N,N-dimethylformamide

Repeated dose toxicity: inhalation

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

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

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

other: Crl:CD-1 (ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Quebec, Canada)
- Age at study initiation: 55 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
- Exposure apparatus: four 9-m3 stainless steel and glass chambers
- System of generating particulates/aerosols: 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 l-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
- Temperature, humidity, pressure in air chamber: During exposure, temperature and relative humidity of the chamber air were monitored continuously. Chamber oxygen content was measured at least twice daily.
- Air flow rate: The chambers were operated in a one-pass flowthrough mode with air flow rates adequate to provide sufficient oxygen for test animals, to prevent contamination from volatiles derived from animal excreta and to enable adequate distribution of DMF. Air flow rates were targeted at 1750 L/min and were recorded at approximately 5-min intervals.
- Treatment of exhaust air: Chamber exhaust was passed through a water scrubber (with scrubbing efficiency of > 95 %) and through charcoal fillers prior to exhausting. Ammonia levels were determined in the control chamber periodically during the study and were within acceptable ranges

TEST ATMOSPHERE
- Brief description of analytical method used: One-microliter aliquots of the samples were analysed with a Hewlett-Packard Model 5890 11 gas chromatograph, equipped with a Hewlett-Packard 3396 integrator. Aliquots were injected into a sample port at a temperature of 155 °C and separated over a 15-m Carbowax 20 M Mega-bore column at 100 °C with helium carrier gas. DMF was detected by nitrogen phosphorous detector at a temperature of 300 °C. The retention time of DMF was approximately 1.7 min. The corresponding peak areas were converted to parts-per-million by external standard calibration.

- Samples taken from breathing zone: yes. Distribution of DMF in the chamber was determined prior to study start and found to be appropriate.
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. During exposures, samples were collected from a representative area of each chamber. Atmosphere samples were collected by withdrawing a known amount of chamber air (via vacuum pump) into a microimpinger containing 1 mL acetone. Samples were also taken at least twice daily from the control chamber to ensure that no DMF was detected

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:

18 months

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. 0.30 mg/L

Dose / conc.:

400 ppm

Remarks:

approx. 1.21 mg/L

No. of animals per sex per dose:

78

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 ul. 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 and 18 month after initiation
- Anaesthetic used for blood collection: Yes (light carbon dioxide anesthesia)
- Animals fasted: No
- How many animals: 10 animals per sex per group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: No

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
HISTOPATHOLOGY: Yes
Lungs, brain, liver, kidneys, 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.

Other examinations:

After 2 weeks, 3 months, and 12 months of testing, five male and five female mice 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 anesthesia and exsanguination 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 nonparametric 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):

Survival in treated male mice was similar to that in the respective control group for all exposure concentrations (56, 68, 60, and 59 % for 0, 25, 100, and 400 ppm, respectively). In females, survival was similar to control at all exposure concentrations (68, 57, 62, and 76 %, respectively). Therefore, compound-related differences in the survival of mice were not evident in this study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male and female mice exposed to 400 ppm generally had higher body weight compared to control values. Similarly, body weight gain was significantly higher for 400 ppm males (20 %) and for 100 and 400 ppm females (16 and 13 %, respectively) during the first 12 months of the study. The higher body weight and body weight gain observed for 100 and 400 ppm males and females 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):

All lesions seen in the eyes of mice in this study were considered to be spontaneous. The most frequent findings were cataracts and corneal mineralization which are common in mice of this strain and age.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no compound-related differences in haematology parameters in either male or female mice at any sampling period.

