end of the study. Hepatic injury was characterized by changes in clinical chemistry values, e.g. increased enzyme activities. Relative liver weights were increased in both sexes. Histological examination revealed an acute to subacute haemorrhagic liver dystrophy with necrosis in both sexes in the two high dose groups. Disturbances in kidney function were characterized by elevated urea (females) and creatinine values, the latter one in both sexes. Relative kidney weights were increased in the males. At 250 and 500 μL/kg bw/day reduced food consumption in the males and at 500 μL/kg bw/day reduced body weight was observed in the males. For the observation of increased relative liver weights in both sexes and of increased relative kidney weights in the males no histopathological correlate was found.

Conclusion

NOAEL of 238 mg/kg bw and LOAEL of 475 mg/kg bw were established for both sexes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
238 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD guideline 451 Carcinogenicity Studies
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Crl:CD BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 47 days
- Fasting period before study: no
- Housing: two animal rooms were used, with males and females being housed together by exposure level
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimatisation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C
- Humidity (%): 50 ± 10 %
- Air changes (per hr): air flow rates were targeted at 1750 L/min
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
DMF was pumped from a glass reservoir to a glass bubbler located in a water bath maintained at 70 ° to 80 °C. Preheated high-pressure air (at approximately 40 psi) was introduced into the bubbler; DMF vapors were swept through a 1-in corrugated Teflon tube into the 4-in-diameter stainless steel duct which supplied the incoming air to the chambers. The generation air was heated by passing through a tube furnace and the Teflon tubing was heated with heat tape to prevent DMF vapors from condensing. The dehumidified air supply to the test chambers was set at approximately 1750 L/min, with the exhaust rate set slightly higher to maintain the chambers under slightly negative pressure. DMF concentration was controlled by adjusting the flow rate into the glass bubbler.

TEST ATMOSPHERE
- Samples taken from breathing zone: yes

VEHICLE
- dehumidified air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
1.The flow rate of the high-pressure air was controlled by a mass flow controller.
2.Chamber atmospheres from each of the three test chambers were analysed at approximately 60-min intervals during each 6-hr exposure period by gas chromatography and compared against a standard curve prepared daily.
3.DMF was detected by nitrogen phosphorous detector at a temperature of 300 °C. The retention time of DMF was approximately 1.7 min.
Duration of treatment / exposure:
2 years
Frequency of treatment:
5 d/w, 6 h/d
Dose / conc.:
25 ppm
Remarks:
approx. 0.08 mg/L
Dose / conc.:
100 ppm
Remarks:
approx. 0.3 mg/L
Dose / conc.:
400 ppm
Remarks:
approx. 1.21 mg/L
No. of animals per sex per dose:
87
Control animals:
yes
Details on study design:
- Dose selection rationale: The high exposure concentration was chosen based on data which demonstrated saturation of DMF metabolism in rats and mice following a single 6-hr exposure to 500 ppm (Hundley et al. 1993) and on previous toxicity data in rats and mice. The concentrations selected were expected to result in no significant life shortening. To obtain a dose response, the lower concentrations were derived from the high concentration by a factor of 4.
- Post-exposure period: none
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once and usually twice daily throughout the study
- Cage side observations included: detection of moribund or dead animals and abnormal behaviour and appearance among animals

