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Administrative data

Description of key information

The relevant studies revealed following results.

 

Oral:

24-months rat drinking water study: NOAEL = 100 mg/kg bw/day (males) based on reduced body weight. Histopathological effects on testes and liver observed at the high dose, exceeding the MTD (1000 mg/kg bw/day). Reliability of NOAEL limited due to limitation of the histopathological assessment to control and high dose. [1979, RL2 (reliability)]

90-days rat feeding study: NOAEL ≥ 60 mg/kg bw/day. No adverse effects observed at the only dose tested. [Kennedy & Sherman, 1986; RL2]

28-days rat gavage study: LOAEL = 290 mg/kg bw/day. A NOEAL was not determinable due to adverse toxic effects at the lowest tested dose (decreased organ weights, uterus atrophy). [1975, RL2]

 

Inhalation:

24-months rat whole body inhalation (vapor): NOAEC = 25 ppm (90 mg/m³) based on body weight, clinical chemistry parameters, histopathological changes in the liver [Malley et al., 1995; RL2]

18-months mouse whole body inhalation (vapor): NOAEC = 25 ppm (90 mg/m³) based on histopathological changes in the liver. [Malley et al., 1995; RL1]

2-weeks mouse whole body inhalation (vapor): NOAEC = 100 ppm (360 mg/m³, males) based on effects on testes. [Valentine et al., 1997; RL2]

 

Dermal:

6-months dog dermal exposure study: NOAEL = 94 mg/kg bw/day based on body weight and clinical chemistry parameters. [Horn, 1961; RL2]

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is comparable to Guideline study 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; limited parameters in clinical chemistry; low number of rats in hematology, urinalysis and clinical chemistry; some organs not included in histopathology; histopathology not extended to low and mid dose groups concerning organs with effects in high dose group).
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
- Name of test material: dimethylacetamide (DMAC)
- Source: Monsanto
No details available.
Species:
rat
Strain:
Long-Evans
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Blues Sruce Farms, New York
- Age at study initiation: 6-7 weeks old
- Housing: individually
- Certified died and distilled water (plus DMAC) ad libitum
- Acclimation period: 2 weeks

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

No further details available.
Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Distilled water was used as vehicle/drinking water; the test solution was prepared weekly and the concentration in drinking water was adjusted by body weight and drinking behaviour.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
DMAC concentration in drinking water was 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 preparation errors. Decreased values were noted in high dose males only during the first weeks (see doses).
Duration of treatment / exposure:
2 years
Frequency of treatment:
daily ad libitum
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
nominal in water,
range of measured mean intake in males: 64-129 mg/kg bw/day,
range of measured mean intake in females: 62-122 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
nominal in water,
range of measured mean intake in males: 177-392 mg/kg bw/day,
range of measured mean intake in females: 190-399 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal in water,
range of measured mean intake in males: 267-1172 mg/kg bw/day,
range of measured mean intake in females: 598-1227 mg/kg bw/day
No. of animals per sex per dose:
70 rats
Control animals:
yes, concurrent vehicle
Details on study design:
At the end of 6 or 12 months of treatment, 10 rats/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 recovery period in satellite groups: 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 by Monsanto in 1990. In this reevaluation all male and female rats of the control and the high dose group were examined in complete histopathology (10 rats each at 6 and 12 months interim sacrifice and all survivors at termination of each sex). However, only a description of non-neoplastic effects was given without detailed tabulated results and without documentation of incidences.
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS, MORTALITY:
Clinical signs and mortality were recorded once daily and the first three months thereafter twice daily.

DETAILED CLINICAL OBSERVATIONS:
Measured once weekly.

OPHTHALMOLOGY:
Parameters were 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 first 14 weeks of exposure, thereafter once monthly and at termination (after fasting).

FOOD CONSUMPTION:
Measured during the pretest, once weekly at weeks 1-14, twice weekly at weeks 15-26, thereafter once monthly.

DRINKING WATER CONSUMPTION:
Determined for 10 rats per dose per sex; pretest value, once weekly at weeks 1-59, once monthly thereafter; test substance intake was calculated from water intake data.

HEMATOLOGY, CLINICAL CHEMISTRY:
Determined 3, 6, 12, 18, 24 months after initiation in 6 rats per dose per sex.
Hematology: hemoglobin, hematocrit, erythrocyte counts, erythrocyte morphology, clotting time, total and differential leukocytes (20 rats/dose/sex 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 groups; after 18 and 24 months urinalysis was performed in all groups (6 rats per dose per sex); parameters: gross appearance, pH, glucose, specific gravity, protein, ketone, bilirubin, occult blood.
Sacrifice and pathology:
GROSS PATHOLOGY:
A complete necropsy was performed.
Organ weights were measured from adrenal glands, brain, testes, ovaries, kidneys, spleen, liver, pituitary gland in 10 rats per dose per sex at the 6 and 12 months interim sacrifice and at termination in all survivors.

HISTOPATHOLOGY:
A histopathological assessment was conducted in the following organs and tissues:
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:
Statistically significant differences were calculated by F-test, Student t-test, Cochran test, Dunnett test; level of significance: p<0.05.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was a slightly increased incidence of alopecia at 1000 mg/kg bw/day in males and females and a slightly increased incidence in yellow staining of the ano-genital area in females of this dose.
Mortality:
no mortality observed
Description (incidence):
There were no effects.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a significant decrease in body weight 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):
There were no statistically significant effects.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
There were technical problems during the first weeks (see also dose range) and decreased palatability (pronounced in high dose males); there was high variability but no test substance-related pattern.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were 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 was reduced in males (only at termination).
See comments below.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were significant effects compared with the concurrent control:
At 100 and 300 mg/kg bw/day alkaline phosphatase activity was reduced in males after 6 and 18 months.
At 1000 mg/kg bw/day reduced alkaline phosphatase activity was noted in males at all intervals.
At 1000 mg/kg bw/day serum GPT was elevated in males and females after 6 months (no effects at other time points).
At >=100 mg/kg bw/day BUN was elevated in males at all intervals.
At 1000 mg/kg bw/day in females there was reduced glucose only after 12 months.
See comments below.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no effects.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were significant effects on organ weights:
At >= 100 mg/kg bw/day relative and absolute liver weight was increased after 6, 12, and 24 months in males and females.
At >= 300 mg/kg bw/day relative and absolute kidney weight was increased in males and females after 12 months and in females after 6 months (no effects after 24 months, no effects in histopathology).
At 1000 mg/kg bw/day relative and absolute kidney weight was increased in males after 6 months (see above).
At >= 100 mg/kg bw/day relative and absolute adrenal weight was increased in males after 6 months; there were no effects after 12 or 24 months (no histopathological effects).
At 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 were noted at necropsy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day the following effects were noted in comparison to the 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 available.
- Pigmentation (brown) of liver cells and reticuloendothelial cells were noted in males and females which was not seen in controls.
- The incidence of small flaccid testis was 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 was noted.
- Hemosiderosis of the spleen was observed in females (but no anemia).
- There was an increased incidence of lymphoid depletion/atrophy in males (questionable relevance).
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male
Remarks on result:
other: no adverse effects observed at this dose level
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
female
Remarks on result:
other: no adverse effects observed at this dose level
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 other: mg/kg bw/day (night be lower; histopathology only on control and high dose animals)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 other: mg/kg bw/day (night be lower; histopathology only on control and high dose animals)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Conclusions:
In a chronic drinking water study reduced body weight gain was reported 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 (provisional) NOAEL was 100 mg/kg bw/day in males and 300 mg/kg bw/day in females.
Executive summary:

In a chronic drinking water study comparable to OECD TG 453 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, 10 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 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 of 1000 mg/kg bw/day. No effects were detected on food and water consumption. The toxicological relevance of effects in hematology and clinical chemistry was questionable. Urinalysis and ophthalmology parameters were unremarkable. 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, pigmentation and vacuolisation were found. The derivation of the NOAEL is questionable due to limitation of the histopathological assessment (only control and high dose).