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Male and female mice exposed to 400 ppm had significantly increased absolute and relative liver weights at the cell proliferation terminations at Day 19 and Day 95. At the cell proliferation euthanasia on Day 363, absolute and relative liver weights were significantly increased in 400 ppm males and slightly increased in 400 ppm females. Male and female mice exposed to 100 ppm exhibited a similar trend toward increased absolute and relative liver weights; however, the differences from control were not statistically significant. At the 18-month euthanasia, 100 and 400 ppm males and 400 ppm females had significantly higher absolute and relative liver weights (Table 2).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross observations at necropsy revealed that male mice exposed to 400 ppm had a higher incidence of large livers and liver deformities.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The increased liver weights are consistent with the microscopic observation of hepatocellular hypertrophy. Compound-related microscopic changes were observed in the livers of both sexes for all three exposure concentrations. The principle effect was minimal to mild centrilobular hypertrophy that progressed to panlobular hypertrophy in some animals. At the 18-month euthanasia, hypertrophy was present in both sexes at all exposure concentrations (Table 6).
In addition, the incidence of individual hepatocellular necrosis (apoptosis) was also increased in both sexes for all three test concentrations (Table 6). Minimal to moderate Kupffer cell hyperplasia with accumulation of lipofuscin and hemosiderin and an increase in the incidence of inflammatory cells in the liver were also observed at all three test concentrations (Table 6). In addition, a dose-related increase in eosinophilic and mixed foci of cellular alteration were observed in both sexes.
Several secondary changes were observed in livers from 100 and 400 ppm mice. These changes included biliary hyperplasia, increased mitotic figures, and multinucleate hepatocytes and are probably due to adaptive or reparative processes rather than a direct compound-related effect.
There were no compound-related lesions observed in the nose or respiratory tract at any exposure concentration.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Exposure of mice to DMF for 18 months did not cause a compound-related increase in tumors. In addition, the incidence of hepatic tumors and testicular tumors was similar to control for all exposure concentrations (Table 7). The incidence of total primary tumors and total benign tumors in 400 ppm males was significantly increased due to higher numbers of lung, liver, and harderian gland tumors. However, the incidences of these tumors individually were not significantly increased, and they are known to have a high spontaneous incidence in male mice (Maita et al., 1988, cited in the original paper). Therefore, exposure of mice for 18 months to DMF was not oncogenic at concentrations up to 400 ppm.

Details on results:

HISTORICAL CONTROL DATA (if applicable)
no effects were seen in the reproductive tissues and organs during this study. The major portal of entry, the respiratory tract, was similarly unaffected. These toxicological data along with epidemiologic study results support the information that was used to establish the existing TLV for DMF at 10 ppm (ACGIH, 1992). It appears that this airborne concentration, along with a program to minimize or eliminate dermal contact would be protective of worker health.

OTHER FINDINGS
There were no statistically or biologically significant differences between treated and control mice in the mean individual cycle length, mean number of estrous cycles, or the number of mice experiencing prolonged estrous.

Dose descriptor:

LOAEC

Effect level:

ca. 25 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: (general toxicity) only minimal changes in liver at this concentration

Dose descriptor:

NOEC

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: oncogenicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

not specified

Table 2: Effect of DMF on Relative^aLiver Weight in Rats and Mice
	DMF (ppm)
	0	25	100	400
Male rats
12 Months^b	2.54 (0.18)	2.73 (0.34)	2.93* (0.32)	3.26* (0.31)
24 Months^c	2.87 (0.45)	2.81 (0.35)	3.28 (0.53)	3.58* (0.73)
Female rats
12 Months^b	2.64 (0.24)	2.70 (0.41)	3.25* (0.40)	3.34* (0.40)
24 Months^c	3.12 (0.67)	3.43 (1.06)	3.33 (0.71)	3.86* (0.61)
Male mice
18 Months^d	5.85 (1.18)	5.94 (1.45)	7.06* (2.04)	7.80* (2.35)
Female mice
18 Months^d	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, 21 and 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, 38, and 36 livers evaluated for 0, 25, 100 and 400 ppm, 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 6:Incidence (%) of Compound-Related Morphological Observations in Mice Exposed to DMF for 18 Months^a