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at every weighing, each animal was individually handled and examined for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: once per week approximately the first 3 month of the study and once every week thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the first exposure and again immediately prior to the final euthanasia.
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 month after initiation
- Anaesthetic used for blood collection: Yes (light carbon dioxide anaesthesia)
- Animals fasted: yes
- How many animals: 10 animals per sex per group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3, 6, 12, 18 and 24 month after initiation
- Animals fasted: Yes
- How many animals: 10 animals per sex per group
- Parameters checked in table [1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: for approximately 14 hr prior to blood collection.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked in table [2] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 3)
Lungs, brain, liver, kidneys, adrenals, ovaries, and testes were weighed wet at necropsy. Organ weight/final body weight ratios were calculated. Organs from animals found dead or euthanized in extremis were not weighed.
Nose, lungs, liver, kidneys, and all gross lesions from animals in the 25 and 100 ppm groups were also processed and examined microscopically. In addition, due to the incidence of endometrial stromal polyps observed in 400 ppm female rats, the uterus from all female rats in the 25 and 100 ppm groups was examined.
Other examinations:
After 2 weeks, 3 months, and 12 months of testing, five male and five female rats from each group were randomly selected and evaluated for cell proliferation in the liver. On the day of termination, animals were injected intraperitoneally with 100 mg/kg 5-bromo-2'-deoxyuridine (BrdU). Approximately 2 hr after BrdU injection, the designated animals were sacrificed by pentobarbital anaesthesia and exsangui nation and necropsied. Livers from animals in 0 and 400 ppm groups were processed and evaluated immunohistochemically. In addition, the livers from all groups were evaluated microscopically.
The estrous cycle was monitored by vaginal smears and recorded daily for each female animal in the 0 and 400 ppm groups from Test Day 107 through Test Day 131. The individual estrous cycle length was evaluated by counting the number of days that followed the day judged to be estrous (characterized by a vaginal smear containing cornified cells) and included the following day judged to be estrous. The mean cycle length, mean number of estrous cycles, number of cycles with prolonged estrous, and the number of animals experiencing a prolonged estrous were determined.
Statistics:
Body weights, body weight gains, organ weights, and clinical laboratory measurements were analysed by a one-way analysis of variance. When the test for differences among test group means (the F test statistic) was significant, pairwise comparisons between test and control groups were made with the Dunnett's test.
Clinical observation incidences were evaluated by the Fisher's exact test with a Bonferroni correction and the Cochran-Armitage test for trend. Survival among groups was evaluated by the Fisher's exact test and the Cochran-Armitage test for trend. The incidences of neoplastic, pre-neoplastic, and compound-related lesions were evaluated by the Fisher's exact test and/or the Cochran-Armitage test for trend. Bartlett's test for homogeneity of variances was performed on the organ weight and clinical laboratory data and, when significant (a = 0.005), was followed by non-parametric procedures.
Data were maintained separately by sex for the purpose of statistical analyses. Except for Bartlett's test, all other significance was judged at a = 0.05.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
Compound-related differences in the survival of rats were not evident in this study. For male rats, survival was 27, 34, 40, and 44 % for 0, 25, 100, and 400 ppm groups, respectively. For female rats, survival was 35, 23, 19, and 39 %, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats exposed to 400 ppm had significantly lower body weight compared to their respective controls. In addition, 100 ppm males had lower body weight from Test Day 674 through the end of the study. Females exposed to 100 ppm exhibited a similar trend; however, the differences in body weight were not statistically significant. Mean body weight gain for 400 ppm male and female rats was lower than controls (22 and 37 %, respectively). Males exposed to 100 ppm also had lower body weight gain (14 %). Females exposed to 25 or 100 ppm had slightly lower body weight gain compared to controls; however, the differences were not statistically significant. Only the lower body weight and body weight gain observed in 400 ppm males and females and 100 ppm males were considered to be compound related.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
An ophthalmologic examination conducted at approximately 24 months showed only spontaneous lesions whose frequencies were within the expected ranges. The most frequent findings were pale ocular fundi and superficial corneal vascularization, which are common in rats of this strain and age. There were no compound-related effects on the eyes that were detected by this evaluation.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no compound-related differences in haematology parameters in either male or female rats at any sampling period
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A compound-related increase in sorbitol dehydrogenase (SDH) activity occurred in males and females exposed to 100 or 400 ppm DMF (Any other information on results incl. tables, Table 1). Although statistical significance was variable, biologically important increases in SDH activity occurred at the 3-, 6-, 12-, and 18-month evaluations. At the 24-month evaluation, the mean SDH value for control males was unusually low, which resulted in an apparent elevation for 25, 100, and 400 ppm males.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean relative liver weights were significantly increased in 100 and 400 ppm females at the 12-month euthanasia and in 400 ppm females at the 24-month euthanasia (Any other information on results incl. tables, Table 2). Mean relative liver weights were also elevated in females euthanized for hepatic cell proliferation studies on Test Days 19, 95, and 363. On Test Day 19, the relative liver weights were elevated in 25, 100, and 400 ppm females. Relative liver weights were also higher in 400 ppm females on Test Day 95 and in 100 and 400 ppm females on Test Day 363. In males, mean relative liver weights were increased at 100 and 400 ppm at the cell proliferation euthanasia on Day 363, at the 12-month interim termination, and at the final euthanasia (Any other information on results incl. tables, Table 2). Since the increased relative liver weights in the 25 ppm females were elevated only on Test Day 19, it was not considered toxicologically significant with regard to establishing a no-observable-effect level since the effect was transient and was not correlated with morphological changes. Although not statistically significant, the higher relative liver weights for females at 100 and 400 ppm on Day 363 and for males at 100 ppm on Day 363 and at the final euthanasia were considered to be compound related based on the morphological findings.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the 12-month interim euthanasia, the incidences of gross lesions were similar for all exposure concentrations. At the 24-month terminal euthanasia, females exposed to 400 ppm had a decreased incidence of grossly observed mammary masses compared to control. The incidences of gross lesions in males were similar for all exposure concentrations at the 24-month euthanasia.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats had several compound-related microscopic effects observed in the liver in the 100 and 400 ppm exposure groups. At the 12-month euthanasia, the incidence of centrilobular hepatocellular hypertrophy was increased in 100 ppm females and in 400 ppm males and females. In addition, males and females exposed to 400 ppm had a higher incidence of hepatocellular single cell necrosis, centrilobular accumulation of lipofuscin/hemosiderin, and clear cell foci at the 12-month euthanasia. At 24 months, 100 and 400 ppm males and females were observed to have an increased incidence of minimal to mild centrilobular hepatocellular hypertrophy and centrilobular accumulation of lipofuscin/hemosiderin (Any other information on results incl. tables, Table 3). In addition, the incidence of focal cystic degeneration was increased in 100 and 400 ppm males which ranged from minimal severity at 100 ppm to moderate severity at 400 ppm. The incidence of clear cell foci was increased in 100 ppm males and in 400 ppm males and females. Eosinophilic foci were also increased in 400 ppm females only. Males and females exposed to 400 ppm also had a significantly higher incidence of minimal to mild hepatocellular single cell necrosis (Table 3). However, basophilic cell foci were decreased in 100 and 400 ppm males and in 400 ppm females at the 24-month euthanasia.
There was a compound-related decrease in several age-related spontaneous lesions in 400 ppm females at the 24-month euthanasia: benign mammary tumors, chronic glomerulonephropathy, and cardiomyopathy. In addition, 400 ppm males and females had a lower incidence of bilateral retinal atrophy.
There was no compound-related lesions noted in the nose or respiratory tract for any exposure concentration.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Exposure to DMF for 2 years did not cause a compound-related increase of tumors in rats. In addition, the incidence of hepatic tumors and testicular tumors in rats exposed to concentrations up to 400 ppm was similar to control values (Any other information on results incl. tables, Table 4). An increased incidence of endometrial stromal polyp of the uterus was observed in females exposed to 400 ppm (1.7 %, 5.1 %, 3.4 %, and 14.8 % for 0, 25, 100, and 400 ppm females, respectively). Endometrial stromal polyps are the most common uterine neoplasm in rats (Leininger and Jokinen, 1990; and Goodman and Hildebrandt, 1987) and they occur with a highly variable incidence.
Details on results:
HISTORICAL CONTROL DATA (if applicable)
The range of historical control incidence for this laboratory is 2.0-15.0 % (for 14 control groups with an average incidence of 6.6 %), and the historical incidence is 1.1-10 % (for 19 control groups) for the animal supplier (Lang, 1992). With such a variable range for this lesion, it would not be unexpected to have a statistically increased incidence in one group compared to another. Therefore, the increased incidence in endometrial stromal polyps in this study is probably a chance variation rather than a compound-related effect. Therefore, exposure of rats for 24 months to DMF was not oncogenic at concentrations up to 400 ppm.

