N,N-dimethylacetamide

Carcinogenicity

Description of key information

ORAL:

A 2-year oral drinking study (1979; RL2: equivalent to OECD 453, non-GLP) in rats (m/f) was performed. Concentration range 100-1000 mg/kg bw/d. No signs for a carcinogenic effect.

INHALATION:

A 2-year inhalation study (Malley, 1995; RL1: equivalent to OECD 453, GLP) in rats (m/f) was performed. Concentration range 25.2-350.5 ppm. No signs for a carcinogenic effect.

An eighteen months inhalation study (Malley, 1995; RL1: equivalent to OECD 453, GLP) in mice (m/f) was performed. Concentration range 25.2-350.5 ppm. No signs for a carcinogenic effect.

DERMAL:

no study available.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

(no details about the test substance; problems in analytical methodology of DMAC in drinking water; limited data on test animals and conditions; some organs not included in histopathology of high dose group and controls; no documentation of details on incidences in the reevaluation [Monsanto 1990])

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Source: Monsanto
no further details available

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blues Sruce Farms, New York
- Age at study initiation: 6-7 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually
- certified died and distilled water (plus DMAC) ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 hrs dark/12 hrs light

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

Distilled water used as vehicle/drinking water; test solution prepared weekly and concentration in drinking water adjusted by body weight and drinking bevaviour.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DMAC concentration in drinking water measured weekly by GC methods. Only 68 out of 114 samples were within 20 % of the nominal concentration. Authors assumed problems with analytical methodology rather than preparations errors. Decreased values in high dose males only during the 1st weeks (see doses).

Duration of treatment / exposure:

2 years

Frequency of treatment:

daily ad libitum

Post exposure period:

no

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

in water. Range of measured mean intake in females and males: 64-122 and 64-129mg/kg bw/day, respectively.

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

in water. Range of measured mean intake in females and males: 190-399 and 177-392 mg/kg bw/day, respectively.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

in water. Range of measured mean intake in females and males: 598-1227 and 267-1172 mg/kg bw/day, respectively.

No. of animals per sex per dose:

70

Control animals:

yes, concurrent vehicle

Details on study design:

At the end of six or twelve months of treatment, ten animals/sex/group (n=9 at 300 mg/kg bw/day) were randomly selected and sacrificed for examinations; at the end of 24 months on test, all of the survivors were killed; immediately after death, the external surface, all orifices, the thoracic, abdominal and pelvic cavities and their associated tissues and organs, the neck and its tissues and organs and the remaining carcass were examined for the presence of gross morphologic abnormalities; rats which died unscheduled deaths during the course of the study were examined similarly.

Post exposure period: no

Histopathology: in the first report (Monsanto 1979) it was stated that a complete histopathology at termination (after 24 months) was performed only in 10 rats/sex of the high dose group and all surviving control rats. In such a case the validity of the study would be limited. Therefore, a reevaluation of histopathology data was started and reported in Monsanto (1990). In this reevaluation all male and female rats of the control and the high dose were examined in complete histopathology (10 rats each at 6 and 12 months interim sacrifice and all survivors at terminatiion of each sex). However, only a description of non-neoplastic effects were given without detailed, tabulated results and without documentation of incidences.

Positive control:

no

Observations and examinations performed and frequency:

Clinical signs and mortality recorded once daily the 1st 3 months thereafter twice daily.
Detailed physical examination once weekly.

Ophthalmology
Parameters measured pretest and 3, 12, and 24 months after initiation in all rats (lids, lacrimal apparatus, cornea, conjunctiva, anterior chamber, lens, humor, retina, optic disc)

Body weight
Measured twice pretest and once weekly during the 1st 14 weeks of exposure, thereafter once monthly and at termination (after fasting).

Food consumption
Measured pretest, once weekly at week 1-14, twice weekly at week 15-26, and thereafter once monthly.

Drinking water consumption
Determined for 10 rats per dose per sex; pretest value, once weekly at week 1-59, once monthly thereafter; test substance intake calculated from water intake data.

Blood sampling 3, 6, 12, 18, 24 months after initiation; 6 rats per dose per sex.
Hematology: hemoglobin, hematocrit, erythrocyte counts, erythrocyte morphology, clotting time, total and differential leukocytes (n=20/sex/dose recommended).
Clinical chemistry: serum GPT, alkaline phosphatase, blood urea nitrogen (BUN), fasting glucose, gamma GT (no data on total protein or albumin).

Urinalysis
In 6 rats per dose per sex parameters were measured 3, 6 and 12 months after initiation in the high dose and control group; after 18 and 24 months urinalysis was performed in all groups (n=6 per sex per dose); parameters: gross appearance, pH, glucose, specific gravity, protein, ketone, bilirubin, occult blood.

Sacrifice and pathology:

Complete necropsy of all rats performed.
Organs weights measured from adrenal glands, brain, testes, ovaries, kidneys, spleen, liver, pituitary gland; n=10 per dose per sex at the 6 and 12 months interim sacrifice; at termination all survivors.

Histopathology of the following organs:
adrenal glands
bone marrow (sternal )
brain (medulia/pons, cerebellar cortex and cerebral cortex)
eye
testes
ovaries
heart (with coronary vessels )
intestine
colon
duodenum
ileum
kidneys
liver
lung
lymph node (mesenteric)
mammary gland (female)
pancreas
pituitary gland
prostate
salivary gland
skeletal muscle
skin
spinal cord (cervical)
spleen
stomach
thyroid/parathyroid glands
urinary bladder
uterus
gross lesions tissue masses

nose, larynx & pharynx, oesophagus, accessory genital organs, muscle, peripheral nerve not included.

Other examinations:

no

Statistics:

Yes, statistically significant differences were calculated by F-test, Students t-test, Cochran test, Dunnett test, level of significance: p<0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

slightly increased incidence of alopecia at 1000 mg/kg bw/day in males and females and slightly increased incidence in yellow staining of anogenital area in females of this dose.