	DMF (ppm)
	0	25	100	400
Lesion    Centrilobular    Hepatocellular    Hypertrophyb
Male	0	8*	41*	52*
Female	0	6	19*	54*
Hepatic single cell necrosis^b
Male	24	59*	68*	87*
Female	29	44*	70*	76*
Hepatic kupffer cell
hyperplasia/pigment
accumulation^b
Male	22	52*	60*	86*
Female	51	57	71*	89*
Hepatic foci of alteration^b
Male: Mixed	0	3	13*	19*
Male: Eosinophilic	2	8	10	8
Female: Mixed	0	0	3	3
Female: Eosinophilic	0	2	5	6

^a Data represent total percentage incidence for both unscheduled and scheduled deaths over the interval 0-18 months.

^b For males exposed to 0, 25, 100 or 400 ppm, the number of livers examined was 60, 62, 60 and 59, respectively. For females exposed to 0, 25,100, or 400 ppm, the number of livers examined was 61, 63, 61 and 63, respectively.

* Statistically significant at P <0.05.

 Table 7: Incidence (%) of Hepatic, Testicular, and Mammary Tumors in Mice Exposed to DMF

		DMF (ppm)
		0	25	100	400
Primary hepatic tumors
Hepatocellular adenomas	(M)^a	22 (13/60)^b	18 (11/62)	18 (11/60)	19 (11/59)
	(F)	0 (0/61)	2 (1/63)	3 (2/61)	2 (1/63)
Hemangioma	(M)	2 (1/60)	0 (0/62)	0 (0/60)	2 (1/59)
	(F)	0 (0/61)	0 (0/63)	2 (1/61)	2 (1/63)
Hepatocellular carcinoma^c	(M)	0 (0/60)	2 (1/62)	7 (4/60)	3 (2/59)
Hemangiosarcoma^c	(M)	0 (0/60)	0 (0/62)	2 (1/60)	3 (2/59)
Primary testicular tumors
Interstitial cell adenoma	(M)	2 (1/59)	0 (0/22)^d	0 (0/25)^d	0 (0/56)
Primary mammary tumors
Adenocarcinoma^e	(F)	3 (2/62)	4 (1/26)^d	12 (3/26)^d	0 (0/58)

^a M, male; F, female.

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

^c This lesion was not observed in females.

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

^e This lesion was not observed in males.

Conclusions:

Exposure of mice to DMF for 18 months did not cause a compound-related increase in tumors. In addition, the incidence of hepatic tumors and testicular tumors was similar to control for all exposure concentrations (Table 7). The incidence of total primary tumors and total benign tumors in 400 ppm males was significantly increased due to higher numbers of lung, liver, and harderian gland tumors. However, the incidences of these tumors individually were not significantly increased, and they are known to have a high spontaneous incidence in male mice (Maita et al., 1988, cited in the original paper). Therefore, exposure of mice for 18 months to DMF was not oncogenic at concentrations up to 400 ppm.

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 mice were approx. 55 days of age at the beginning of the study. The animals were exposed by whole-body exposure to DMF vapors at concentrations of 0, 25, 100 and 400 ppm. The concurrent control (0 ppm) was exposed to dehumidified air alone. Examinations on body weight, organ weights, ophthalmoscopy, and a complete necropsy including microscopically examination were carried out. Haematology was investigated at 3, 6, 12 and 18 months in each 10 male and 10 female animals/group. Estrous cycle evaluation was done in all female animals in the 0 and 400 ppm groups from test day 107 to test day 131. Cell proliferation in the liver was investigated after 2 weeks, 3 months and 12 months in five randomly selected animals per sex and group. Livers from animals in the 0 and 400 ppm groups were immunohistochemically evaluated.