Conclusion: In a chronic drinking water study reduced body weight gain was reported 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 (provisional) NOAEL was 100 mg/kg bw/day in males and 300 mg/kg bw/day in females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
chronic
Species:
rat
System:
other: hepatobiliary, male reproductive system
Organ:
liver
testes

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is comparable to Guideline study with acceptable restrictions (tabulated details of results not presented for all parameters).
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no
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 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 were relocated every 2 weeks
- Sentinel animals (not exposed) were kept in the same room for detection of pathogens in blood.
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: Not applicable (vapour)60 mg/m3
Details on inhalation 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)
Dose / conc.:
25 ppm (nominal)
Remarks:
90 mg/m³,
analytical conc. range: 22-28 ppm, mean: 25.2 ppm
Dose / conc.:
100 ppm (nominal)
Remarks:
360 mg/m³,
analytical conc. range: 85-115 ppm, mean: 101.0 ppm
Dose / conc.:
350 ppm (nominal)
Remarks:
1260 mg/m³,
analytical conc. range: 300-390 ppm, mean: 350.5 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 (rat at 150 ppm), toxicity data after repeated inhalation in rats.
- 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 (18 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 consisted of 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 and 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:
Cell proliferation in the liver was 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 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:
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:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related 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, 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.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related effects were found concerning neoplastic effects
Other effects:
no effects observed
Description (incidence and severity):
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.
Details on results:
No treatment-related effects were found concerning the parameters clinical signs, survival, hematology, ophthalmology, liver cell proliferation, urinalysis and neoplastic effects. 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. 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. No carcinogenic activity was found.
Dose descriptor:
NOAEC
Effect level:
25 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: 90 mg/m³
Dose descriptor:
LOAEC
Effect level:
100 ppm
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: 360 mg/m³
Dose descriptor:
LOAEC
Effect level:
100 ppm
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: 360 mg/m³
Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Toxic effects in rats after chronic inhalation exposure for 2 years (Means ± Standard Deviation)

Parameter

0 ppm

25 ppm

100 ppm

350 ppm

SDH in males after 3 months

6.2 ± 2.5

6.7 ± 1.8

9.7 ± 5.2

14.2 ± 4.5*

SDH in males after 6 months

5.6 ± 2.0

6.6 ± 2.8

7.2 ± 3.4

12.6 ± 5.7*

SDH in females after 3 months

5.7 ± 1.5

5.8 ± 0.9

6.6 ± 1.8

8.7 ± 2.6*

Rel. liver weight (%) in males after 24 months

2.95 ± 0.23

3.01 ± 0.41

3.59 ± 1.34

3.95 ± 0.84

Rel. liver weight (%) in females after 12 months

2.68 ± 0.23

2.97 ± 0.38

3.30 ± 0.47*

3.42 ± 0.32*

Incidence (%) hepatic focal cystic degeneration in males

26

38

44*

50*

Incidence (%) biliary hyperplasia in males

57

73

67

79*

Incidence (%) accumulation of pigments in Kupffer cells in males

2

6

8

34*

Incidence (%) accumulation of pigments in Kupffer cells in females

3

3

13

25*

Incidence (%) hepatic peliosis in males

5

3

11

13*

Rel. kidney weight (%) in males after 24 months

0.76 ± 0.06

0.83 ± 0.26

0.95 ± 0.34

1.29 ± 0.73*

Incidence (%) severe progressive nephropathy in males after 25 months

15

15

19

32*

SDH: Sorbitol Dehydrogenase Activity in U/L (no significant effects after 12, 18, or 24 months);

*: significant, p <= 0.05

Conclusions:
In chronic inhalation studies in rats the liver was the target organ. Effects on body weight, clinical chemistry parameters, organ weight parameters, and/or morphological changes were detected in males and females at ≥100 ppm (360 mg/m³; NOAEC 25 ppm or 90 mg/m³). According to the authors, the test substance was not oncogenic in rats under the experimental conditions (see also Section 7.7).
Executive summary:

In a chronic inhalation study comparable to OECD TG 453 groups of 87 male and 87 female Crl:CD® BR rats were exposed 6 h per day, 5 days per week, to 0, 25, 100, 350 ppm (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 (10 rats per dose per sex) for evaluation of toxic effects. For exclusive measurement of liver cell proliferation further interim sacrifices (5 rats per dose per sex) were performed 0.5, 3, and 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 and neoplastic effects. 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 sorbitol 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 (1260 mg/m³) resulted in decreased body weight, increased serum sorbitol 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. No treatment related effects were detected in testes (compare with subacute toxicity).

Conclusion: In chronic inhalation toxicity studies in rats the liver was the target organ. Effects on body weight, clinical chemistry parameters, organ weight parameters, and/or morphological changes were detected in males and females at >=100 ppm (360 mg/m³; NOAEC: 25 ppm or 90 mg/m³).

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to Guideline.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no
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 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:
whole body
Vehicle:
air
Details on inhalation 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, no post exposure period
Frequency of treatment:
6 h/day, 5 days/week (18 months)
(not during holidays, no details)
Dose / conc.:
25 ppm (nominal)
Remarks:
90 mg/m³,
analytical conc. range: 22-28 ppm, mean: 25.2 ppm
Dose / conc.:
100 ppm (nominal)
Remarks:
360 mg/m³,
analytical conc. range: 85-115 ppm, mean: 101.0 ppm
Dose / conc.:
350 ppm (nominal)
Remarks:
1260 mg/m³,
analytical conc. range: 300-390 ppm, mean: 350.5 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:
no effects observed
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 comment: 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 deceased 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
Description (incidence and severity):
There were no test substance-related effects.
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:
effects observed, non-treatment-related
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, 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 tumor 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:
LOAEC
Effect level:
100 ppm
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: 360 mg/m³
Dose descriptor:
LOAEC
Effect level:
350 ppm
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
other:
Remarks on result:
other: 1260 mg/m³
Dose descriptor:
NOAEC
Effect level:
25 ppm
Sex:
male
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: 90 mg/m³
Dose descriptor:
NOAEC
Effect level:
100 ppm
Sex:
female
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: 360 mg/m³
Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
In chronic inhalation studies in mice the liver was the target organ, effects were detected in males at 100 ppm (360 mg/m³; NOAEC: 25 ppm or 90 mg/m³) and in females at 350 ppm (1260 mg/m³; NOAEC: 100 ppm or 360 mg/m³).
Executive summary:

Groups of 78 male and 78 female CD-1 mice were exposed in an inhalation study comparable to OECD TG 453 6 h per day, 5 days per week, to 0, 25, 100, or 350 ppm (0, 90, 360, 1260 mg/m³). The exposure period lasted 18 months and no post exposure observation period followed. Interim sacrifice (5 mice per dose per sex) was performed 0.5, 3, and 12 months after initiation of the study for assessment 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). There were no test substance-related effects observed in the testes (compare with subacute studies).

Conclusion: In chronic inhalation studies in mice the liver was the target organ, effects were detected in males at 100 ppm (360 mg/m³; NOAEC: 25 ppm or 90 mg/m³) and in females at 350 ppm (1260 mg/m³; NOAEC: 100 ppm or 360 mg/m³).

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to Guideline with acceptable restrictions (only males; no clinical chemistry; weight of adrenal, brain and thymus not determined).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
(14-day)
Deviations:
yes
Remarks:
no clinical chemistry; no adrenal, brain and thymus weights; only males
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
- Name of test material: dimethylacetamide (DMAC)
- Analytical purity: 99.8 %
- Impurities: 0.2 % water
- Source: DuPont Fibers, Richmond, VA

No further details available.
Species:
mouse
Strain:
other: Crl:CD-1(ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 35 days old
- Weight at study initiation: 23 to 33 g
- Housing: singly or in pairs in 7" x 4" x 5" suspended, stainless-steel, wire-mesh cages
- Diet: Purina certified rodent chow 5002; ad libitum, except during exposure
- Water: ad libitum, except during exposure
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2 °C
- Humidity: 50 ± 10 %
- Air changes: no data
- Photoperiod: 12 hours dark/12 hours light

IN-LIFE DATES: May 15, 1989 to June 9, 1989 (main inhalation study); June 19, 1989 to July 14,
1989 (follow-up inhalation study); August 19, 1989 to August 31, 1989 (inhalation pharmacokinetic
study)
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Not applicable (vapour)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test atmospheres of DMAC were generated for a 20-L cylindrical glass exposure chamber by bubbling conditioned, filtered houseline air (0.3-5.0 L/min) through midget glass impingers containing DMAC. The vapour generators were placed within heated water baths maintained at 28 °C. The resulting vapour was mixed with dilution air (34 L/min) and swept through glass tubing into the top of the glass exposure chambers. The desired atmospheric concentration of DMAC was attained by adjusting the flow of air through the vapour generator. Chamber atmospheres were exhausted through a water-containing scrubber and an MSA activated charcoal/HEPA cartridge filter prior to discharge into the fume hood. Each exposure chamber had a dispersion plate located at the chamber inlet, which was used to increase turbulence and promote uniform distribution of the test substance. The procedural control groups were exposed whole-body to conditioned, filtered houseline air only (32 L/min) in an identical 20-L cylindrical glass exposure chamber.
- Temperature and humidity in air chamber: 23 ± 2 °C; 50 ± 10 %

TEST ATMOSPHERE
- Brief description of analytical method used: HP Model 5890 gas chromatograph
- Samples taken from breathing zone: Yes