Mortality:

no mortality observed

Description (incidence):

no effects

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease at 1000 mg/kg bw/day in males and females and at 300 mg/kg bw/day in males.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no statistically significant effects

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

technical problems during the first weeks (see also dose range) and decreased palatability (pronounced in high dose males); high variability but no test substance-related pattern.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

no treatment related effects

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

at >= 100 mg/kg bw/day the hemoglobin concentrations and erythrocyte counts were reduced in females after 6 months of exposure (no effects after 3, 12, 18, or 24 months);
at >=300 mg/kg bw erythrocyte counts in males were increased (only at termination)
at 1000 mg/kg bw/day blood clotting time reduced in males (only at termination)
see comments below under - Any other information on results incl. tables.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

significant effects compared with the concurrent control
at 100 & 300 mg/kg bw/day alkaline phosphatase reduced in males after 6 and 18 months
at 1000 mg/kg bw/day reduced alkaline phosphatase activity in males at all intervals
at 1000 mg/kg bw/day serum GPT elevated in males and females after 6 months (no effects at other time points)
at >=100 mg/kg bw/day BUN elevated in males at all intervals
at 1000 mg/kg bw/day in females reduced glucose only after 12 months
see comments below under - Any other information on results incl. tables.

Urinalysis findings:

no effects observed

Description (incidence and severity):

no effects

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

>=100 mg/kg bw/day: rel. and absolute liver weight was increased after 6, 12, and 24 months in males and females
>=300 mg/kg bw/day: rel & abs. kidney weight increased in males and females after 12 months and in females after 6 months (no effects after 24 months, no effects in histopathology)
1000 mg/kg bw/day: rel & abs. kidney weight increased in males after 6 months (see above)
>=100 mg/kg bw/day: rel. and abs. adrenal weight increased in males after 6 months, no effects after 12 or 24 months (no histopathological effects)
1000 mg/kg bw/day testis weight was reduced (not statistically significant) after 12 months, significant at termination.

Gross pathological findings:

no effects observed

Description (incidence and severity):

no treatment related effects

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

at 1000 mg/kg bw/day in comparison to control group (no data about low and mid dose level; no details about incidences available):

- hypertrophy/hyperplasia of the central lobular hepatocytes (minimal to moderate in severity) was seen in numerous males and females but not in controls
- the incidence of liver cell vacuolisation and liver cell degeneration was increased in males and females; no data was given on statistical significance or toxicological relevance; no data about low and mid dose level is available
- pigmentation (brown) of liver cells and reticuloendothelial cells in males and females which was not seen in controls
- incidence of small flaccid testis increased as well as degeneration/atrophy of the germinal epithelium (minimal to marked in severity) in comparison to control
- secondary to effects in testis atrophy of prostate
- hemosiderosis of the spleen in females (but no anemia)
- increased incidence of lymphoid depletion/atrophy in males (questionable relevance)

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

at 1000 mg/kg bw/day in comparison to control group (no data about low and mid dose level; no details about incidences available):

-The incidence of thymomas in females was slightly increased: 0/50 in the control group, 1/50 (2 %) in the 100 mg/kg group, 0/50 in the 300 mg/kg group and 3/50 (6 %) in the high-dose group . These tumours, which occur spontaneously in Long-Evans rats with a somewhat variable incidence, showed no dose-response relationship and are not thought to be related to treatment

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no treatment related increase in the incidences of neoplasms in any organ

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: based on reduced body weight observed at 300 mg/kg bw/day (nominal)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: based on reduced body weight, liver cell degeneration, observed at 300 mg/kg bw/day (nominal)

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: reduced body weight

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: reduced body weight, liver cell degeneration

Remarks on result:

other: Effect type: toxicity (migrated information)

The results were not discussed by the authors.

Comment: statistically significant effects in hematology and clinical chemistry were not evaluated concerning the toxicological relevance. No data were given on the range of historical value for this rat strain in this or other laboratories. Furthermore, only 6 rats per dose per sex were evaluated concerning clinical chemistry (n=10 recommended) and hematology (n=20 recommended) for some time points only 4 -5 blood samples were assessed (e.g. clinical chemistry 12 months, males, control or n=5 for hematology 12 months, males, control). In summary, the relevance of these effects is questionable.

Comments on histopathology: Hypertrophy of the liver (correlated with increased liver weight) seems to be related to increased metabolism of the test substance and is considered not to be an adverse effect, however, increased incidences in liver cell degeneration and vacuolisation as well as increased pigmentation might be adverse in view on the increased liver weight. Unfortunately, the liver was not histopathologically examined at the low and mid dose level complicating the derivation of an NOAEL. This problem is also unsolved concerning the effects in the testes (atrophy, degeneration, reduced organ weight). The other effects reported in histopathology are not considered to be of toxicological relevance. In the reevaluation of the histopathology (Monsanto 1990) the authors did not present data on statistical significance.

Conclusions:

Conclusion: In a chronic drinking water study no carcinogenic activity was detected but toxic effects like reduced body weight gain at >= 300 mg/kg bw/day in males and at 1000 mg/kg bw/day in females; the high dose induced liver cell degeneration in males and females and testis atrophy in males; the NOAEL was 100 mg/kg bw/day in males and 300 mg/kg bw/day in females. No carcinogenic activity was observd in this long-term drinking water study.