OTHER FINDINGS
Rats—Estrous Cycle Evaluation
There were no statistically or biologically significant differences in the mean individual cycle length, mean number of estrous cycles, or the number of rats experiencing prolonged estrous compared to their respective control groups. Therefore, there were no compound-related effects detected in this study on the estrous cycles of rats exposed to concentrations up to 400 ppm.

SORBITOL DEHYDROGENASE ACTIVITY (SDH)
A compound-related increase in sorbitol dehydrogenase (SDH) activity occurred in males and females exposed to 100 or 400 ppm DMF (Any other information on results incl. tables, Table 1). Although statistical significance was variable, biologically important increases in SDH activity occurred at the 3-, 6-, 12-, and 18-month evaluations. At the 24-month evaluation, the mean SDH value for control males was unusually low, which resulted in an apparent elevation for 25, 100, and 400 ppm males. There were no compound-related differences in haematology parameters in either male or female rats at any sampling period.
Dose descriptor:
NOEC
Effect level:
25 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight changes, clinical chemistry changes
Dose descriptor:
LOEC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: hepatotoxic effects
Critical effects observed:
not specified

Table 1:  Effect of DMF on Sorbitol Dehydrogenase Activity in Male and Female Ratsa

3 Months 6 Months 12 Months 18 Months 24 Months
Concentration (ppm) Males
0 7.0b(3.3) 10.4 (7.5) 10.9 (4.8) 6.5 (2.1) 2.0 (0.9)
25 9.8 (5.5) 11.5 (6.1) 18.9 (17.6) 9.7 (3.3) 4.4 (2.3)*
100 35.0 (26.4)* 23.0 (17.9) 33.6 (33.1)* 19.8 (10.6)* 18.3 (24.3)*
400 22.6 (18.7)* 19.4 (10.8) 21.7 (12.5)* 19.3 (15.8)* 9.7 (8.1)*
Concentration (ppm) Females
0 11.5 (2.8) 20.9 (24.9) 6.6 (2.8) 6.0 (1.5) 5.7 (6.9)
25 11.0 (3.3) 7.7 (3.0) 7.6 (3.3) 14.8 (11.1)* 9.0 (11.0)
100 17.4 (6.0)* 18.4 (9.0) 17.3 (6.3)* 9.7 (4.3)* 4.9 (3.4)
400 30.9 (15.5)* 27.8(18.0) 23.8 (13.0)* 23.2 (25.0)* 12.9 (13.7)

Table 2: Effect of DMF on RelativeaLiver Weight in Rats and Mice
DMF (ppm)
0 25 100 400
Male rats  
12 Monthsb 2.54 (0.18) 2.73 (0.34) 2.93* (0.32) 3.26* (0.31)
24 Monthsc 2.87 (0.45) 2.81 (0.35) 3.28 (0.53) 3.58* (0.73)
Female rats  
12 Monthsb 2.64 (0.24) 2.70 (0.41) 3.25* (0.40) 3.34* (0.40)
24 Monthsc 3.12 (0.67) 3.43 (1.06) 3.33 (0.71) 3.86* (0.61)
Male mice  
18 Monthsd 5.85 (1.18) 5.94 (1.45) 7.06* (2.04) 7.80* (2.35)
Female mice  
18 Monthsd 5.59 (0.92) 5.71 (0.95) 5.99 (1.45) 6.35* (0.78)

a % of body weight.

b Livers evaluated from 10 rats/sex/concentration.

c For males n =17,19,21and 26 livers evaluated for 0, 25, 100 and 400ppm, respectively. For females n = 22, 14, 12, and 23 livers evaluated for 0, 25, 100, and 400 ppm, respectively.

d For males n =31,42, 3 8, and 36 livers evaluated for 0,25, 100 and 400ppm, respectively. For females n = 42, 35, 36 and 47 livers evaluated for 0, 25, 100, and 400 ppm, respectively.