Results: Survival in treated male and female mice was similar to that in the concurrent control group (male animals: 56, 68, 60 and 59 % for 0, 25, 100 and 400 ppm, respectively; female animals: 68, 57, 62 and 76 %, respectively). There were no compound-related differences in haematology parameters and no significant differences with respect to estrous cycle evaluations or ophthalmoscopy. In male animals exposed to 100 and 400 ppm, and in female mice at 400 ppm a significant increase in absolute and relative liver weights together with hepatocellular hypertrophy was observed. Microscopy revealed hepatic changes (minimal to mild hepatocellular hypertrophy) in all treated groups with the incidence being dose-related. Individual hepatocellular necrosis was seen in all groups with the incidence being greater in the DMF-treated groups. Minimal to moderate Kupffer cell hyperplasia with accumulation of lipofuscin and hemosiderin was also observed in all groups again with the incidence being greater in the DMF-treated animals. A dose-related increase in mixed foci in the liver was seen in the males and a higher incidence of eosinophilic foci was seen in both sexes of the treated groups when compared to the concurrent control animals. Cell-labelling indices in the liver showed no compound-related effect at any exposure level. No compound-related lesions were observed in the nose or respiratory tract at any exposure level. The incidence of hepatic and testicular tumors was similar to control for all exposure concentrations.

Discussion and conclusion: The exposure of mice to DMF over a time period of 18 months was not oncogenic at concentrations up to 400 ppm. Therefore, the no-observable-effect level (NOEL) for oncogenicity was 400 ppm in 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. However, due the findings at 25 ppm (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 incidence of hepatic kupffer cell hyperplasia and pigment accumulation) that occurred in most cases in a dose-related manner, a clear NOAEC could not be determined and consequently, the LOAEC for mice was considered to be 25 ppm (general toxicity).

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: inhalation

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:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

not specified

Species:

mouse

Strain:

other: Crl:CD-1 (ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Quebec, Canada
- Age at study initiation: 55 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

Vehicle:

unchanged (no vehicle)

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:

18 months

Frequency of treatment:

5 d/w; 6 h/d

Post exposure period:

no

Dose / conc.:

25 ppm

Remarks:

about 0.08 mg/L

Dose / conc.:

100 ppm

Remarks:

about 0.30 mg/L

Dose / conc.:

400 ppm

Remarks:

about 1.21 mg/L

No. of animals per sex per dose:

78

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 and 18 month after initiation
- Anaesthetic used for blood collection: Yes (light carbon dioxide anesthesia)
- Animals fasted: No
- How many animals: 10 animals per sex per group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: No

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, 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.

Other examinations:

After 2 weeks, 3 months, and 12 months of testing, five male and five female mice 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 exsanguination 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):

Survival in treated male mice was similar to that in the respective control group for all exposure concentrations (56, 68, 60, and 59 % for 0, 25, 100, and 400 ppm, respectively). In females, survival was similar to control at all exposure concentrations (68, 57, 62, and 76 %, respectively). Therefore, compound-related differences in the survival of mice were not evident in this study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male and female mice exposed to 400 ppm generally had higher body weight compared to control values. Similarly, body weight gain was significantly higher for 400 ppm males (20 %) and for 100 and 400 ppm females (16 and 13 %, respectively) during the first 12 months of the study. The higher body weight and body weight gain observed for 100 and 400 ppm males and females 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):

All lesions seen in the eyes of mice in this study were considered to be spontaneous. The most frequent findings were cataracts and corneal mineralization which are common in mice of this strain and age.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no compound-related differences in haematology parameters in either male or female mice at any sampling period.

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Male and female mice exposed to 400 ppm had significantly increased absolute and relative liver weights at the cell proliferation terminations at Day 19 and Day 95. At the cell proliferation euthanasia on Day 363, absolute and relative liver weights were significantly increased in 400 ppm males and slightly increased in 400 ppm females. Male and female mice exposed to 100 ppm exhibited a similar trend toward increased absolute and relative liver weights; however, the differences from control were not statistically significant. At the 18-month euthanasia, 100 and 400 ppm males and 400 ppm females had significantly higher absolute and relative liver weights (Table 2).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross observations at necropsy revealed that male mice exposed to 400 ppm had a higher incidence of large livers and liver deformities.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

hepatocellular and centrilobular hypertrophy (for more information, please see: 'Details on results')