Executive summary:

Monsanto (1979) performed a 2-year oral drinking water study (equivalent to OECD 453, non-GLP) in male and female rats. Seventy animals were exposed to the test substance diluted in water daily for two years at concentrations of 100, 300, and 1000 mg/kg bw/day. The water was available ad libitum. A concurrent vehicle control was included. Animals were examined at least once daily for clinical signs and mortality. Complete necropsy was conducted of all animals at the end of the study. Clinical signs, effects on body weight, hematology, clinical biochemistry, organ weights, and non-neoplastic histopathological findings were reported to be treatment related. Non-treatment related effect included changes in water consumption and neoplastic histopathological findings. The NOAEC for carcinogenicity based on the results obtained in this study was concluded to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Purity: >99.9 %
- Impurities: water, monomethyl acetamide, dimethylformamide, peroxides, iron
- Source: DuPont
- Batch No.: H-18842
no further details available

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: approximately 43 days old
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually
- Certified and irradiated diet: ad libitum (not during exposure)
- Tap water: ad libitum (not during exposure)
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25 °C
- Humidity: 40-60 %
- Air changes: no data
- Photoperiod: 12 hours dark/12 hours light
- Cage racks relocated every 2 weeks
- Sentinel animals (not exposed) were kept in the same room for detection of pathogens in blood.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Stainless-steel and glass chambers (4 m³) were operated in a onepass flow through mode at 1 L/min; chamber temperature (mean) was 23, 22, 23, and 24°C at control, low, mid and high dose level, respectively; the relative humidity was 40, 41, 40, and 40 %, respectively, and mean airflow ranged between 730 to 1050 L/min.

Vapour was generated separately by metering the liquid chemical into a glass J-tube filled with glass beads; heated air (approximately 100-130 °C) was blown through the glass beads to evaporate DMAC; resulting vapour was diluted to the desired concentrations with filtered conditioned (dehumidified) air for each of the three test chambers; chamber concentrations were controlled by varying the test substance flow rates into the J-tubes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber atmosphere was analyzed by gas chromatography at approximately 30-min intervals during each 6-h exposure period; the atmospheric concentration of DMAC was determined by comparing the detector response of the chamber samples to that of liquid standards using standard curves.

Duration of treatment / exposure:

24 months

Frequency of treatment:

- 6 h/day, 5 days/week (24 months)
(not during holidays, no details)

Post exposure period:

No

Dose / conc.:

25.2 ppm (analytical)

Remarks:

range: 22-28 ppm

Dose / conc.:

101 ppm (analytical)

Remarks:

range: 85-115 ppm

Dose / conc.:

350.5 ppm (analytical)

Remarks:

range: 300-390 ppm

No. of animals per sex per dose:

87 animals

Control animals:

yes, sham-exposed

Details on study design:

- Post-exposure period: None
- Dose selection rationale: Saturation in toxicokinetic experiments (150 ppm), toxicity data after repeated inhalation.
- Liver cell proliferation was tested in sub-groups (5 rats per sex per dose) after 0.5, 3, or 12 months of exposure (interim sacrifice).

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL SIGNS, BODY WEIGHT:
All rats were weighed once per week for the first 3 months and once every other week thereafter; at every weighing, each animal was individually handled and examined for clinical signs of toxicity; cage-side examinations were conducted at least once and usually twice daily throughout the study.

OPHTHALMOSCOPIC EXAMINATION:
Examination was conducted by a veterinary ophthalmologist prior to the first exposure and again immediately prior to sacrifice (24 months); at least 1 h before each examination, 1 or 2 drops of 1 % atropine sulfate solution (pretest) or 1 % tropicamide (final euthanization) was placed in each eye of every animal; both eyes were examined by focal illumination and indirect ophthalmoscopy.

HEMATOLOGY:
Ten rats per sex per dose were randomly selected for evaluations after 3, 6, 12, 18 and 24 (only males after 24 months) months of testing; blood samples were collected from the orbital sinus of each fasted rat while the animal was under light carbon dioxide anesthesia; hematological parameters examined at each sampling time were erythrocyte, leukocyte, differential leukocyte, and platelet counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin concentration; reticulocyte counts and bone marrow smears (after sacrifice only).

CLINICAL CHEMISTRY:
The same blood samples as for hematology were used; serum from rats was evaluated for activities of 5'-nucleotidase, alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, and concentrations of blood urea nitrogen, total protein, albumin, globulin (calculated), creatinine, cholesterol, glucose, calcium, sodium, potassium, phosphate, chloride, and total bilirubin.

URINALYSIS:
Urine was collected from each rat (10 rats per dose per sex) for approximately 14 hours prior to blood collection in metabolism cages. Urine volume, pH, and osmolality were measured; urine was also evaluated for the presence of glucose, protein, bilirubin, urobilinogen, ketone, and occult blood; urine colour. Transparency was recorded and sediment from each urine sample microscopically examined.

Sacrifice and pathology:

NECROPSY, GROSS PATHOLOGY:
All rats that were found dead, accidentally killed, or were euthanized in extremis were necropsied; all surviving animals were euthanized by pentobarbital overdose followed by exsanguination and necropsied after 24 months of testing, females after 23.5 months. Interim sacrifice of rats used for clinical chemistry and hematology was conducted after 12 months (10 rats per dose per sex).

ORGAN WEIGHTS:
Lungs, brain, liver, kidneys, and testes were weighed wet at necropsy; organ weight/final body weight ratios were calculated; organs from animals found dead or sacrificed in extremis were not weighed.

HISTOPATHOLOGY:
The following tissues were collected from all animals: skin, bone marrow (femur, sternum), lymph nodes (mandibular, mesenteric), spleen, thymus, aorta (thoracic), heart, trachea, lungs (inflated), nose (4 cross sections, including paranasal sinuses), larynx/pharynx, salivary glands, esophagus, stomach, liver, pancreas, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), prostate, kidneys, urinary bladder, pituitary, thyroid, parathyroid, adrenals, testes, epididymides, seminal vesicles, mammary gland, ovaries, uterus, vagina, brain (including sections of medulla/pons, cerebellar cortex, cerebral cortex), spinal cord (cervical, thoracic, lumbar), peripheral nerve (sciatic), muscle (thigh), bones (femur, sternum), eyes, exorbital lacrimal glands, harderian glands, and all gross lesions.
All tissues were fixed in 10 % neutral-buffered formalin except testes, epididymides, eyes, and skin with mammary gland (fixed in Bouin's solution). The lungs were inflated with formalin at the time of necropsy.
All tissues collected from animals in the 350 ppm and control groups, and from animals that were found dead (tissue integrity permitting), or were euthanized in extremis, were further processed to slides, stained with hematoxylin and eosin, and examined microscopically; lungs, liver, kidneys, and all gross lesions from animals in the 25 and 100 ppm groups were also processed and examined microscopically.