*Statistically significant at P <0.05.

 Table 3: Incidence (%) of Compound-Related Morphological Observations in Rats Exposed to DMF for 24 Monthsa

DMF (ppm)
  0 25 100 400
Lesion
  Centrilobular
  Hepatocellular
  Hypertrophyb
       
Male 0 0 5* 30*
Female 0 0 3* 40*
Hepatic single cell necrosis*      
Male 2 2 3 30*
Female 0 0 5* 18*
Hepatic accumulation of
   lipofuscin/hemosiderinb
     
Male 4 4 17* 58*
Female 8 7 22* 61*

Hepatic foci of alterations

 

 

 

Male: clear cell

11

8

22*

35*

Male: eosinophilic

33

36

24

45

Female: clear cell

5

5

14

24*

Female: eosinophilic

22

12

25

40*

a Data represent total percentage incidence for both unscheduled and scheduled deaths for the interval 12-24 months.

b The number of livers examined was 57, 59, 58, and 60 for 0, 25, 100 and 400 ppm males, respectively. For females exposed to 0, 25, 100 or 400ppm, the number of livers examined was 60, 59, 59 and 62, respectively.

* Statistically significant at P <0.05.

 

Table 4: Incidence (%) of Hepatic, Testicular and Mammary Tumors in Rats Exposed to DMF

 

DMF (ppm)

0

25

100

400

Primary hepatic tumors

 

Hepatocellular adenoma

(M)a

2 (1/57)b

2 (1/59)

5 (3/58)

3 (2/60)

(F)

0 (0/60)

2 (1/59)

0 (0/59)

0 (0/60)

Hepatocellular carcinoma

(M)

0 (0/57)

0 (0/59)

0 (0/58)

2 (1/60)

(F)

0 (0/57)

0 (0/59)

0 (0/59)

0 (0/59)

Primary testicular tumors

 

Testicular interstitial cell adenomas

(M)

9 (5/57)

7 (3/44)c

0 (0/41)c

10 (6/60)

Testicular mesothelioma

(M)

0 (0/57)

0 (0/44)c

0 (0/44)c

2 (1/60)

Primary mammary tumors

 

Fibroadenoma

(M)

2 (1/44)

8 (3/37)c

11 (4/38)c

3 (1/32)

Adenomad

(F)

55 (33/60)

64 (34/53)c

63 (34/54)c

37(23/62)*

(F)

2 (1/60)

2 (1/53)

4 (2/54)

2 (1/62)

a M, male; F, female.

b Numerator represents number of tumors, and the denominator represents number of tissues examined.

c For the 25 and 100 ppm concentrations, non-target organ tissues (such as testes and mammary gland) were examined only in animals which died prior to scheduled sacrifice or had grossly observable lesions.

d This lesion was not observed in males.

Conclusions:
DMF produced compound-related morphological effects only in liver and was not oncogenic in rat and mouse under experimental conditions of this study.
Executive summary:

Study design

This fully reliable study was performed according to OECD TG 451 Carcinogenicity Study. The carcinogenic effect of the test substance was investigated in groups of 78 male and 78 female young adult mice. The rats were approx. 47 days of age at the beginning of the study. They were exposed to DMF vapors by whole body exposure at dose levels of 0, 25, 100 and 400 ppm for two years. The concurrent control group animals (0 ppm) were exposed to dehumidified air alone. Clinical pathology was investigated at 3, 6, 12, 18 and 24 months in each 10 male and 10 female /group. At 12 months interim sacrifice of 10 male and 10 female animals per group took place, thus again 10 rats per sex and group had to be selected for the 18 and 24 months examinations. After 2 weeks, 3 months and 12 months of testing cell proliferation in the liver was evaluated in 5 randomly selected rats per sex and group. An immunohistochemical evaluation was done on livers from animals of the 0 ppm and 400 ppm groups. Estrous cycle evaluation was done in all female animals of the control and the high dose group from test day 107 through test day 131. Moreover, examinations on body weight, organ weights, ophthalmoscopy, urinalysis and a complete necropsy including microscopical examinations were carried out.