Chamber oxygen levels were monitored with a BioMarine model 3100R oxygen analyzer. Chamber temperatures were measured continually with a mercury thermometer, and relative humidity was measured once during each exposure with a Belfort model 566 psychrometer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Air samples were taken (using acetone as trapping solvent) and analysed at approximately 60-min intervals to determine the test substance concentration in each test chamber, and analysed with replicate injections on a HP Model 5890 gas chromatograph equipped with a flame ionization detector. The atmospheric concentrations of the test substance were determined by comparing the detector response of the impinger samples with the test substance standard curves prepared prior to each exposure.
Duration of treatment / exposure:
2 weeks (10 exposures)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
30 ppm (analytical)
Remarks:
30 ± 4 ppm
corresponding to 110 mg/m³;
calculated with the molar mass = 87.12 g/mol
Dose / conc.:
100 ppm (analytical)
Remarks:
100 ± 10 ppm
corresponding to 360 mg/m³;
calculated with the molar mass = 87.12 g/mol
Dose / conc.:
310 ppm (analytical)
Remarks:
310 ± 30 ppm
corresponding to 1120 mg/m³;
calculated with the molar mass = 87.12 g/mol
Dose / conc.:
490 ppm (analytical)
Remarks:
490 ± 39 ppm
corresponding to 1760 mg/m³;
calculated with the molar mass = 87.12 g/mol
Dose / conc.:
700 ppm (analytical)
Remarks:
700 ± 65 ppm
corresponding to 2520 mg/m³;
calculated with the molar mass = 87.12 g/mol
No. of animals per sex per dose:
10 male mice
Control animals:
yes, sham-exposed
Details on study design:
- First experiment: 4 groups were exposed to target concentrations of 0 (control group 1), 30, 100 and 300 ppm.
- Second experiment: 3 groups were exposed to target concentrations of 0 (control group 2), 500 and 700 ppm.
- Section schedule rationale (if not random): After the 10th exposure, blood samples were collected from surviving mice for hematological analyses, and 5 mice per group were killed for pathologic examination.
- Post-exposure recovery period in satellite groups: The remaining mice were weighed and observed during a 14-day post exposure recovery period and then subjected to the same hematologic and pathologic examinations.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily, before each exposure; during and immediately after each exposure; daily, weekends included when warranted by the condition of the mice during the 14-day recovery period

BODY WEIGHT:
- Time schedule: daily; before each exposure; daily, weekends included when warranted by the condition of the mice during the 14-day recovery period

FOOD CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HEMATOLOGY:
- Time schedule for collection of blood: After the 10th exposure (collected from the orbital sinus), and from the remaining mice of each group on the 14th day of recovery
- Anaesthetic used for blood collection: Carbon dioxide
- Animals fasted: No data
- How many animals: 10 mice (unless the number was reduced by mortality) per group
- Parameters checked: erythrocytes, leukocytes, platelets, hemoglobin concentration, hematocrit, relative numbers of neutrophils, band neutrophils, lymphocytes, atypical lymphocytes, monocytes, eosinophils, and basophils; mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC) and mean corpuscular hemoglobin (MCH)

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS:
The weights of lungs, liver, kidneys, spleen, and testes were recorded.

HISTOPATHOLOGY:
The following organs/tissues were fixed/processed for microscopic examination:
Liver, kidneys, urinary bladder, heart, lungs, nose, pancreas, thymus, spleen, adrenal glands, thyroid glands, mesenteric lymph nodes, trachea, larynx/pharynx, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, sternum (bone and marrow), testes, epididymides, brain, and eyes.
Statistics:
Mean body weights and body weight gains for each test group were compared to controls during the exposure and recovery periods. Data were statistically analysed by a one-way analysis of variance. Exposure group values were compared to controls by the least significant difference and Dunnett's tests when the ratio of variance indicated a significant among-to-within group variation. Significant differences were judged at the 0.05 probability level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No compound-related clinical signs of toxicity were noted in the groups exposed to 30, 100, or 310 ppm throughout the study. At concentrations of 490 ppm or more, however, clinical signs including exophthalmos, laboured or irregular respiration, weakness or lethargy, incapacitation, and tremors were typically seen either prior to exposure or upon unloading mice immediately after exposure. While some clinical signs were suggestive of central nervous system involvement, these signs were noted only in moribund mice. Mice which survived the exposure period did not exhibit test substance-related clinical signs during the recovery period.
Mortality:
mortality observed, treatment-related
Description (incidence):
Exposure to 490 or 700 ppm test substance was not well-tolerated in mice. 2/10 mice from the 490 ppm group were killed in extremis within 6 days of study initiation while 8/10 mice exposed to 700 ppm were either killed in extremis or found dead during the exposure period (up to study day 12). Two additional mice, one each from the 30 and 700 ppm groups, were accidentally killed immediately after the 10th exposure during the collection of the blood sample. All other mice from the remaining groups survived until the scheduled termination date.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights and body weight gains of mice exposed to 30, 100, 310, or 490 ppm were comparable to control values during the exposure and recovery periods (occasional increases in daily mean body weights or weight gains were considered to be due to normal biological variation and not test substance-related). In the 700 ppm group, mean body weights and body weight gains were significantly lower than controls during the exposure period. Individual weight losses were noted within this group after 1 day of exposure and became significantly lower than controls by the 3rd day of exposure; weight losses persisted until animals were either found dead or killed in extremis. The high number of mortalities in the 700 ppm group prevented valid comparison of body weights with controls during the recovery period (see Table 1).
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Mice from the 30, 100, and 310 ppm groups did not have any biologically-significant hematological findings following either the exposure or recovery periods.
Although effects in red blood cell parameters and platelet counts were found in individual mice from the 490 and 700 ppm groups, considerable variability in the measured values was noted and may have obviated any statistical differences among the groups. One of 2 mice from the 700 ppm group that survived until the 10th exposure had decreased erythrocyte and platelet counts, hemoglobin concentration, and hematocrit. At the same sampling period, the other mouse in this group had a decreased platelet count; a marked rebound in platelet count occurred in this mouse after the 14-day recovery period. Two of 8 surviving mice from the 490 ppm group had mild to moderate decreases in erythrocyte and platelet counts, hemoglobin concentration, and hematocrit. Four of the other 6 mice from the 490 ppm group had mild to moderate decreases in platelet counts after the 10th exposure.
By the end of the recovery period, all 4 surviving mice from the 490 ppm group had erythrocyte parameters and platelet counts which were similar to controls. Other changes in hematological parameters noted were considered to be within the range of expected biological variation and were not test substance-related (see Table 2).
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No significant absolute or relative (to body weight) organ weight differences were found in mice exposed to 30, 100, or 310 ppm when compared to controls. In particular, the mean absolute and relative testes weights of mice from the 310 ppm group were approximately 15 % lower than control values after the 10th exposure. However, organ weight differences were present in the testes, liver, lungs, and spleen of mice exposed to 490 ppm; an insufficient number of mice were available for statistical evaluation of organ weight data from the 700 ppm group.
After the 10th exposure, mean absolute and relative testes weights in the 490 ppm group mice were lower than controls although only the relative testes weight was significantly different. After a 14-day recovery period, mean relative and absolute testes weights of mice from the 490 ppm group were significantly lower than controls.
Mean absolute and relative liver weights were slightly higher than controls in the 490 ppm group after the 10th exposure but only the relative liver weight was significantly different. By the end of the recovery period, both mean relative and absolute liver weights were significantly greater than controls.
Mean absolute and relative lung weights in the 490 ppm group were lower than control values after the 10th exposure but were similar to controls by the 14th day of recovery. Mean absolute spleen weights were higher than controls by the end of the recovery period (see Table 3).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related gross lesions were found in mice from the 700 ppm group. These lesions consisted of small testes in the only surviving mouse killed at the end of the recovery period and small spleens in 2 of 9 mice killed in extremis or found dead by the 10th exposure. All other gross changes were considered to be incidental and not related to the test substance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Although there were no treatment-related microscopic changes present in mice exposed to the test substance at 30 or 100 ppm, microscopic lesions were found in the testes of mice from the 310, 490, and 700 ppm groups and in the liver, bone marrow, lymphoid organs, and adrenal glands of mice exposed to 490 or 700 ppm.
Testicular lesions were observed after exposure in 2 of 5 mice from the 310 ppm group, 3 of 6 mice from the 490 ppm group, and 9 of 9 mice from the 700 ppm group. The testicular lesions were considered to be concentration-related with respect to incidence and severity and were usually associated with reduced number of sperm and increased germinal epithelium in the epididymides. Among mice found dead or killed in extremis, the lesions in the seminiferous tubules were primarily acute and necrotizing; however, in mice killed by design on study day 12, the lesions were less severe and were characterized by testicular atrophy with residual degeneration. Moderate tubular atrophy without significant regeneration was found in the single surviving mouse from the 700 ppm group after the 14-day recovery period. Minimal to mild bilateral tubular atrophy with regeneration was noted in each of the 3 affected mice killed 14 days after the last exposure to 490 ppm. The unilateral testicular atrophy-degeneration and Leydig cell hyperplasia noted in the remaining mouse from this group were considered to be spontaneous degenerative lesions and not test substance-related.
Minimal to mild hepatocellular necrosis was noted in 2 of 6 mice from the 490 ppm group and 4 of 9 mice from the 700 ppm group. Necrosis occurred as either randomly distributed foci or in centrilobular regions, primarily in mice found dead or killed in extremis during the exposure period. Hepatocellular hypertrophy and/or cytoplasmic hyalinization was observed in 1 of 6 mice exposed to 490 ppm and 5 of 9 mice exposed to 700 ppm by the 10th exposure. No test substance-related liver changes were noted after a 14-day recovery period.
Lymphoid atrophy and/or necrosis were present in one or more lymphoid organs (i.e., mesenteric lymph node, spleen, or thymus) in 5 of 6 mice from the 490 ppm group and in 9 of 9 mice from the 700 ppm group by the 10th exposure. The severity of lymphoid atrophy/necrosis was generally concentration-related. No significant microscopic changes occurred in lymphoid organs neither in mice exposed to 310 ppm or less, nor in mice from any group by the end of the recovery period.
Bone marrow hypoplasia was found in 1 of 6 mice from the 490 ppm group and in 8 of 9 mice exposed to 700 ppm by the 10th exposure. The hypoplasia involved both myeloid and erythroid elements although megakaryocyte numbers appeared unaffected. Lesion severity was generally minimal to mild however more severe changes were found in mice killed in extremis. No alterations in bone marrow were found in mice killed after the 14-day recovery period. Minimal adrenal cortical necrosis was present in 1 of 6 mice exposed to 490 ppm and 5 of 9 mice exposed to 490 or 700 ppm by the 10th exposure. No test substance-related effects were seen in mice following the 14-day recovery period.
Minimal adrenal cortical necrosis was present in 1 of 6 mice exposed to 490 ppm and 5 of 9 mice exposed to 490 or 700 ppm by the 10th exposure. No test substance-related effects were seen in mice following the 14-day recovery period.
All other microscopic changes were considered to be incidental or spontaneous lesions common to mice of this strain and age.
Dose descriptor:
NOEC
Effect level:
100 ppm (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effect observed at this dose
Remarks on result:
other: 360 mg/m³
Remarks:
(separate entries for publication/study report)
Dose descriptor:
LOAEC
Effect level:
310 ppm (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: 1120 mg/m³
Remarks:
(separate entries for publication/study report)
Critical effects observed:
yes
Lowest effective dose / conc.:
310 ppm (analytical)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Mean body weights (g):