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Exposure of mice to DMF for 18 months did not cause a compound-related increase in tumors. In addition, the incidence of hepatic tumors and testicular tumors was similar to control for all exposure concentrations (Table 7). The incidence of total primary tumors and total benign tumors in 400 ppm males was significantly increased due to higher numbers of lung, liver, and harderian gland tumors. However, the incidences of these tumors individually were not significantly increased, and they are known to have a high spontaneous incidence in male mice (Maita et al., 1988, cited in the original paper). Therefore, exposure of mice for 18 months to DMF was not oncogenic at concentrations up to 400 ppm.

Details on results:

HISTOPATHOLOGY: NON-NEOPLASTIC
The increased liver weights are consistent with the microscopic observation of hepatocellular hypertrophy. Compound-related microscopic changes were observed in the livers of both sexes for all three exposure concentrations. The principle effect was minimal to mild centrilobular hypertrophy that progressed to pan lobular hypertrophy in some animals. At the 18-month euthanasia, hypertrophy was present in both sexes at all exposure concentrations (Table 6).
In addition, the incidence of individual hepatocellular necrosis (apoptosis) was also increased in both sexes for all three test concentrations (Table 6). Minimal to moderate Kupffer cell hyperplasia with accumulation of lipofuscin and hemosiderin and an increase in the incidence of inflammatory cells in the liver were also observed at all three test concentrations (Table 6). In addition, a dose-related increase in eosinophilic and mixed foci of cellular alteration were observed in both sexes.
Several secondary changes were observed in livers from 100 and 400 ppm mice. These changes included biliary hyperplasia, increased mitotic figures, and multinucleate hepatocytes and are probably due to adaptive or reparative processes rather than a direct compound-related effect.
There were no compound-related lesions observed in the nose or respiratory tract at any exposure concentration.

HISTORICAL CONTROL DATA (if applicable)
no effects were seen in the reproductive tissues and organs during this study. The major portal of entry, the respiratory tract, was similarly unaffected. These toxicological data along with epidemiologic study results support the information that was used to establish the existing TLV for DMF at 10 ppm (ACGIH, 1992). It appears that this airborne concentration, along with a program to minimize or eliminate dermal contact would be protective of worker health.

OTHER FINDINGS
There were no statistically or biologically significant differences between treated and control mice in the mean individual cycle length, mean number of estrous cycles, or the number of mice experiencing prolonged estrous.

Relevance of carcinogenic effects / potential:

Exposure of mice to DMF for 18 months did not cause a compound-related increase in tumors. In addition, the incidence of hepatic tumors and testicular tumors was similar to control for all exposure concentrations (Table 7). The incidence of total primary tumors and total benign tumors in 400 ppm males was significantly increased due to higher numbers of lung, liver, and harderian gland tumors. However, the incidences of these tumors individually were not significantly increased, and they are known to have a high spontaneous incidence in male mice (Maita et al., 1988, cited in the original paper). Therefore, exposure of mice for 18 months to DMF was not oncogenic at concentrations up to 400 ppm.

Dose descriptor:

NOEC

Effect level:

400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: carcinogenicity

 Table 2: Effect of DMF on Relative^aLiver Weight in Rats and Mice

	DMF (ppm)
	0	25	100	400
Male rats
12 Months^b	2.54 (0.18)	2.73 (0.34)	2.93* (0.32)	3.26* (0.31)
24 Months^c	2.87 (0.45)	2.81 (0.35)	3.28 (0.53)	3.58* (0.73)
Female rats
12 Months^b	2.64 (0.24)	2.70 (0.41)	3.25* (0.40)	3.34* (0.40)
24 Months^c	3.12 (0.67)	3.43 (1.06)	3.33 (0.71)	3.86* (0.61)
Male mice
18 Months^d	5.85 (1.18)	5.94 (1.45)	7.06* (2.04)	7.80* (2.35)
Female mice
18 Months^d	5.59 (0.92)	5.71 (0.95)	5.99 (1.45)	6.35* (0.78)
^a% of body weight.
^bLivers evaluated from 10 rats/sex/concentration.
^cFor males n =17, 19, 21 and 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.
^dFor males n =31, 42, 38, 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 6: Incidence (%) of Compound-Related Morphological Observations in Mice Exposed to DMF for 18 Months^a
	DMF (ppm)
	0	25	100	400
Lesion    Centrilobular    Hepatocellular    Hypertrophyb
Male	0	8*	41*	52*
Female	0	6	19*	54*
Hepatic single cell necrosis^b
Male	24	59*	68*	87*
Female	29	44*	70*	76*
Hepatic kupffer cell
hyperplasia/pigment
accumulation^b
Male	22	52*	60*	86*
Female	51	57	71*	89*
Hepatic foci of alteration^b
Male: Mixed	0	3	13*	19*
Male: Eosinophilic	2	8	10	8
Female: Mixed	0	0	3	3
Female: Eosinophilic	0	2	5	6

^aData represent total percentage incidence for both unscheduled and scheduled deaths over the interval 0-18 months.
^bFor males exposed to 0, 25, 100 or 400 ppm, the number of livers examined was 60, 62, 60 and 59, respectively.
For females exposed to 0, 25,100, or 400 ppm, the number of livers examined was 61, 63, 61 and 63, respectively.
* Statistically significant at P <0.05.

Table 7: Incidence (%) of Hepatic, Testicular, and Mammary Tumors in Mice Exposed to DMF
		DMF (ppm)
		0	25	100	400
Primary hepatic tumors
Hepatocellular adenomas	(M)^a	22 (13/60)^b	18 (11/62)	18 (11/60)	19 (11/59)
	(F)	0 (0/61)	2 (1/63)	3 (2/61)	2 (1/63)
Hemangioma	(M)	2 (1/60)	0 (0/62)	0 (0/60)	2 (1/59)
	(F)	0 (0/61)	0 (0/63)	2 (1/61)	2 (1/63)
Hepatocellular carcinoma^c	(M)	0 (0/60)	2 (1/62)	7 (4/60)	3 (2/59)
Hemangiosarcoma^c	(M)	0 (0/60)	0 (0/62)	2 (1/60)	3 (2/59)
Primary testicular tumors
Interstitial cell adenoma	(M)	2 (1/59)	0 (0/22)^d	0 (0/25)^d	0 (0/56)
Primary mammary tumors
Adenocarcinoma^e	(F)	3 (2/62)	4 (1/26)^d	12 (3/26)^d	0 (0/58)

^aM, male; F, female.

^bNumerator represents number of tumors, and the denominator represents number of tissues examined.

^cThis lesion was not observed in females.

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

^eThis lesion was not observed in males.

Conclusions:

DMF was not carcinogenic under the 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 animals/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 relation-ship 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.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: inhalation

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:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

GLP compliance:

not specified

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

Vehicle:

unchanged (no vehicle)

Details on 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 I -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

Post exposure period:

no

Dose / conc.:

25 ppm (nominal)

Remarks:

about 0.08 mg/L

Dose / conc.:

100 ppm (nominal)

Remarks:

about 0.3 mg/L

Dose / conc.:

400 ppm (nominal)

Remarks:

about 1.2 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 [No.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 exsanguination 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

Dermal irritation (if dermal study):

not examined

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 opthalmologic 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 (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 (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 (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:

effects observed, non-treatment-related

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):

centrilobular hepatocellular hypertrophy (for more information see: 'Details on result')

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 (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:

HISTOPATHOLOGY: NON-NEOPLASTIC
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 (Table 3). In addition, the incidence of focal cystic degeneration was increased in 100 and 400 ppm males which range 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.

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.