Results and discussion

There were no compound-related differences in the survival of the animals in the present study (for male rats survival was 27, 34, 40 and 44 % for 0, 25, 100 and 400 ppm, respectively. For female rats survival was 35, 23, 19 and 39 %, respectively). Ophthalmologic examinations, haematology and urinalysis revealed no compound-related effects in the rats. Moreover, no compound- related effects were seen on the estrous cycles of rats exposed up to 400 ppm DMF. Body weight and body weight gain were reduced in both sexes of the 400 ppm group and in the male animals of the 100 ppm group. Serum sorbitol dehydrogenase activity was increased in the animals of the 100 and 400 ppm groups. These animals also showed increased mean relative liver weights and centrilobular hepatocellular hypertrophy as well as an increased centrilobular accumulation of lipofuscin/hemosiderin. At 400 ppm there was also an increased incidence of hepatocellular single cell necrosis. The incidence of clear cell foci was increased in 100 ppm males and in both sexes of the highest dose group. An increased incidence of eosinophilic foci was seen in the 400 ppm females. Cell-labelling indices for hepatocytes were not statistically significant different between control and 400 ppm rats, however, rates were slightly higher for 400 ppm males at 2 weeks and 3 months but not at 12 months. An increased incidence of endometrial stromal polyp of the uterus (14.8 %) occurred in the females of the 400 ppm group. According to the authors endometrial stromal polyps are the most common uterine neoplasm in rats. Moreover, the incidence showed no clear dose-response relationship and was in the range of historical control incidences for the respective laboratory (2.0-15.0 %). Thus, the authors concluded, that the increased incidence is probably a chance variation rather than a compound-related effect. There were no compound-related lesions noted in the nose or respiratory tract for any exposure concentration. The incidences of hepatic tumors and testicular tumors in rats exposed up to 400 ppm DMF were similar to control values.

Conclusion: Exposure to DMF for 2 years did not cause a compound-related increase of tumors in rats. According to the authors, the NOEC in rats is 25 ppm (common toxicity) and the NOEC for oncogenicity is 400 ppm

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
80 mg/m³
Study duration:
chronic
Species:
other: mouse and rat
Quality of whole database:
There are a lot of studies of different quality levels and from different literature sourses available for DMF.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

A non-GLP in vivo study was conducted similar to OECD TG 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) (BASF, 1977).

In this key oral sub-acute study, Sprague–Dawley rats received 250, 500, 1000 and 2000 μL DMF/ kg bw (about 238, 475, 950 and 1900 mg/kg bw/day) by gavage on 5 days/week.

In the highest dose group all animals died, mostly at the beginning of the study. At 1000 μL/kg bw/day all animals were affected by reduced food consumption and reduced body weight, males already at the beginning, females at the end of the study. Hepatic injury was characterized by changes in clinical chemistry values, e.g. increased enzyme activities. Relative liver weights were increased in both sexes. Histological examination revealed an acute to subacute hemorrhagic liver dystrophy with necrosis in both sexes in the two high dose groups. Disturbances in kidney function were characterized by elevated urea (females) and creatinine values, the latter one in both sexes. Relative kidney weights were increased in the males. At 250 and 500 μL/kg bw/day reduced food consumption in the males and at 500 μL/kg bw/day reduced body weight was observed in the males. For the observation of increased relative liver weights in both sexes and of increased relative kidney weights in the males no histopathological correlate was found. NOAEL of 238 mg/kg bw and LOAEL of 475 mg/kg bw were established for both sexes.

 

In a supporting 90 -day feeding study, Charles River CD strain rats received 200, 1000 and 5000 ppm DMF (about 12, 60 and 300 mg/kg bw/day) (TSCATS: OTS 0520880, 1960; TSCATS: OTS 0571664, 1960; TSCATS: OTS 0572893, 1960). This study was conducted in accordance with national standard methods with acceptable restrictions.

40 male and 40 female weanling rats were observed during a six-day pre-test period. At the end of this period 6 male and 6 female animals were assigned to 4 groups, one control group and 3 dose groups. Dose levels chosen were 200, 1000 and 5000 ppm. Body weight and food consumption were determined at least once weekly. Routine hematological examinations were performed for all animals on study days 30, 60 and 90. Alkaline phosphatase activity was also determined at these time points. At the end of the study for evaluation of liver function enzyme activities and phospholipid and cholesterol content were measured in the serum. At sacrifice the animals were submitted to gross and microscopic pathological appraisal and to organ weight determination (brain, liver, kidney, adrenal, lung, spleen and testis). For histology the following organs were preserved: organs that were weight and ovary, heart, pancreas, stomach and small intestine.

All treated animals survived the feeding study. At a dose of 1000 ppm DMF relative liver weights were slightly increased without a histopathological correlate. Leucocytosis was observed after 60 days of DMF feeding. The red blood cell count was slightly lower in the 1000 ppm group in comparison to the control. Hypercholesterolemia was observed in the females and elevated phospholipid values were seen in 2/6 females at 1000 ppm. In both sexes at 5000 ppm body weight gain was depressed during the entire study period (statistically significant only in male animals), food consumption was lower during the first 5-6 weeks when compared to control animals and food efficiency were also lower during the first two weeks of the study. Moreover slight anemia, leucocytosis, hypercholesterolemia and elevated phospholipid concentration together with mild liver injury (the latter finding in 3/6 males and 5/6 females) and increased relative liver weights were observed in both sexes of the 5000 ppm group. The increase in relative liver weights in both sexes at 1000 and 5000 ppm were dose-related. No effects were observed in animals of the lowest dose group. In conclusion, the liver was the predominant organ of DMF toxicity. NOAEL of 200 ppm and LOAEL of 1000 ppm were established for both sexes.