Other examinations:

LIVER CELL PROLIFERATION:
Cell proliferation in the liver measured (5 rats per sex per dose after 0.5, 3, or 12 months of exposure, see Malley et al., 1995) after labelling with bromodeoxyuridine (BrdU). 1000 nuclei per animal evaluated for S-phase; no further examinations of these animals.

Statistics:

One-way analysis of variance, Fisher's exact test with a Bonferroni correction and the Cochran-Armitage test for trend, Bartlett's test and Kruskal-Wallis and Mann-Whitney U test.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related adverse clinical signs of toxicity in either males or females at any dose level.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related effect on mortality occurred throughout the study; the low survival of control females (sacrifice of all females after 23.5 months of exposure) did not affect the interpretation or conclusions of the study .

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were substance-related effects on body weight and/or body weight gain in males and females at 350 ppm. There was a significant decrease at >= study day 350 in females and >= study day 547 in males. A slight decrease was also noted in 100 ppm males (less than 10 %).

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test substance-releted effects (common lesions were equally distributed among groups) after 24 months of exposure.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology parameters in either male or female rats at any dose level.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were several treatment-related changes in clinical chemistry parameters; male and female rats exposed to 350 ppm had significantly increased serum sorbital dehydrogenase (SDH) activity at the 3-month evaluation (14.2 versus 6.2 U/L in control (males) and 8.7 versus 5.7 U/L (females)) and also at the 6-month evaluation for 350 ppm males (12.6 versus 5.6 U/L).
Serum cholesterol concentrations were significantly increased in 100 and 350 ppm females at the 3-, 6-, and 12-month evaluations, and in 25 ppm females at the 6-month evaluation. The increased cholesterol concentration in 100 and 350 ppm females was considered by the authors to be biologically significant.
Serum glucose concentration was also significantly higher for 100 and 350 ppm females at the 3-, 6-, and 12-month evaluations.
The changes in serum cholesterol and serum glucose for 100 and 350 ppm females were considered to be indicative of a compound-related, toxicologically important change in energy metabolism.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on urinalysis parameters in either males or females at any exposure concentration.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects on liver weight parameters were observed at 350 ppm in males and at 100 and 350 ppm in females at termination of the main study; at the 12-month interim euthanization, 100 and 350 ppm females had significantly higher mean relative liver weight (not statistically significant increase in absolute liver weights [14 .5 and 18.5 %] for 100 and 350 ppm females) but no treatment-related changes in absolute or relative organ weight for males at any exposure concentration at the 12-month interim sacrifice.
At the 24-month sacrifice, 350 ppm males had significantly higher absolute and relative liver and kidney weights. In addition, although not statistically significant, absolute and relative liver weights were also higher for 100 ppm males and were considered by the authors to be test substance related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross morphological changes were similar to control in females for all dose levels. Males at 350 ppm had an increased incidence of kidneys with gross changes (indicative of chronic progressive nephropathy), small testes, and large parathyroid glands. The morphological changes in the kidney were considered by the authors to be treatment-related, and the changes in the testes and parathyroid were considered to be secondary to the effects on the kidney.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related microscopic changes at the 12-month interim sacrifice. After 24 months in males at >= 100 ppm increased hepatic focal cystic degeneration and increased incidence of hepatic peliosis were noted. Males at 350 ppm showed an increased incidence of biliary hyperplasia and an increased incidence of pigment in the Kupffer cells (hemosiderin and lipofuscin). There was increased severity of chronic progressive nephropathy (incidence unchanged; severe nephropathy incidence 15, 15, 19, and 32 % for 0, 25, 100, and 350 ppm males, respectively).
In females at >=100 ppm an increased incidence of pigment in the Kupffer cells (hemosiderin and lipofuscin) was noted.
No effects were detected in the respiratory tract. The authors did not report treatment-related effects on testes.
No increase in the incidences of tumours was detected in any treatment group with exception of the occurrence of squamous cell papillomas in the stomach of two (3.1 %) females from the 350 ppm group (statistically significant). This effect was not considered to be test substance-related (incidences in historical controls 0-3.3 %; no other lesions known to precede squamous cell papillomas).

Other effects:

no effects observed

Description (incidence and severity):

LIVER CELL PROLIFERATION:
There were no increases in the rate of hepatic cell proliferation for either 350 ppm males or females at any of the time points evaluated.

Dose descriptor:

NOAEC

Effect level:

350 ppm

Sex:

male/female

Basis for effect level:

other: (1260 mg/m³)

Dose descriptor:

NOAEC

Effect level:

25 ppm

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Conclusions:

Inhalation exposure of rats to DMAC for 24 months did not cause a treatment-related increase in tumour incidences at concentrations up to 350 ppm (1260 mg/m³) but toxic effects in males and females at 100 ppm (360 mg/m³) (NOAEC: 25 ppm, 90 mg/m³). No carcinogenic effects were observed.