Relevance of carcinogenic effects / potential:

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 (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.

Dose descriptor:

NOEC

Effect level:

400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Carcinogenicity

 Table 1: Effect of DMF on Sorbitol Dehydrogenase Activity in Male and Female Rats^a

	3 Months	6 Months	12 Months	18 Months	24 Months
Concentration (ppm)	Males
0	7.0^b (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)
^a10 Rats/sex/concentration were sampled at each time point.
^bMean and standard deviation. Units are u/liter.
^*Statistically significant atP <0.05.

 Table 2: Effect of DMF on Relative^aLiver Weight in Rats and Mice

	DMF (ppm)
	0	25	100	400
Male rats
12 Months^b	2.54 (0.18)	2.73 (0.34)	2.93* (0.32)	3.26* (0.31)
24 Months^c	2.87 (0.45)	2.81 (0.35)	3.28 (0.53)	3.58* (0.73)
Female rats
12 Months^b	2.64 (0.24)	2.70 (0.41)	3.25* (0.40)	3.34* (0.40)
24 Months^c	3.12 (0.67)	3.43 (1.06)	3.33 (0.71)	3.86* (0.61)
Male mice
18 Months^d	5.85 (1.18)	5.94 (1.45)	7.06* (2.04)	7.80* (2.35)
Female mice
18 Months^d	5.59 (0.92)	5.71 (0.95)	5.99 (1.45)	6.35* (0.78)

 ^a% of body weight.

 ^bLivers evaluated from 10 rats/sex/concentration.

 ^cFor 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.

 ^dFor males n =31, 42, 38, 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 Months^a

	DMF (ppm)
	0	25	100	400
Lesion   Centrilobular   Hepatocellular   Hypertrophy^b
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/hemosiderin^b
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*

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

 ^bThe 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)
Adenoma^d	(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)

 ^aM, male; F, female.

 ^bNumerator represents number of tumors, and the denominator represents number of tissues examined.

 ^cFor 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.

 ^dThis lesion was not observed in males.

 * Statistically significant at P <0.05

Conclusions:

DMF was not carcinogenic under the 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 animals/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 microscopically 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 relation-ship 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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 451 Carcinogenicity Studies

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethylformamide

EC Number:

200-679-5

EC Name:

N,N-dimethylformamide

Cas Number:

68-12-2

Molecular formula:

C3H7NO

IUPAC Name:

N,N-dimethylformamide

Details on test material:

N,N-dimethylformamide, 99.9 % pure

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 ppm

Remarks:

approx. 0.08 mg/L

Doses / concentrations 2

Dose / conc.:

100 ppm

Remarks:

approx. 0.3 mg/L

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

Dose descriptor:

LOEC

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: hepatotoxic effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1:  Effect of DMF on Sorbitol Dehydrogenase Activity in Male and Female Rats^a

	3 Months	6 Months	12 Months	18 Months	24 Months
Concentration (ppm)	Males
0	7.0^b(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 Relative^aLiver Weight in Rats and Mice
	DMF (ppm)
	0	25	100	400
Male rats
12 Months^b	2.54 (0.18)	2.73 (0.34)	2.93* (0.32)	3.26* (0.31)
24 Months^c	2.87 (0.45)	2.81 (0.35)	3.28 (0.53)	3.58* (0.73)
Female rats
12 Months^b	2.64 (0.24)	2.70 (0.41)	3.25* (0.40)	3.34* (0.40)
24 Months^c	3.12 (0.67)	3.43 (1.06)	3.33 (0.71)	3.86* (0.61)
Male mice
18 Months^d	5.85 (1.18)	5.94 (1.45)	7.06* (2.04)	7.80* (2.35)
Female mice
18 Months^d	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 Months^a

	DMF (ppm)
	0	25	100	400
Lesion   Centrilobular   Hepatocellular   Hypertrophy^b
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/hemosiderin^b
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)
Adenoma^d	(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.

Applicant's summary and conclusion

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