Male Wistar rats were dosed with N,N-dimethylformamide (DMF) in drinking water at four concentration levels (0, 0.1, 0.5 and 1.0 g/L) for 2 or 7 weeks (Elovaara et al., 1983).

The weight gain in the DMF-treated rats did not differ from the controls. A dose-related increase of relative liver weights was observed. In liver and kidneys increased amounts of reduced glutathione and increased activity of microsomal UDP Glucuronosyl Transferase and of Ethoxycumarine O-Demethylase were observed. Cytochrome P-450 and NADPH-Cytochrome C-Reductase activity in the liver were not influenced by the treatment. Oxidative N-demethylation of DMF by hepatic microsomes in vitro was not enhanced by oral treatment. No DMF-dependent formaldehyde liberation in vitro could be detected under conditions where formaldehyde liberation from N,N-dimethylnitrosamine could be demonstrated. However, the endogenous rate of formaldehyde generation by liver microsomes isolated from DMF-treated rats was enhanced with the highest oral dose of DMF. The daily intake of DMF lowered the activities of both formaldehyde and propionaldehyde dehydrogenases in the liver soluble fraction. No inhibition of these dehydrogenases was shown in vitro by DMF (≤ 10 mM) or by its main urinary metabolite N-methylformamide (≤ 10 mM). The observed impairment of aldehyde oxidation in liver and kidneys of the rat after the DMF intake could explain the mechanism behind the alcohol intolerance observed in man after DMF exposure.

Repeated inhalation exposures

A fully reliable study was performed according to OECD TG 451 Carcinogenicity Study in rats and mice. In this key chronic inhalation studies Crl:CD BR rats were exposed over a period of 2 years and Crl:CD-1 (ICR)BR mice were exposed for 18 months at concentrations of 25, 100 and 400 ppm (about 80, 300 and 1210 mg/m³) 5 d/w and 6 h/d (Malley et al., 1994). In the rats body weight and body weight gain were reduced in both sexes at 400 ppm and in the male animals at 100 ppm. Moreover, the animals in these groups showed increased enzyme activity (serum sorbitol dehydrogenase), increased liver weights and some histopathological findings in the liver. There was no compound related increase of tumors. Estrous cycles were not altered in the females. Similar findings were observed in mice . At 400 ppm liver weights were increased in both sexes and at 100 ppm in the males. At all concentrations tested minimal to mild hepatocellular hypertrophy was observed (incidence being dose-related). Individual hepatocellular necrosis together with some other histopathological findings (minimal to moderate kupffer cell hyperplasia with pigment accumulation of lipofuscin and hemosiderin) were seen in all groups (also control, incidence being greater in N,N-dimethylformamide-treated animals). A compound-related increase in tumors was not observed and there was no effect on estrous cycles in female mice. According to the authors, a NOEC (no-observable -effect level) was not achieved in mice due to morphological changes seen in the liver at all three test concentrations, nevertheless they expected the NOEC to be close to 25 ppm due to the minimal changes observed at this concentration. These minimal changes included a slightly (for the males significantly) increased incidence of hepatocellular hypertrophy, dose-related and statistically significantly increased incidence of hepatic single cell necrosis in both sexes, and dose-related (for the males significantly) increased incidences of hepatic kupffer cell hyperplasia and pigment accumulation. For rats, the NOEC is 25 ppm (80 mg/m³) based on the body weight changes, clinical chemistry changes and hepatotoxic effects observed at 100 and 400 ppm.

The other sub-chronic inhalation studies confirmed these findings (NTP, 1992 (Lynch et al., 2003) and Senoh et al., 2003):

In the NTP study (1992), groups of 30 rats (Fischer 344) and 10 mice (B6C3F1) of each sex were exposed to vapor concentrations of DMF at 0 (chamber controls), 50, 100, 200, 400, or 800 ppm, 6 h (plus T90) per day, 5 days/week, for 13 weeks. Each group of 30 rats was subdivided into a base study group (10) and two additional subgroups of 10 for evaluations of cardiac physiology and renal function, respectively. Animals were observed twice daily for mortality and moribundity. Body weights were measured weekly and at necropsy. In rats, haematology and serum chemistry analyses, urinalyses, renal function, and cardiac physiology (blood pressure and electrocardiograms) studies were performed. Additionally, sperm morphology and vaginal cytology evaluations were performed on rats and mice exposed at 0, 50, 200, or 800 ppm DMF. A complete necropsy was performed on all animals. Organs and tissues were examined for gross lesions. Tissues from all control and 800 ppm DMF exposed animals were examined microscopically whereby livers were examined in rats and mice of both sexes from all treatment groups.