Executive summary:

Malley et al. (1995) conducted a 2-year whole body inhalation study (equivalent to OECD 453, GLP) in male and female rats. Eighty-seven animals were exposed 6 hours/day, 5 days/week for 24 months to either 25.2, 101.0 and 350.5 ppm (analytical) of the test substance. Control animals were sham exposed, a positive control was not included. Animals were analyzed for any signs of toxicity. No clinical signs were observed and no treatment-related mortality. Treatment-related findings were found on body weight changes (350 ppm test group), on clinical biochemistry (all test groups), on organ weight (100 and 350 ppm test group), and on gross pathology (350 ppm test group). Histopathological findings were reported as non-treatment-related. No treatment-related increase in tumor incidences were observed and the NOAEC for carcinogenicity was concluded to be 350 ppm.

Reference 2

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Purity: >99.9 %
- Impurities: water, monomethyl acetamide, dimethylformamide, peroxides, iron
- Source: DuPont
- Batch No.: H-18842
no further details available

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: approximately 49 days old
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually
- Certified and irradiated diet: ad libitum (not during exposure)
- Tap water: ad libitum (not during exposure)
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25 °C
- Humidity: 40-60 %
- Air changes: no data
- Photoperiod: 12 hours dark/12 hours light
- Cage racks relocated every 2 weeks
- Sentinel animals (not exposed) were kept in the same room for detection of pathogens in blood.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Stainless-steel and glass chambers (4 m³) were operated in a onepass flow through mode at 1 L/min; chamber temperature (mean) was 23, 22, 23, and 24°C at control, low, mid and high dose level, respectively; the relative humidity was 40, 41, 40, and 40 %, respectively, and mean airflow ranged between 730 to 1050 L/min.

Vapour was generated separately by metering the liquid chemical into a glass J-tube filled with glass beads; heated air (approximately 100-130 °C) was blown through the glass beads to evaporate DMAC; resulting vapour was diluted to the desired concentrations with filtered conditioned (dehumidified) air for each of the three test chambers; chamber concentrations were controlled by varying the test substance flow rates into the J-tubes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber atmosphere was analyzed by gas chromatography at approximately 30-min intervals during each 6-h exposure period; the atmospheric concentration of DMAC was determined by comparing the detector response of the chamber samples to that of liquid standards using standard curves.

Duration of treatment / exposure:

18 months

Frequency of treatment:

- 6 h/day, 5 days/week (18 months)
(not during holidays, no details)

Post exposure period:

no

Dose / conc.:

25.2 ppm (analytical)

Remarks:

range: 22-28 ppm

Dose / conc.:

101 ppm (analytical)

Remarks:

range: 85-115 ppm

Dose / conc.:

350.5 ppm (analytical)

Remarks:

range: 300-390 ppm

No. of animals per sex per dose:

78 mice

Control animals:

yes, sham-exposed

Details on study design:

- Post-exposure period: none
- Dose selection rationale: Saturation in toxicokinetic experiments (300 ppm), toxicity data after repeated inhalation.
- Liver cell proliferation was tested in sub-groups (5 mice per sex per dose) after 0.5, 3, or 12 months of exposure (interim sacrifice).

Positive control:

No

Observations and examinations performed and frequency:

CLINICAL SIGNS, BODY WEIGHT:
All mice weighed once per week for the first 3 months and once every other week thereafter; at every weighing, each animal was individually handled and examined for clinical signs of toxicity; cage-side examinations conducted at least once and usually twice daily throughout the study.

OPHTHALMOSCOPIC EXAMINATION:
Conducted by a veterinary ophthalmologist prior to the first exposure and again immediately prior to sacrifice (18 months); at least 1 h before each examination, 1 or 2 drops of 1 % atropine sulfate solution (pretest) or 1 % tropicamide (final euthanization) placed in each eye of every animal; both eyes examined by focal illumination and indirect ophthalmoscopy.

HEMATOLOGY:
Ten mice per sex per dose were randomly selected for evaluations after 3, 6, 12 and 18 months of testing; blood samples were collected from the orbital sinus of each unfasted mouse while the animal was under light carbon dioxide anesthesia; hematological parameters examined at each sampling time were erythrocyte, leukocyte, differential leukocyte, and platelet counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin concentration.

Sacrifice and pathology:

NECROPSY, GROSS PATHOLOGY:
All mice that were found dead, accidentally killed, or were euthanized in extremis were necropsied; all surviving animals were euthanized by pentobarbital overdose followed by exsanguination and necropsied after 18 months of testing. A complete necropsy was performed.

ORGAN WEIGHTS:
Lungs, brain, liver, kidneys, and testes were weighed wet at necropsy; organ weight/final body weight ratios calculated; organs from animals found dead or sacrificed in extremis were not weighed.

HISTOPATHOLOGY:
The following tissues were collected from all animals: skin, bone marrow (femur, sternum), lymph nodes (mandibular, mesenteric), spleen, thymus, aorta (thoracic), heart, trachea, lungs (inflated), nose (4 cross sections, including paranasal sinuses), larynx/pharynx, salivary glands, esophagus, stomach, liver, pancreas, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), gallbladder, kidneys, urinary bladder, pituitary, thyroid, parathyroid, adrenals, testes, epididymides, seminal vesicles, mammary gland, ovaries, uterus, vagina, brain (including sections of medulla/pons, cerebellar cortex, cerebral cortex), spinal cord (cervical, thoracic, lumbar), peripheral nerve (sciatic), muscle (thigh), bones (femur, sternum), eyes, exorbital lacrimal glands, harderian glands, and all gross lesions.
All tissues were fixed in 10 % neutral-buffered formalin except testes, epididymides, eyes, and skin with mammary gland (fixed in Bouin's solution). The lungs were inflated with formalin at the time of necropsy.
All tissues collected from animals in the 350 ppm and control groups, and from mice that were found dead (tissue integrity permitting), or were euthanized in extremis, were further processed to slides, stained with hematoxylin and eosin, and examined microscopically; lungs, liver, kidneys, and all gross lesions from animals in the 25 and 100 ppm groups were also processed and examined microscopically.