Test substance (ppm)

Day 1

Day 2

Day 3

Day 5

Day 8

Day 12

Recovery day 7

Recovery day 14

0 (control 1)

27 ± 1

27 ± 1

27 ± 1

29 ± 1

31 ± 1

31 ± 2

34 ± 1

36 ± 1

0 (control 2)

25 ± 1

25 ± 1

25 ± 1

26 ± 1

28 ± 2

28 ± 2

31 ± 1

34 ± 1

30

27 ± 1

27 ± 2

27 ± 2

28 ± 3

30 ± 2

31 ± 2

34 ± 2

37 ± 2

100

27 ± 1

27 ± 1

28 ± 1

30 ± 1

32 ± 1

33 ± 2

35 ± 1

36 ± 2

310

28 ± 1

27 ± 1

27 ± 1

29 ± 1

31 ± 1

31 ± 2

35 ± 1

36 ± 2

490

25 ± 1

25 ± 2

26 ± 2

27 ± 2

28 ± 3

29 ± 4

32 ± 2

36 ± 1

700

25 ± 2

24 ± 2

23* ± 4

23* ± 4

24* ± 4

24 ± 3

31

37

* statistically different from control, p < 0.05

Table 2: Hematologic findings

Test substance

Test day (D)

Recovery (R)

Erythrocyte count

Haemoglobin

Haematocrit

Platelet count

ppm

 

x10E06/µL

g/dL

%

x10E03/µL

0 (control 1)

D 12

9.17 ± 0.3

14.4 ± 0.3

50 ± 1

1787 ± 483

R 14

9.13 ± 0.31

14.4 ± 0.5

49 ± 1

1412 ± 69

0 (control 2)

D 12

9.58 ± 0.46

15.5 ± 0.4

50 ± 1

910 ± 167

R 14

8.55 ± 0.41

14.3 ± 0.5

44 ± 1

1180 ± 146

30

D 12

9.14 ± 0.58

14.4 ± 0.9

49 ± 3

1799 ± 534

R 14

9.29 ± 0.48

14.7 ± 0.8

49 ± 3

1286 ± 191

100

D 12

9.24 ± 0.54

14.8 ± 0.8

50 ± 2

1764 ± 260

R 14

9.22 ± 0.64

14.6 ± 0.6

44 ± 3

1434 ± 182

310

D 12

9.08 ± 0.32

14.6 ± 0.6

49 ± 2

1542 ± 302

R 14

9.15 ± 0.12

14.9 ± 0.4

49 ± 1

1212 ± 302

490

D 12

8.92 ± 0.71

14.6 ± 1.0

46* ± 3

646* ± 209

R 14

8.77 ± 0.70

14.3 ± 0.6

45 ± 2

1313 ± 352

700

D 12

8.02* ± 0.99

12.7* ± 1.8

41* ± 6

620 ± 138

R 14

8.88

14.2

44

2535

* statistically different from control, p < 0.05

Table 3: Organ weight (g) and organ to body weight

Test substance

Test day (D)

Recovery (R)

Testes

Liver

Lung

ppm

Abs. (g)

Rel. (g/100 g)

Abs. (g)

Rel. (g/100 g)

Abs. (g)

Rel. (g/100 g)

0

(control 1)

D 12

0.21

0.70

1.51

4.89

0.22

0.72

R 14

0.23

0.65

1.94

5.38

0.22

0.61

0

(control 2)

D 12

0.20

0.70

1.47

5.01

0.23

0.81

R 14

0.18

0.54

1.81

5.38

0.23

0.67

30

D 12

0.20

0.65

1.40

4.55

0.21

0.70

R 14

0.23

0.61

2.05

5.50

0.25

0.67

100

D 12

0.20

0.62

1.67

5.10

0.22

0.67

R 14

0.24

0.61

2.16

5.89

0.22

0.61

310

D 12

0.18

0.59

1.52

5.01

0.22

0.68

R 14

0.22

0.62

2.06

5.72

0.23

0.64

490

D 12

0.14*

0.49*

1.71

5.98*

0.18*

0.65*

R 14

0.13*

0.35*

2.20*

6.04*

0.22

0.61

* statistically different from control, p < 0.05

Abs.: Absolute organ weight (g)

Rel.: Organ weight to body weight (g/100 g bw)

Conclusions:
Under the conditions of this study, the no-observable-effect concentration (NOEC) for repeated exposure to the test substance in male mice was 100 ppm. Despite the fact no significant body or organ weight changes were noted at 310 ppm and that testicular atrophy may occur spontaneously in mice, based on the qualitatively similar testicular pathology found in mice exposed at this and higher concentrations, 310 ppm was considered to be a minimal effect level for testicular injury. The toxicological significance of testicular injury was minimized by the use of prepubertal mice in this study.
At lethal exposure concentrations of 490 and 700 ppm clinical signs, body weight decrease, effects on hematological parameters, histopathological alterations in testes (atrophy, degeneration) and liver (necrosis), lymphoid atrophy, hypoplasia in bone marrow, and necrosis in adrenal cortex were observed.
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability) for the target organs examined.
Executive summary:

In a subacute inhalation study comparable to OECD TG 412 10 male Crl:CD-1(ICR)BR mice (35 days old) per dose level were exposed 6 h per day, 5 days per week, for 2 weeks to 0, 30 ± 4, 100 ± 10; 310 ± 30, 490 ± 39, 700 ± 65 ppm (means ± SD; 0, 110, 360, 1120, 1760, 2520 mg/m³) and sacrificed after the last exposure (5 mice) or after 2 weeks recovery period (5 mice).

No effects were detected at 100 ppm. At 310 ppm a slight (15 %) decrease in testis weight (not statistically significant) was found and histopathological effects like reduced number of sperm and increased germinal epithelium in the epididymides were noted. At >= 490 ppm clinical signs (including mortality) were obvious, the body weight decreased, and effects on hematological parameters were seen; histopathological alterations in testis (atrophy, degeneration) and liver (necrosis), lymphoid atrophy, hypoplasia in bone marrow, and necrosis in adrenal cortex were reported.

Conclusion: There were histopathological effects in testis of 35-days old mice after subacute inhalation at a dose level of 310 ppm (1120 mg/m³); the NOAEC was 100 ppm (360 mg/m³).

DISCUSSION:

Repeated exposure to 490 or 700 ppm of the test substance was associated with mortality, clinical signs of central nervous system involvement only in moribund animals, hematological changes suggestive of adverse effects on the hematopoietic system, organ weight changes in the liver and testes, and microscopic changes in the liver, testes, adrenal cortex, and lymphoid organs. The interpretation of the toxicological significance of these findings was complicated by the fact that most of these effects were noted at exposure concentrations associated with dead or moribund animals.

Increased mean relative liver weights were found in mice exposed to 490 ppm, yet microscopic evidence of hepatocellular hypertrophy was found in only 1 of 6 mice. Hepatocellular hypertrophy was a more consistent finding in mice exposed to 700 ppm, yet could not be fully evaluated in this group since liver weight data were not available for comparison. Although no microscopic evidence of hepatic injury was found in mice after a 14-day recovery period, liver weights remained significantly elevated in the 490 ppm group. These findings suggest that the threshold concentration for liver change is approximately 490 ppm and that based on the normal liver morphology observed after a 14-day recovery period, the hepatic changes appear reversible. Also noted in the 490 and 700 ppm groups were dose-related atrophy/necrosis of lymphoid organs and bone marrow hypoplasia. While endogenous corticosteroid release

attributable to stress has been associated with lymphocytolysis, it cannot be determined whether the changes in the lymphoid organs were direct effects of the test substance or secondary effects attributable to stress. The reductions in red blood cell and platelet counts, hemoglobin concentration, and hematocrit in mice from the 490 and 700 ppm groups were consistent, in theory, with the observed bone marrow hypoplasia.