Subchronic inhalation exposure to DMF produced hepatotoxicity characterized by statistically significant increased liver weights at all exposure concentrations, statistically significant increased activities of serum enzymes and other markers of liver function and histopathological changes in the liver with rats exhibiting more severe lesions. The liver was the only target organ identified and no histopathological changes were observed in any other organs or tissues in either species. Relative liver weights were significantly increased at all DMF concentrations in both sexes and both species. Activities of serum sorbitol dehydrogenase (SDH) were statistically increased in male and female rats (200 to 800 ppm) on study days 4, 24, and 91 (13 weeks). Activities of alanine aminotransferase (ALT) and isocitrate dehydrogenase (ICD) were statistically increased in both sexes of rats exposed to 800 ppm DMF at all time points. Cholesterol (CHOL) levels were statistically increased in male and female rats (50–800 ppm) at all sampling time points. Levels of total bile acids (TBA) were statistically increased in both sexes of rats (400–800 ppm) on days 24 and 91. Centrilobular hepatocellular necrosis (minimal to moderate) was seen in rats of both sexes exposed at 400 and 800 ppm, with the lesions more severe in females. Centrilobular hepatocellular hypertrophy (minimal to mild) was found in all groups of DMF-exposed male mice, and in female mice exposed at 100–800 ppm. No treatment-related hematological or renal function changes were observed in rats and no adverse effects on male reproductive endpoints were found. No evidence of cardiotoxicity was observed in rats exposed to DMF, which does not support the limited evidence of DMF-induced cardiotoxicity in the literature. Adverse effects on the estrous cycle were observed in female rats exposed at 800 ppm but not at lower concentrations of DMF. For rats of both sexes the no-observed-adverse-effect concentration (NOAEC) for microscopic liver lesions was 200 ppm based on the absence of microscopic liver lesions at this and lower exposure concentrations, although liver enzymes and liver weights were increased at all DMF exposure concentrations (50–800 ppm). The NOAEC was 50 ppm for female mice, but an NOAEC based upon the absence of microscopic liver injury was not determined in male mice.

A highly reliable GLP study was conducted according to OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study) and OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study).

In this supporting study, F344 rats and BDF1 mice of both sexes were exposed to DMF by inhalation (6 h/d × 5 d/wk) to 100, 200, 400, 800 or 1,600 ppm DMF for 2 weeks, and 50, 100, 200, 400 or 800 ppm DMF for 13 weeks (Senoh et al., 2003). Three male and 7 female rats died during the 2-week exposure to 1,600 ppm DMF, but no death of the exposed rats or mice occurred under any other exposure conditions. Massive, focal and single cell necrosis were observed in the liver of DMF-exposed rats and mice. The massive necrosis associated with the centrilobular fibrosis occurred at the highest exposure concentration. The single cell necrosis was associated with fragmentation of the nucleoli as well as an increased mitotic figure. The 13-week exposures of rats and mice to DMF were characterized by increases in the relative liver weight and the incidence of the centrilobular hepatocellular hypertrophy as well as increased serum levels of AST, ALT, LDH, total cholesterol and phospholipid. Lower confidence limits of the benchmark dose yielding the response with a 10 % extra risk (BMDL10) were determined for the relative liver weight and the incidence of hepatocellular hypertrophy of the 13-week exposed animals. For the increased relative liver weight, the BMDL10 value resulted in 1.1 and 13.1 ppm for male and female rats, and 1.1 ppm for male mice, respectively. Nevertheless, the BMDL10 value for the relative liver weight of female mice was not determined because of insignificant changes in the relative liver weight throughout the range of exposure concentrations. For the hepatocellular hypertrophy, the BMDL10 value resulted in 68.5 and 191 ppm for male and female rats, and 17.5 and 372.5 ppm for male and female mice, respectively. These BMDL10 values for hepatocellular hypertrophy are consistent with the finding by Lynch et al. 2003 that the NOAEL of hepatocellular hypertrophy were 50 and 200 ppm for female mice and rats of both sexes, respectively.

In a supporting study (non-GLP, non-OECD), Cynomolgus monkeys were exposed to DMF by inhalation during 13 weeks (TSCATS, 1990 Report No. 86 -910000212). The aim was to determine the target organ effects, concentration response, a NOAEL, to measure selected pharmacokintetic parameters, evaluate potential toxic effects on the male and female reproductive system, examine differences in response between sexes and to evaluate potential specimen differences in toxic responses (comparison with literature data) following exposure to DMF vapors. A total of 20 male and 12 adult female monkeys were required for this study. Three monkeys/sex/exposure group were exposed to the three concentrations of DMF (30, 100 or 500 ppm) or filtered room air (concurrent control). In addition, two males per exposure group were designated as the post-exposure group. The post-exposure group was held for 13 additional weeks with no exposure and was then necropsied.

The effects of the test substance were studied in groups of 5 male and 3 female monkeys (two males/group served as additional animals for the post-exposure period). There were no early deaths in this study and all animals were sacrificed on their scheduled day of necropsy. There were no treatment-related findings in the 13 week inhalation study except possible alterations in the menstrual cycle of DMF exposed females. The menstrual cycle of 1 low dose group female, 2 mid dose females and all high dose females were altered in length. According to the authors, the subchronic exposure of cynomolgus monkeys to DMF did not cause any adverse health effects (liver function, sperm production, and sperm motility appeared unaffected). With respect to the possible increase in mensis length with exposure to DMF and its relevance, the experts conclusions were that while the data are suggestive of an effect, there is no confirmed evidence that DMF caused an effect on menstrual cycle because of the monkeys recent importation history and lack of pre-exposure data. NOAEL of 500 ppm was established for male and female animals.