Other examinations:

LIVER CELL PROLIFERATION:
Cell proliferation in the liver was measured (5 mice per sex per dose after 0.5, 3, or 12 months of exposure) after labelling with bromodeoxyuridine (BrdU). 1000 nuclei per animal were evaluated for S-phase; no further examinations of these animals.

Statistics:

One-way analysis of variance, Fisher's exact test with a Bonferroni correction and the Cochran-Armitage test for trend, Bartlett's test and Kruskal-Wallis and Mann-Whitney U test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Diarrhea was noted in males of the mid and high dose but no morphological changes which correlated with the increased incidence of diarrhea were observed. These effects were transient and did not affect body weight or survival; authors result: not adverse. The incidence of ruffled fur was increased in females at 350 ppm; authors comment: no morphological changes which correlated with this increased incidence were seen and there were no effects on survival, and no adverse effects on body weight, and therefore, it was not considered to be adverse.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No treatment-related mortality occurred throughout the study. A slight decrease in survival was found in high dose females: 80 and 60 % survival for control and 350 ppm females, respectively. Authors comment: no pathological findings in 350 ppm females, not treatment related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects on body weight or body weight gain were noted. However, the mid and high dose groups tended to higher body weight (significant in males at 100 ppm); the effects were not dose dependent and not correlated with any pathological alterations and thus considered not to be treatment related.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related changes in any parameter in either males or females were noted at any exposure concentration; statistically significant differences were within the expected range of normal biological variation or did not exhibit dose-response relationships and were not considered to be treatment-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No effects were detected in males at any dose level. 350 ppm females had significantly increased absolute and relative liver weight compared to control values; these effects were most likely the result of enzyme induction associated with metabolism of the test substance; absolute and relative kidney weights were also increased in 350 ppm females, and relative kidney weights were increased in 25 ppm females; however, there were no morphological changes in the kidney associated with the higher kidney weights and effects did not follow a dose-response relationship; authors comment: not treatment-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross morphological changes were similar to control in both males and females for all dose levels.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Male mice exposed to 100 or 350 ppm and females at 350 ppm had an increased incidence of pigment accumulation (hemosiderin and lipofuscin) in the Kupffer cells of the liver (statistically significant only for 100 and 350 ppm males; biologically significant in 350 ppm females).
350 ppm males had an increased incidence of minimal to mild hepatocellular hypertrophy (enzyme induction was suggested by the authors but a regeneration effect was not excluded); 350 ppm females had a statistically significant increased incidence of minimal to mild individual hepatocellular necrosis which was also seen in males exposed to 100 or 350 ppm (biologically significant apoptosis). The incidence but not the severity of retinal atrophy was significantly increased in 350 ppm females (6.6, 12.9, 10.5, 34.5 % for 0, 25, 100, and 350 ppm, respectively). Authors comment: the increased incidence of retinal atrophy was secondary to the treatment-related effects on liver function rather than a direct compound-related effect on the retina. No effects were detected in the respiratory tract. The authors did not report effects on testis.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No increase in tumour incidences was observed at any exposure concentration in either males or females, especially in testis (target organ in sub-acute inhalation studies) and liver (target organ in this study, see also Section 7.5.2). However, 350 ppm females had a significantly higher incidence of lymphoma compared to controls (5, 2, 5, 15 % for 0, 25, 100, and 350 ppm, respectively). The historical control range for lymphoma at this laboratory was 3.3 to 23.8 %, and the average incidence for historical controls was 15.5 %. Since the incidence of lymphoma in 350 ppm females was nearly identical to the average incidence of the historical controls, and a dose-response relationship was not present, it was not considered by the authors to be a compound-related effect.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

LIVER CELL PROLIFERATION:
No treatment-related effects on cell proliferation of liver cells were detected in BrdU-labelled liver tissue at any dose level in the corresponding sub-groups. Increased cell proliferation in 350 ppm males at study day 26 (not after 3 or 12 months) was considered by the authors not to be treatment-related.

Dose descriptor:

NOAEC

Effect level:

350 ppm

Sex:

male/female

Basis for effect level:

other: (1260 mg/m³) no treatment-related increase in tumour incidences

Dose descriptor:

LOAEC

Effect level:

100 ppm

Sex:

male

Basis for effect level:

other: (360 mg/m³) increased centrilobular hepatocellular hypertrophy, increased hepatic single cell necrosis, increased pigmentation of Kupffer cells

Dose descriptor:

LOAEC

Effect level:

350 ppm

Sex:

female

Basis for effect level:

other: (1260 mg/m³) increased liver weight, increased hepatic single cell necrosis, increased pigmentation of Kupffer cells, increased bilateral diffuse retinal atrophy

Remarks on result:

other: Effect type: toxicity (migrated information)

Conclusions:

Inhalation exposure of mice to DMAC for 18 months did not cause a treatment-related increase in tumour incidences at concentrations up to 350 ppm (1260 mg/m³) but toxic effects in males at 100 ppm (360 mg/m³) (NOAEC: 25 ppm, 90 mg/m³) and in females at 350 ppm (NOAEC: 100 ppm, 360 mg/m³). No carcinogenic effects were observed.

Executive summary:

Malley et al. (1995) conducted an eighteen months whole body inhalation study (equivalent to OECD 453, GLP) in male and female mice. Seventy-eight animals were exposed 6 hours/day, 5 days/week for 18 months to either 25.2, 101.0 and 350.5 ppm (analytical) of the test substance. Control animals were sham exposed, a positive control was not included. Animals were analyzed for any signs of toxicity. Effects on clinical signs, mortality, body weight, hematology, organ weights, and histopathology were observed, but were deemed to be not treatment related. The test substance did not cause an increase in tumor incidences and thus, a NOAEC of 350 ppm was concluded.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 260 mg/m³

Study duration:

chronic

Species:

rat

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

no further information

Justification for classification or non-classification

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011, classification for carcinogenicity is not warranted.