Conceivably, stress-related effects may also be responsible for the adrenal cortical necrosis noted in the 490 and 700 ppm groups by the 10th exposure. The role of stress in mediating the hematologic changes appeared minimal, however, since nonspecific elevations in neutrophil counts are generally associated with stress. But such elevations were not noted in this study. The absence of microscopic changes in the bone marrow and lymphoid organs after the 14-day recovery period suggests these effects are reversible. Microscopic evidence of testicular and epididymal injury were observed in mice exposed to 310, 490, and 700 ppm. Although the incidence and severity of testicular changes appeared dose-related, the nature of the lesion varied depending upon the total dose. At 310 ppm, seminiferous tubule degeneration and oligospermia were considered minimal, occurred in only 2 of 5 mice by the 10th exposure, and were not observed after a 14-day recovery period. In contrast, testicular atrophy and oligospermia were more severe in the 490 and 700 ppm groups and at 700 ppm, necrosis of germinal epithelium was also noted. Despite the fact no significant body or organ weight changes were noted at 310 ppm and that testicular atrophy may occur spontaneously in mice, based on the qualitatively similar testicular pathology found in mice exposed at this and higher concentrations, 310 ppm was considered to be a minimal effect level for testicular injury. The toxicological significance of testicular injury was minimized by the use of prepubertal mice in this study.

In a supplemental 10-day inhalation study, no deaths occurred, yet microscopic testicular changes were seen among older mice (approximately 61 days after study start) only at 480 ppm, the highest concentration tested. In that study, the testicular lesions were similar to those described in this report except that based on both sperm counts and microscopic examination, oligospermia was not present. Absolute and relative testes weights of mice from the 480 ppm subgroup that was used to evaluate tissue pathology and sperm counts were significantly lower than the controls. These data suggest that the less sexually-mature mice were more sensitive to the test substance-induced testicular injury than older mice. It may also be noted that the 10-day inhalation pharmacokinetic toxicity study with the test substance in rats demonstrated that rats are less susceptible to the test substance testicular injury than mice. In that study, no effects on testicular weights, gross or microscopic pathology, or sperm counts were found at 480 ppm, the highest concentration tested.

Overall, these studies show that testicular toxicity may be accentuated in sexually-immature animals.

Thus, the incidence, severity and effect concentrations for the testicular abnormalities described may have been dependent upon the age of the mice at the start of exposure.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
90 mg/m³
Study duration:
chronic
Species:
rat
System:
other: hepatobiliary, male reproductive system
Organ:
liver
testes

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is comparable to Guideline study with acceptable restrictions (tabulated details of results not presented for all parameters).
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no
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 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 were relocated every 2 weeks
- Sentinel animals (not exposed) were kept in the same room for detection of pathogens in blood.
Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: Not applicable (vapour)60 mg/m3
Details on inhalation 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)
Dose / conc.:
25 ppm (nominal)
Remarks:
90 mg/m³,
analytical conc. range: 22-28 ppm, mean: 25.2 ppm
Dose / conc.:
100 ppm (nominal)
Remarks:
360 mg/m³,
analytical conc. range: 85-115 ppm, mean: 101.0 ppm
Dose / conc.:
350 ppm (nominal)
Remarks:
1260 mg/m³,
analytical conc. range: 300-390 ppm, mean: 350.5 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 (rat at 150 ppm), toxicity data after repeated inhalation in rats.
- 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 (18 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 consisted of 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 and 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:
Cell proliferation in the liver was 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 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:
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:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related 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, 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.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related effects were found concerning neoplastic effects
Other effects:
no effects observed
Description (incidence and severity):
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.
Details on results:
No treatment-related effects were found concerning the parameters clinical signs, survival, hematology, ophthalmology, liver cell proliferation, urinalysis and neoplastic effects. 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. 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. No carcinogenic activity was found.
Dose descriptor:
NOAEC
Effect level:
25 ppm
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: 90 mg/m³
Dose descriptor:
LOAEC
Effect level:
100 ppm
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: 360 mg/m³
Dose descriptor:
LOAEC
Effect level:
100 ppm
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: 360 mg/m³
Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Toxic effects in rats after chronic inhalation exposure for 2 years (Means ± Standard Deviation)

Parameter

0 ppm

25 ppm

100 ppm

350 ppm

SDH in males after 3 months

6.2 ± 2.5

6.7 ± 1.8

9.7 ± 5.2

14.2 ± 4.5*

SDH in males after 6 months

5.6 ± 2.0

6.6 ± 2.8

7.2 ± 3.4

12.6 ± 5.7*

SDH in females after 3 months

5.7 ± 1.5

5.8 ± 0.9

6.6 ± 1.8

8.7 ± 2.6*

Rel. liver weight (%) in males after 24 months

2.95 ± 0.23

3.01 ± 0.41

3.59 ± 1.34

3.95 ± 0.84

Rel. liver weight (%) in females after 12 months

2.68 ± 0.23

2.97 ± 0.38

3.30 ± 0.47*

3.42 ± 0.32*

Incidence (%) hepatic focal cystic degeneration in males

26

38

44*

50*

Incidence (%) biliary hyperplasia in males

57

73

67

79*

Incidence (%) accumulation of pigments in Kupffer cells in males

2

6

8

34*

Incidence (%) accumulation of pigments in Kupffer cells in females

3

3

13

25*

Incidence (%) hepatic peliosis in males

5

3

11

13*

Rel. kidney weight (%) in males after 24 months

0.76 ± 0.06

0.83 ± 0.26

0.95 ± 0.34

1.29 ± 0.73*

Incidence (%) severe progressive nephropathy in males after 25 months

15

15

19

32*

SDH: Sorbitol Dehydrogenase Activity in U/L (no significant effects after 12, 18, or 24 months);

*: significant, p <= 0.05

Conclusions:
In chronic inhalation studies in rats the liver was the target organ. Effects on body weight, clinical chemistry parameters, organ weight parameters, and/or morphological changes were detected in males and females at ≥100 ppm (360 mg/m³; NOAEC 25 ppm or 90 mg/m³). According to the authors, the test substance was not oncogenic in rats under the experimental conditions (see also Section 7.7).
Executive summary:

In a chronic inhalation study comparable to OECD TG 453 groups of 87 male and 87 female Crl:CD® BR rats were exposed 6 h per day, 5 days per week, to 0, 25, 100, 350 ppm (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 (10 rats per dose per sex) for evaluation of toxic effects. For exclusive measurement of liver cell proliferation further interim sacrifices (5 rats per dose per sex) were performed 0.5, 3, and 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 and neoplastic effects. 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 sorbitol 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 (1260 mg/m³) resulted in decreased body weight, increased serum sorbitol 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. No treatment related effects were detected in testes (compare with subacute toxicity).

Conclusion: In chronic inhalation toxicity studies in rats the liver was the target organ. Effects on body weight, clinical chemistry parameters, organ weight parameters, and/or morphological changes were detected in males and females at >=100 ppm (360 mg/m³; NOAEC: 25 ppm or 90 mg/m³).

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to Guideline.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no
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 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:
whole body
Vehicle:
air
Details on inhalation 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, no post exposure period
Frequency of treatment:
6 h/day, 5 days/week (18 months)
(not during holidays, no details)
Dose / conc.:
25 ppm (nominal)
Remarks:
90 mg/m³,
analytical conc. range: 22-28 ppm, mean: 25.2 ppm
Dose / conc.:
100 ppm (nominal)
Remarks:
360 mg/m³,
analytical conc. range: 85-115 ppm, mean: 101.0 ppm
Dose / conc.:
350 ppm (nominal)
Remarks:
1260 mg/m³,
analytical conc. range: 300-390 ppm, mean: 350.5 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:
no effects observed
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 comment: 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 deceased 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
Description (incidence and severity):
There were no test substance-related effects.
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:
effects observed, non-treatment-related
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, 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 tumor 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:
LOAEC
Effect level:
100 ppm
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: 360 mg/m³
Dose descriptor:
LOAEC
Effect level:
350 ppm
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
other:
Remarks on result:
other: 1260 mg/m³
Dose descriptor:
NOAEC
Effect level:
25 ppm
Sex:
male
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: 90 mg/m³
Dose descriptor:
NOAEC
Effect level:
100 ppm
Sex:
female
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: 360 mg/m³
Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
In chronic inhalation studies in mice the liver was the target organ, effects were detected in males at 100 ppm (360 mg/m³; NOAEC: 25 ppm or 90 mg/m³) and in females at 350 ppm (1260 mg/m³; NOAEC: 100 ppm or 360 mg/m³).
Executive summary:

Groups of 78 male and 78 female CD-1 mice were exposed in an inhalation study comparable to OECD TG 453 6 h per day, 5 days per week, to 0, 25, 100, or 350 ppm (0, 90, 360, 1260 mg/m³). The exposure period lasted 18 months and no post exposure observation period followed. Interim sacrifice (5 mice per dose per sex) was performed 0.5, 3, and 12 months after initiation of the study for assessment 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). There were no test substance-related effects observed in the testes (compare with subacute studies).