In a disregarded study*, carcinogenicity and chronic toxicity of N,N-Dimethylformamide (DMF) were examined by inhalation exposure of groups of 50 rats and 50 mice of both sexes to DMF vapor at a concentration of 0, 200, 400 or 800 ppm (v/v) for 6 h/d, 5 d/wk, for 104 wk (Senoh et al., 2004). In rats, incidences of hepatocellular adenomas and carcinomas significantly increased in the 400 and 800 ppm-exposed groups and in the 800 ppm-exposed group, respectively. The hepatocellular adenoma did not increase significantly in the 400 ppm exposed female rats, but its incidence exceeded a range of historical control data in the Japan Bioassay Research Center (JBRC). In mice, incidences of hepatocellular adenomas and carcinomas significantly increased in all the DMF-exposed groups. Incidence of hepatoblastomas significantly increased in the 200 and 400 ppm-exposed male mice, and 4 cases of hepatoblastomas in the 400 ppm-exposed female mice and the 800 ppm-exposed male mice exceeded the range of historical control data of the JBRC. Incidences of altered cell foci increased in the liver of exposed rats and mice in an exposure concentration-related manner, and those foci were causally related to the hepatocellular tumors. Liver weights increased in both rats and mice exposed to DMF at 200 ppm and above. Increased levels of γ-GTP, ALT, AST and total bilirubin in exposed rats of both sexes and AST and ALT in exposed mice of both sexes were noted. It was concluded that 2-yr inhalation exposure to DMF increased incidences of hepatocellular adenomas and carcinomas in rats and incidences of hepatocellular adenomas, carcinomas and hepatoblastomas in mice, and that hepatocarcinogenicity of DMF was more potent in mice than in rats.

In a disregarded study*, Male Wistar rats were exposed by inhalation to N,N-dimethylformamide (DMF) at 0 (control), 200 or 400 ppm (v/v) for 6 hr/day, 5 days/week and 4 weeks, and each inhalation group received DMF-formulated drinking water at 0, 800, 1,600 or 3,200 ppm (w/w) for 24 hr/day, 7 days/week and 4 weeks (Ohbayashi et al., 2008). Both the combined inhalation and oral exposures and the single-route exposure through inhalation or ingestion induced centrilobular hypertrophy and single-cell necrosis of hepatocytes, increased plasma levels of alanine aminotransferase (ALT), increased percentage of proliferating cell nuclear antigen (PCNA)-positive hepatocytes without glutathione-S-transferase placental form (GST-P)-positive liver foci, and increased relative liver weight. Those hepatic parameters of the DMF-induced effects were classified into hypertrophyc, necrotic and proliferative responses according to the pathological characteristics of affected liver. While magnitudes of the hypertrophic and necrotic responses were linearly increased with an increase in amounts of DMF uptake in the single-route exposure groups, those dose-response relationships tended to level off in the combined-exposure groups. Saturation of the hypertrophic and necrotic responses at high dose levels might be attributed to suppression of the metabolic conversion of DMF to its toxic metabolites. Percentage of PCNA-stained hepatocytes classified as the proliferative response was increased more steeply in the combined-exposure groups than in the single-route exposure groups. It was suggested that the proliferative response of hepatocytes to the combined exposures would be greater than that which would be expected under an assumption of additivity for the component proliferative responses to the single-route exposures through inhalation and ingestion.

In a disregarded study*, a group of 50 male F344 rats, 6 -week old, was exposed by inhalation to 0 (clean air), 200 or 400 ppm (v/v) of DMF vapor-containing air for 6 h/day and 5 days /week during a 104 week period, and each inhalation group was given ad libitum DMF-formulated drinking water at 0, 800 or 1600 (w/w) for 104 weeks (Ohbayashi et al., 2009). The study was designed in order to examine hepatocarcinogenic effect of combined inhalation and oral exposures of rats to DMF.

Incidences of hepatocellular adenomas and carcinomas and their combined incidences were significantly increased in the combined-exposure groups compared with the untreated control group or each of the inhalation-alone and oral-alone groups. Incidences of hepatocellular adenomas and carcinomas induced by the combined exposures were greater than the sum of the two incidences of the hepatocellular adenomas and carcinomas induced by the single-route exposures through inhalation and ingestion. The combined exposures enhanced tumor malignancy. The hepatocarcinogenic effect of the combined exposures is greater than the effect that would be expected under assumption that two effects of single-route exposures through inhalation and drinking are additive.

* The doses selected exceeded the maximum tolerated dose (MTD), which was exacerbated by probable exposure to an aerosol during atmosphere generation. In addition, the selection of test system used for this study may have contributed to increased tumor incidence observed. No historical data is available on the rat strain


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The best documented study with the lowest NOAEL.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The study with the longest duration.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

The classification is not warranted according to the criteria of Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008.