Additional information

In the OECD SIDS document (2001; SIAM 13) on N,N-dimethylacetamide (DMAC) data were presented on this endpoint which were also documented in this report but more details were presented and an evaluation of the validity of these reports. The evaluation of results is comparable to the OECD SIDS document.

In a study comparable to OECD Guideline 451 (Malley et al., 1995; RL1) groups of 78 male and 78 female CD-1 mice were exposed 6 h per day, 5 days per week, to 0, 25, 100, 350 ppm (ca. 0, 90, 360, 1260 mg/m³). The exposure period lasted 18 months and no post exposure observation period followed. Interim sacrifice (n=5 per dose per sex) was performed 0.5, 3, or 12 months after initiation of the study for studying of liver cell proliferation. No treatment-related effects were found concerning the parameters body weight, hematology, ophthalmology, liver cell proliferation, and necropsy. At a dose level of 350 ppm (1260 mg/m³) females showed clinical signs like ruffled fur and the survival was reduced. Furthermore, hepatocellular necrosis, increased pigmentation of Kupffer cells, and increased liver weight were detected in high dose females and the incidence of retinal atrophy was increased (presumably secondary to other effects). Liver cell necrosis and increased pigmentation of Kupffer cells was seen in males even at a dose level of 100 ppm (360 mg/m³). No increase in tumour incidences were observed at any exposure concentration in either males or females.

Conclusion: Inhalation exposure of mice to DMAC for 18 months did not cause a treatment-related increase in tumour incidences at concentrations up to 350 ppm (1260 mg/m³) but toxic effects in males at 100 ppm (360 mg/m³) (NOAEC 25 ppm, 90 mg/m³) and in females at 350 ppm (NOAEC 100 ppm, 360 mg/m³).

Groups of 87 male and 87 female Crl:CD® BR rats (Malley et al., 1995; RL1: study comparable to OECD 451) were exposed 6 h per day, 5 days per week, to 0, 25, 100, 350 ppm (ca. 0, 90, 360, 1260 mg/m³). The exposure period lasted 24 months and no post exposure observation period followed. Interim sacrifice was conducted after 12 months (n=10 per dose per sex) for the evaluation of toxic effects. For exclusive measurement of liver cell proliferation further interim sacrifice (n=5 per dose per sex) was performed 0.5, 3, or 12 months after initiation of the study. No treatment-related effects were found concerning the parameters clinical signs, survival, hematology, ophthalmology, liver cell proliferation, urinalysis. In female rats a dose level of >=100 ppm (360 mg/m³) resulted in increased serum cholesterol and serum glucose levels, increased liver weight, and increased pigmentation of Kupffer cells in the liver. At 350 ppm females revealed also increased serum sorbital dehydrogenase activity and decreased body weight. In males a dose level of >=100 ppm induced increased liver weight, increased incidences of hepatic focal cystic degeneration and of hepatic peliosis. In male rats the high dose level of 350 ppm resulted in decreased body weight, increased serum sorbital dehydrogenase activity; increased kidney weight combined with increased severity of nephropathy; increased pigments in Kupffer cells and increased incidence of biliary hyperplasia. No carcinogenic activity was found. The squamous cell papillomas in the stomach of two (3.1%) females from the 350 ppm group (statistically significant) were not considered to be compound related.

Conclusion: Inhalation exposure of rats to DMAC for 24 months did not cause a treatment-related increase in tumours at concentrations up to 350 ppm (1260 mg/m³) but toxic effects in males and females at 100 ppm (360 mg/m³) (NOAEC 25 ppm, 90 mg/m³).

In a drinking water study in rats (1979, RL2: comparable to Guideline with acceptable restrictions: no details about the test substance; problems in analytical methodology of DMAC in drinking water; limited data on test animals and conditions; some organs not included in histopathology of high dose group and controls; no documentation of details on incidences in the reevaluation) 70 Long-Evans rats per dose per sex were treated with 0, 100, 300, or 1000 mg/kg bw/day. At the end of six or twelve months of treatment, ten animals/sex/group were randomly selected and sacrificed for examinations; at the end of 24 months on test, all of the survivors were killed. Except a slight increase in alopecia in high dose rats no relevant clinical signs were observed. The body weight was reduced in males at >=300 mg/kg bw/day and in females at the high dose level. No effects were detected on food and water consumption. The toxicological relevance of effects in hematology and clinical chemistry is questionable. Urinalysis and ophthalmology were unremarkable. No increase in the incidences of tumours were detected in any organ but nonneoplastic effects. At 1000 mg/kg bw/day reduced testes weight and atrophy/degeneration were seen. In males and females of the high dose group increased incidence of liver cell degeneration and pigmentation and vacuolisation of liver cells were found. The derivation of the NOAEL is questionable due to limitation of the histopathology.

Conclusion: In a chronic drinking water study no carcinogenic activity was detected but toxic effects like reduced body weight gain at >= 300 mg/kg bw/day in males and at 1000 mg/kg bw/day in females: the high dose induced liver cell degeneration in males and females and testis atrophy in males; the NOAEL was 100 mg/kg bw/day in males and 300 mg/kg bw/day in females.

In the following, two Japanese carcinogenicity studies (Anonymous, 2013) are summarized which are considered as supplementary data.