Conclusion: In chronic inhalation studies in mice the liver was the target organ, effects were detected in males at 100 ppm (360 mg/m³; NOAEC: 25 ppm or 90 mg/m³) and in females at 350 ppm (1260 mg/m³; NOAEC: 100 ppm or 360 mg/m³).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is comparable to Guideline with acceptable restrictions (only 2 animals per dose; limited parameters in hematology and clinical chemistry; limited documentation of urinalysis; only 3 organs examined in histopathology [liver, kidney, skin]; partly limited documentation [details on test substance, analytical results or animals]).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
- Name of test material: dimethylacetamide (DMAC)
No details available.
Species:
dog
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
No details available.
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
The dose was applied to the shaved skin. Licking was prevented.

REMOVAL OF TEST SUBSTANCE
- Washing: After 5 hours of exposure.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
a) 6 months at the two lower dose levels
b) 6 weeks at the two higher dose levels
Frequency of treatment:
Once daily for 5 h (washing); 5 days per week
Dose / conc.:
0.1 other: mL/kg bw/day
Remarks:
94 mg/kg bw/day
Dose / conc.:
0.32 other: mL/kg bw/day
Remarks:
300 mg/kg bw/day
Dose / conc.:
1 other: mL/kg bw/day
Remarks:
940 mg/kg bw/day
Dose / conc.:
4 other: mL/kg bw/day
Remarks:
3760 mg/kg bw/day
No. of animals per sex per dose:
a) 2 (1 m, 1 f) in the two low dose groups plus control
b) 2 males in the two high dose groups plus control
Control animals:
other: yes, unspecified
Details on study design:
- Post-exposure recovery period in satellite groups: Presumably no post exposure observation period.
Positive control:
No
Observations and examinations performed and frequency:
CLINICAL SIGNS:
Clinical signs were recorded (no details).

BODY WEIGHT:
The animals were weighed at weekly intervals.

HEMATOLOGY:
- Time schedule for collection of blood: Initial and after 1, 2, 3, 6 months in a) (no details), weekly in b).
- Parameters examined: Hemoglobin, sedimentation rate, hematocrit, total and differential white blood cell counts.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Initial and after 1, 2, 3, 6 months in a) (no details), weekly in b).
- Parameters examined: Blood urea nitrogen (BUN), bromsulphthalein (BSP) dye retention, thymol turbidity, alkaline phosphatase.

URINALYSIS:
Urine analysis was performed but no details given .
Sacrifice and pathology:
GROSS PATHOLOGY:
Gross autopsies were performed upon the animal that died and on the remaining animals sacrificed at termination of the study.

HISTOPATHOLOGY:
Sections of liver, kidney and skin were processed and examined.
Other examinations:
No
Statistics:
No data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Anorexia, depression, weakness, ataxia, abdominal tenderness, diarrhea and frank jaundice were present in the dogs on the 4 mL/kg bw/day level just prior to death
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
No significant microscopic findings were seen in the skin at 0.1 mL, except for slight thickening and/or inflammatory reaction.
Local effects (ulcer) were observed in 1/2 dogs at 0.32 mL/kg bw/day after 4 month. Administration to this site was stopped, and the ulcer gradually healed over a period of 1 month . The skin of both dogs showed marked scaliness.
Mild to moderate skin irritation at ≥ 1 mL/kg bw/d (at termination of the studies the dogs showed fissuring and thick scales, which resembled eschars).
Mortality:
mortality observed, treatment-related
Description (incidence):
At 4 mL/kg bw/day one dog died after 15 applications and the other was sacrificed moribund at day 16.
At 1 mL/kg bw/day both dogs were sacrificed moribund after 6 weeks of treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dogs at both high dose levels showed weight loss. At 0.32 mL/kg bw/d there was reduced body weight during the initial exposure period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At ≥1.0 mL/kg bw/d there was increased hematocrit and hemoglobin, and leukocytosis.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At ≥0.32 mL/kg bw/d the activity of alkaline phosphatase was increased. There was altered BSP retention.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Pale or yellow liver was noted at necropsy.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Fatty degeneration of the liver was noted at ≥1 mL/kg bw/day (940 mg/kg bw/day); there were no effects noted in the kidneys.
Dose descriptor:
NOAEL
Effect level:
0.1 other: mL/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose
Remarks on result:
other: 94 mg/kg bw/day
Dose descriptor:
LOAEL
Effect level:
0.32 other: mL/kg bw/day
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
Remarks on result:
other: 300 mg/kg bw/day
Critical effects observed:
not specified
Conclusions:
In chronic studies toxic effects were detected in dogs after dermal exposure to DMAC at dose levels >=300 mg/kg bw/day. The dermal NOAEL was 94 mg/kg bw/day.
Executive summary:

In a chronic dermal toxicity study comparable to guideline (OECD TG 452) 2 dogs per dose level received 0, 0.10, 0.32, 1.0, or 4.0 mL/kg bw/day corresponding to 0, 94, 300, 940, 3760 mg/kg bw/day to clipped skin. The dermal exposure was conducted openly with washing after 5 h exposure at 5 days per weeks. The exposure period was 6 weeks for the two higher dose levels (lethal effects) and 6 months for the two lower dose levels.

Mortality was observed at the two higher dose levels; dogs died or were sacrificed moribund (after 6 weeks at 1 mL/kg bw/day). At a dose level of 0.32 mL/kg bw/day local effects (ulcer), decrease in body weight, increased activity of alkaline phosphatase, and altered BSP retention were seen. Fatty degeneration of the liver was detected in histopathology at >=1 mL/kg bw/day (940 mg/kg bw/day). No data were available on histopathology of the testes (compare with other repeated dose studies in mouse and rat).

Conclusion: The dermal NOAEL was 94 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
94 mg/kg bw/day
Study duration:
subchronic
Species:
dog

Mode of Action Analysis / Human Relevance Framework

no further information

Additional information

A bright set of studies is available for the endpoint repeated dose toxicity for DMAC. In this endpoint summary only relevant studies are mentioned.

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. However, further studies relevant for evaluation were found which were not included in the OECD SIDS (2001). This might be related to the fact that the initial version of the OECD SIDS document was prepared for SIAM3 (1995). Delegates of SIAM3 criticised lacking information and these comments could still not be resolved at the meeting of SIAM12.

Oral exposure

In a chronic drinking water study (Monsanto, 1979, RL2: comparable to Guideline study with acceptable restrictions, e.g. no details about the test substance; problems in analytical methodology of DMAC in drinking water; limited data on test animals and conditions; limited parameters in clinical chemistry; low number of rats in hematology, urinalysis and clinical chemistry; some organs not included in histopathology; histopathology not extended to low and mid dose groups concerning organs with effects in high dose group) 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, 10 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 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 of 1000 mg/kg bw/day. High dose exceeded MTD as BW decrease was more than 10%. No effects were detected on food and water consumption. The toxicological relevance of effects in hematology and clinical chemistry was questionable. Urinalysis and ophthalmology parameters were unremarkable. 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, pigmentation and vacuolisation were found. The derivation of the NOAEL is questionable due to limitation of the histopathological assessment.

Conclusion: In a chronic drinking water study reduced body weight gain was reported at >=300 mg/kg bw/day in males and at 1000 mg/kg bw/day in females; the high dose, exceedintg the MTD induced liver cell degeneration in males and females and testis atrophy in males; the (provisional) NOAEL was 100 mg/kg bw/day in males and 300 mg/kg bw/day in females.

In a feeding study 6 CD rats per sex received 0 or 60 mg/kg bw/day for 93 - 94 days (Kennedy and Sherman,1986; RL2: only one dose, partly insufficient documentation; incomplete histopathology). Clinical signs (mortality), body weight gain, food consumption, hematology, and clinical chemistry were recorded. At termination macroscopic and microscopic pathology were performed of the main organs including liver and testes. No toxic effects were detected except a slight leukocytosis and slight anaemia. However, the toxicological relevance of the hematology data is questionable (no details including historical range).

Conclusion: Rats receiving in a subchronic feeding study 60 mg/kg bw/day showed no effects except a slight leukocytosis and anaemia (questionable toxicological relevance). 

 

Groups of 10 male and 10 female Sprague-Dawley rats (BASF, 1975, RL2: test procedure in accordance with national standard methods but partly limited documentation, e.g. about test substance and organ weights; histopathological results not available; FOB not performed) were gavaged with the test substance at 0, 290, 590, 1170, or 2350 mg/kg bw/day for 4 weeks, 5 days per week. Clinical signs like ruffled coat and reduced general condition were seen at 1170 mg/kg bw/day as well as significant decrease of body weight (> -10% compared to control). Lethal effects were observed at the high dose level of 2350 mg/kg bw/day. Increased total lipids in males and females were detected in clinical chemistry as well as reduced activity of alkaline phosphatase at >=290 mg/kg bw/day (questionable relevance). Significant reduction of absolute heart weight was noted at >=290 mg/kg or >= 590 mg/kg in males or females, respectively; reduced relative heart weight in males and females at >=590 mg/kg bw/day. In females relative and absolute adrenal weight was reduced at >=290 mg/kg bw/day. Reduced testis, uterus and ovar weights were observed at >=1170 mg/kg bw/day. Increased liver weight was found in males and females (aboslute >= 1170 mg/kg; relative >= 590 mg/kg). Histopathological data are not available.

Conclusion: In rats the subacute oral application of 290 mg/kg bw/day (LOAEL) resulted in adverse effects which needed verification by histopathology.

In a pilot subacute drinking water study (Monsanto, 1976, RL2, limited parameters: clinical signs, body weight gain, water and food consumption, necropsy data; details of results not documented) no toxic effects were detected in male and female Long-Evans rats even at the high dose level of 1000 mg/kg bw/day.

 

Dermal exposure

In a chronic dermal toxicity study (Horn, 1961; RL2: comparable to Guideline with acceptable restrictions: only 2 animals per dose; limited parameters in hematology and clinical chemistry; limited documentation of urinalysis; only 3 organs examined in histopathology [liver, kidney, skin]; partly limited documentation [details on test substance, analytical results or animals]), 2 dogs per dose level received 0, 0.10, 0.32, 1.0, or 4.0 mL/kg bw/day (0, 94, 300, 940, 3760 mg/kg bw/day) to the clipped skin (open; 5 days/weeks; washing after 5 h exposure) for 6 weeks at the two higher dose levels (lethal effects) and for 6 months at the two lower dose levels. Mortality was obvious at the two higher dose levels; dogs died or were sacrificed moribund (after 6 weeks at 1 mL/kg bw/day). At a dose level of 0.32 mL/kg bw/day local effects (ulcer), decrease in body weight, increased activity of alkaline phosphatase, and altered BSP retention were seen. Fatty degeneration of the liver was detected in histopathology at >=1 mL/kg bw/day (940 mg/kg bw/day). No data were available on histopathology of the testes (compare with other repeated dose studies in mouse and rat).

Conclusion: In chronic studies toxic effects were detected in dogs after dermal exposure at dose levels >=300 mg/kg bw/day; the NOAEL was 94 mg/kg bw/day.

 

In male rats subacute dermal exposure (Monsanto, 1973, RL4) resulted in reduced body weight at a dose level of 1000 mg/kg bw/day, while no relevant effects were found at 500 mg/kg bw/day (limited parameters examined). These results were confirmed in a second trial where reduced body weight was noted in male rats at 1000 mg/kg bw/day after an exposure period of 56 days. However, the reliability of these two studies is not assignable, as they were performed at IBT, where numerous irregularities in study conduct and documentation of non-acute studies were detected (OECD, 2005: Manual for the Assessment of Chemicals. Chapter 3: Data Evaluation).

 

Local effects after dermal application were only reported by Horn (1961) but not in any other reliable study with dermal application. Moreover, DMAC was not irritating to the rabbit skin and there are no indications for irritations to human skin. Therefore, the findings reported by Horn (1961) are conspicuous and are more likely related to the extensive shaving/washing procedures within this study, and not directly related to the treatment with the test substance.

 

Inhalation exposure

In a chronic inhalation study comparable to Guideline groups of 87 male and 87 female Crl:CD® BR rats were exposed 6 h per day, 5 days per week, to 0, 25, 100, 350 ppm (0, 90, 360, 1260 mg/m³; Malley et al., 1995; RL2). The exposure period lasted 24 months and no post exposure observation period followed. Interim sacrifice was conducted after 12 months (10 rats per dose per sex) for evaluation of toxic effects. For exclusive measurement of liver cell proliferation further interim sacrifices (5 rats per dose per sex) were performed 0.5, 3, and 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 and neoplastic effects. 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 sorbitol 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 (1260 mg/m³) resulted in decreased body weight, increased serum sorbitol 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. No treatment related effects were detected in testes (compare with subacute toxicity).

Conclusion: In chronic inhalation toxicity studies in rats the liver was the target organ. Effects on body weight, clinical chemistry parameters, organ weight parameters, and/or morphological changes were detected in males and females at >=100 ppm (360 mg/m³; NOAEC: 25 ppm or 90 mg/m³).

 

Groups of 78 male and 78 female CD-1 mice were exposed in further experiments of the same working group (Malley et al., 1995; RL1: comparable to Guideline study but no clinical chemistry and no urinalysis; tabulated details of results not presented) 6 h per day, 5 days per week, to 0, 25, 100, or 350 ppm (0, 90, 360, 1260 mg/m³). The exposure period lasted 18 months and no post exposure observation period followed. Interim sacrifice (5 mice per dose per sex) was performed 0.5, 3, and 12 months after initiation of the study for assessment 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). There were no test substance-related effects observed in the testes (compare with subacute studies).

Conclusion: In chronic inhalation studies in mice the liver was the target organ, effects were detected in males at 100 ppm (360 mg/m³; NOAEC: 25 ppm or 90 mg/m³) and in females at 350 ppm (1260 mg/m³; NOAEC: 100 ppm or 360 mg/m³).

 

In a subacute inhalation study (DuPont, 1983, RL2: comparable to OECD 412; some clinical chemistry parameters recommended in OECD 412 not measured; no details in the result section except organ weights) groups of 10 male and 10 female Crl:CD rats were exposed 6 h per day, 5 days per week, for 2 weeks to 0, 100, 288, or 622 ppm (0, 360, 1040, 2240 mg/m³). Rats were sacrificed after 10 exposures (5 rats per sex per dose) or after a 2 week recovery period (other half of each group). Clinical signs, body weight gain, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic and microscopic pathology data were recorded. No NOAEC was identified. Local nasal effects were found at >=100 ppm in males and females; additionally decreased leukocyte counts after the recovery period were detected in females, and in males decreased mean corpuscular volume and increased serum cholesterol were noted (effects of questionable relevance without historical data). At 288 ppm there were nasal effects and liver hypertrophy (males and females), atrophy of testis (males); serum cholesterol increase and decrease in neutrophils (females), relative liver and kidney weight increase (males and females), relative spleen weight decrease (females) but no clinical signs or effects on body weight. At 622 ppm rats died after 4 exposures (no further treatment); pathology of survivors revealed liver hepatocellular hypertrophy and necrosis, lymphocytic depletion in thymus and spleen, hypocellularity in bone marrow, local (ingestion of test substance suggested) inflammation of the stomach and intestine, and testicular atrophy. Comment: No details are available about local effects in the nasal cavity comparing incidences with concurrent and historical controls.

Conclusion: Local effects in the nasal cavity were found at 100 ppm (360 mg/m³) and systemic effects on liver and testis at 288 ppm (1040 mg/m³). No NOAEC was identified. The local effects were not confirmed in the chronic inhalation toxicity studies in mice and rats. Furthermore, the available human data do not indicate a respiratory irritation potential. This leads to the conclusion that the local effects reported in the present study are conspicuous and should not be considered as reliable.

 

In a sub-acute inhalation study (Valentine et al., 1997c; RL2: study restricted to clinical signs, body weight gain, necropsy data and effects on testis but meets scientific standards) 13 male Crl:CD-BR rats (control n=9) per dose level were exposed 6 h per day, 5 days per week, for 2 weeks to 0, 52, 150, 300, or 480 ppm (0, 190, 540, 1080, 1730 mg/m³) and sacrificed after the last exposure. No clinical signs were detected and necropsy revealed no effects. However, at the high dose level a significant decrease in body weight was found and slight effects on sperm counts and testis weight were noted at 480 ppm (no effects at 300 ppm) which were not statistically significant.

Conclusion: Significant effects on body weight gain and slight but statistically not significant effects on testis weight and sperms counts were reported in rats after subacute inhalation at a dose level of 480 ppm (1730 mg/m³); the NOAEC was 300 ppm (1080 mg/m³).

 

In a subacute inhalation study comparable to Guideline (Valentine et al., 1997a; RL2, clinical chemistry and adrenal weight not measured

) 10 male Crl:CD-1(ICR)BR mice (35 days old) per dose level were exposed 6 h per day, 5 days per week, for 2 weeks to 0, 30 ± 4, 100 ± 10; 310 ± 30, 490 ± 39, 700 ± 65 ppm (means ± SD; 0, 110, 360, 1120, 1760, 2520 mg/m³) and sacrificed after the last exposure (5 mice) or after 2 weeks recovery period (5 mice). No effects were detected at 100 ppm. At 310 ppm a slight (15 %) decrease in testis weight (not statistically significant) was found and histopathological effects like reduced number of sperm and increased germinal epithelium in the epididymides were noted. At >= 490 ppm clinical signs (including mortality) were obvious, the body weight decreased, and effects on hematological parameters were seen; histopathological alterations in testis (atrophy, degeneration) and liver (necrosis), lymphoid atrophy, hypoplasia in bone marrow, and necrosis in adrenal cortex were reported.

Conclusion: There were histopathological effects in testis of 35-days old mice after subacute inhalation at a dose level of 310 ppm (1120 mg/m³); the NOAEC was 100 ppm (360 mg/m³).