In an inhalation study comparable to OECD Guideline 451 (Anonymous, 2013; RL2: with acceptable restrictions: inadequate air change rate during exposure, highest test dose exceeded the MTD) groups of 50 male and 50 female F344 rats were exposed 6 hours per day, 5 days per week, to 0, 18, 90 and 450 ppm (ca. 0, 65, 324 and 1620 mg/m³) for an exposure period of 24 months. No treatment related effects were observed on the viability of the animals. Reduced body weight (84%) and body weight gain (78%) were observed in males at the highest dose when compared to the control group values. In females does at 450 ppm, the body weight gain was reduced to 85% of the respective control group value. Male animals of the 450 ppm group showed a decreasing trend in food consumption throughout the study course while this trend was seen in female animals only until week 7 with gradual recovery thereafter. Altered haematology parameters were observed in both sexes at the dose level of 450 ppm (mainly decreases in MCV, MCH). Relevant changes in clinical chemistry parameters (e.g. increases in cholesterol, triglycerides, phospholipids, gamma-GTP, total bilirubin, high urine nitrogen, creatinine) were observed for both sexes at 90 and 450 ppm and mainly related to liver and kidney toxicity. Urinalysis showed decreased positivity for ketone bodies in males and an increased positivity for protein in females. In male animals gross pathology revealed an increased number of liver nodules at the highest dose and granulated alterations in the kidney. The increases in liver and kidney weights in males and females at 90 and/or 450 ppm correlated with adverse non-neoplastic lesions at histopathology in liver and kidney described as adipose liver degeneration and chronic nephropathy. Increased incidences of hepatocellular adenoma and carcinoma, and liver tumours consisting of hepatocellular adenoma and carcinoma were observed in male rats at 450 ppm. In female rats, no increase in tumour incidence was observed.

During the exposure period the air change rate was inadequately low (6 changes/hr instead of at least 10). This raises concerns on the reliability of the study as it may have resulted in higher dermal, oral and inhalation exposure of the animals in comparison to exposure conditions with the recommended air change rate.

The highest dose of 450 ppm caused excessive toxicity indicated by a more than 10 % reduction in body weight gain and severe impact on liver function. Significant hepatotoxicity was also seen at the mid dose level of 90 ppm. According to OECD TG 451 the highest dose should elicit some evidence of toxicity, e.g. depression of body weight gain by approximately 10%. OECD draft guidance document No. 116 states that body weight gain should be decreased by no more than 10% and that the highest dose should be selected to identify toxic effects while avoiding severe toxicity. Considering the OECD guideline and guidance, the effects observed at 450 ppm show that the maximum tolerated dose (MTD) was exceeded at this dose level. Excessive dosing can result in tumour formation secondary to cytotoxicity i.a. as a consequence of detoxification mechanisms.

Based on the above, the high dose level of 450 ppm is not useful for the assessment of the carcinogenic potential of DMAC in rats since this concentration was in excess of the MTD.

 

Conclusion: The NOAEC for toxic effects is 18 ppm (65 mg/m³) and the NOAEC for carcinogenicity is 90 ppm (324 mg/m³) in males and 450 ppm (1620 mg/m³) in females. It is concluded that DMAC is not carcinogenic, when dose levels do not exceed the MTD.

Groups of 50 male and 50 female B6D2F1 mice (Anonymous, 2013; RL2: study comparable to OECD Guideline 451 with acceptable restrictions: uncommon sensitive mouse strain used, inadequate air change rate during exposure, in male animals highest dose exceeded MTD) were exposed 6 hours per day, 5 days per week, to 0, 12, 60 and 300 ppm (ca. 0, 43, 216 and 1080 mg/m³) for an exposure period of 24 months. No treatment related effects were observed on the viability of the animals. In male animals, body weight and body weight gain were reduced to 91% and 78% of the control group, respectively, at 300 ppm. No relevant effects on body weight/body weight gain were observed in female animals. At the highest dose level of 300 ppm food consumption showed an increasing trend early at administration for both sexes and also at late phases for females. Haematology parameters such as platelets and neutrophil ratio were increased and MCHC and haemoglobin were decreased. Female mice showed increased platelet counts. Clinical chemistry parameters such as AST, ALT, ALP, LDH and gamma-GTP were increased at 300 ppm, which can be linked to hepatic toxicity. Urinalysis showed decreased positivity for protein and ketone bodies in males at 300 ppm. In male and female mice, liver weights were increased at the highest dose level 300 ppm. Gross pathology revealed increases in liver nodules for both sexes at 300 ppm, and additionally, deformed kidneys at 300 ppm in male animals. Histopathology showed an increase in eosinophilic small foci and a decreased incidence of aggregated foci of inflammatory cells at 300 ppm in male and female mice. In the kidney of male mice higher incidences of non-neoplastic lesions were found already at doses of 60 ppm. Increased incidences of hepatocellular adenoma (both sexes) and carcinoma (females only), and liver tumours consisting of hepatocellular adenoma and carcinoma (both sexes) were observed at 300 ppm.

During the exposure period the air change rate was inadequately low (6 changes/hr instead of at least 10). This raises concerns on the reliability of the study as it may have resulted in higher dermal, oral and inhalation exposure of the animals in comparison to exposure conditions with the recommended air change rate.

The reduction in body weight gain by 22 % seen in male mice at 300 ppm and the observed effects indicative of liver toxicity suggest that the concentration of 300 ppm might has exceeded the MTD, especially in male mice.

The uncommon B6D2F1/Crlj mouse strain was used for this inhalation study, whereas OECD TG 451 states that commonly used laboratory animal strains should be used. The response of this hybrid strain to known carcinogens/non-carcinogens is unknown. The B6D2F1 mouse has been shown to have different genetic mutations in hepatocellular tumors compared to other mouse strains, and has been shown to have a high prevalence of hepatocellular neoplasia in control animals compared to other mouse strains (Takahashi et al., 2002 Toxicologic Pathology, vol 30: pp 599-605; Kushida et al., 2006 Toxicologic Pathology, vol 34: pp237-242). Thus, it is likely that this strain is more sensitive than typical mouse strains with regard to liver tumour formation, especially as a secondary effect to exposure to substances known to cause liver toxicity.

Conclusion: The NOAEC for toxic effects and for carcinogenicity is 60 ppm (216 mg/m³). The liver tumours found at the highest dose level of 300 ppm were likely a result of the sensitivity of the uncommon mouse strain used in the study in combination with the known liver toxicity induced by DMCA.