 

In a further subacute inhalation study older male mice (61 days old) were used (Valentine et al., 1997b; RL2). This study was restricted to the assessment of clinical signs, body weight gain, necropsy data and effects on testis. Twelve male Crl :CD-1(CR)BR (control n=9; 61-days old) mice per dose level were exposed 6 h per day, 5 days per week, for 2 weeks to 0, 52, 150, 300, or 480 ppm (0, 190, 540, 1080, 1730 mg/m³) and sacrificed after the last exposure. No clinical signs and no effects on body weight gain were detected and necropsy revealed no effects. However, at the high dose level a significant decrease in testis weight was found as well as minimal to mild bilateral degeneration and atrophy of seminiferous tubules in 3 out of 9 mice at 480 ppm (no effects at 300 ppm).

Conclusion: Significant effects on testis weight (but not sperm counts) as well as histopathological alteration in testis were reported in 61-day old mice after subacute inhalation of the test substance at a dose level of 480 ppm (1730 mg/m³); the NOAEC was 300 ppm (1080 mg/m³).

In a subacute inhalation study (Kinney et al., 1993; RL2: limited to clinical signs, body weight gain and effects on liver, testis and respiratory tract) 15 male Crl:CD(SD) BR rats per dose level were exposed 3, 6, or 12 h per day, 5 days per week, for 2 weeks to 0, 10, 30, 100, or 300 ppm (0, 36, 110, 360, 1080 mg/m³). Specified parameters were measured after 10 exposures or after a 2 week recovery period. No clinical signs were detected. Dose dependent effects in clinical chemistry were found like increased serum cholesterol levels or increased total serum protein concentrations (LOAEL at 30 ppm for 12 h/d), the effects were reversible after recovery. No effects on organ weights (liver and testes) were reported and also no effects at necropsy. Histopathology, however, revealed changes in the liver but only at the 300 ppm level and exposure for 12 h (liver changes consisted of hepatocellular hypertrophy together with margination of hepatocellular cytoplasmic contents and hepatic cellular cytoplasmic lipid-like vacuolation). These changes were present in all of the rats examined in this group (graded as between slight to moderate in severity). These changes persisted after the recovery period but were less severe. No treatment related changes were seen in the testes and nasal passages. In contrast to other subacute studies (whole body inhalation exposure including dermal absorption) only the inhalation route was investigated (nose-only) in this study. The toxicological relevance of altered clinical chemistry parameters is questionable without effects in histopathology.

Conclusion: In subacute nose only experiments in rats morphological effects on liver cells were detected after repeated inhalation exposure to 300 ppm (1080 mg/m³) and 12 h exposure per day, no such effects were seen at 100 ppm (360 mg/m³).

 

Supplementary inhalation studies

In a one-generation reproduction toxicity study (Ferenz et al., 1986; RL2: comparable to Guideline study but MTD also not reached; no histopathology of reproductive organs) 10 males and 20 females per group were exposed via the inhalation route 6 h per day, 5 days per week, for 10 weeks (prebreeding), then 7 days per week for 7-8 weeks (breeding, gestation, lactation) to 0, 30, 100, or 300 ppm (group I-IV, respectively; nominal concentrations corresponding to 0, 110, 360, 1080 mg/m³). In additional groups only males (group V) or only females (group VI) were exposed to 300 ppm. The exposure period started when rats were 35 days old and ended for the males upon completion of the breeding period (after 63 exposures), and for the females and offspring at 21 days post partum (after 89-104 exposures). Half of the adult males in each group were sacrificed after the breeding period; the other half after a recovery period of 20 days. Dams were sacrificed on day 21 post partum. Adverse effects on fertility or developmental toxicity were not observed. In the P and F1 generation no treatment related clinical signs were observed and necropsy revealed also no treatment related effects. During the first 3 weeks of exposure a significant decrease in body weight was measured in P males of group V (300 ppm); this effect was not found in males of group IV (300 ppm) or in any other test group. No effects were found on absolute liver weights in P animals except a slight increase in treatment groups (not significant); in group IV the relative liver weight of males and females was significantly increased but not in group V and VI (also 300 ppm); no effects were reported in recovery males (one half of males of each group sacrificed after a post exposure observation period of 20 days). Body weight in exposed male and female F1 rats was significantly reduced at 300 ppm.

Conclusion: No effects on reproductive parameters, but effects on body weight and liver were found in rats after repeated inhalation exposure to DMAC at a dose level of 300 ppm (1080 mg/m³).

The subchronic inhalation study of Wang et al. (1989, RL2) was related to male fertility, but scientific standards were met concerning investigated parameters of repeated dose toxicity like clinical signs, body weights, clinical chemistry, necropsy data, testis and liver weights, histopathology of liver, testis, and kidney. Twelve male Sprague-Dawley rats per concentration were exposed 6 h per day, 5 days per week to 0, 40, 120, or 400 ppm (0, 140, 430, 1400 mg/m³; whole body). The study was terminated at week 15 after 69 exposures (no post exposure observation period). No clinical signs were recorded in any group and no effects on body weight gain. Clinical chemistry (limited parameters) revealed also no alterations. Liver weight was increased at >=120 ppm (no effects on testis) but histopathology revealed no effects in any of the three studied organs. Since the increase in liver weight was not accompanied by effects in clinical chemistry and histopathology the toxicological relevance is questionable.

Conclusion: There were no effects on liver and testis in male rats inhalation exposed for 15 weeks at up to 400 ppm (1400 mg/m³).

 

In the study of Horn (1961; RL2: comparable to Guideline but only 2 animals per dose; limited parameters in hematology and clinical chemistry; limited documentation of urinalysis; only 3 organs examined in histopathology [liver, kidney, skin]; partly limited documentation) two dogs per dose level were exposed 6 h per day, 5 days per week, for 6 months to 0, 40, 64, 103, or 195 ppm (corresponding to 0, 140, 230, 370, 700 mg/m³). No effects were found concerning clinical signs, body weight, urinalysis or hematological parameters. Clinical chemistry revealed increased alkaline phosphatase activity at 195 ppm and altered bromsulphthalein retention at >=103 ppm. In histopathology fatty liver cell degeneration was found at >=103 ppm.

Conclusion: In a chronic inhalation study toxic effects were detected in the liver of dogs at concentration >=103 ppm (370 mg/m³); the NOAEC was 64 ppm (230 mg/m³).

In the same publication by Horn, 1961 (RL3; limited documentation; pneumonia also in control rats which might influence results.) 20 rats per dose level were exposed 6 h per day, 5 days per week, for 6 months to 0, 40, 64, 103, or 195 ppm (corresponding to 0, 140, 230, 370, 700 mg/m³). Hematology, clinical chemistry and urinalysis were performed during the application period; after necropsy at the end of the study, the animals were examined histopathologically (at least for damage of liver and lungs). No effects were found concerning clinical signs, body weight, urinalysis or hematological parameters. In histopathology fatty liver cell degeneration was found at >=103 ppm.

Conclusion: In a chronic inhalation study toxic effects were detected in the liver of rats at concentration >=103 ppm (370 mg/m³). However, the study was not taken into account, as pneumonia was observed also in control rats which might influence the results.

Two inhalation studies in rats and mice comparable to OECD Guideline 451 (Anonymous 2013; RL2: with acceptable restrictions: inadequate air change rate during exposure, highest dose exceeded the MTD, uncommon mouse strain used) were performed with an exposure of 6 hours/day, 5 days/week for an exposure period of 2 years. The studies are described in detail in the carcinogenicity section. Dose levels for exposure of rats were 0, 18, 90 and 450 ppm (ca. 0, 65, 324 and 1620 mg/m³) and for mice doses of 0, 12, 60 and 300 ppm (ca. 0, 43, 216 and 1080 mg/m³) were applied. The liver was identified as the target organ. The NOAEC for toxic effects in rats was 18 ppm (65 mg/m³) and the NOAEC for toxic effects in mice was 60 ppm (216 mg/m³).

 

Inhalation and/or dermal exposure of humans

Exposure of workers to N,N-dimethylacetamide (DMAC) in an acrylic fiber plant was measured over a 1-year study period, by full-shift (12 h) personal air monitoring of DMAC and biological monitoring for levels of DMAC, N-methylacetamide (NMAC) and acetamide in post-shift spot urine samples (Spies et al., 1995; see Section 7.10.2; RL2: study meets scientific standards but low number of workers in cohort, only 21 workers in high exposure group; no exposure data of control group reported [minor restriction]). Evidence of liver toxicity was assessed by serum clinical chemistry tests (total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and y-glutamyl transpeptase) at least once during the study period for all 127 male workers in the 2 study departments and for 217 male in-plant controls with no previous or current exposure to DMAC. Additional serum clinical chemistry tests were conducted at weekly intervals for 3 weeks in workers showing increased DMAC (>132 mg/g creatinine) or NMAC (>60 mg/g creatinine) levels in urine (trigger values). DMAC-exposed workers were classified in groups reflecting either high exposure (if one biomonitoring result exceeded one of the trigger values), or unspecified exposure (trigger value was not reached). Control employees were classified as not exposed. Mean DMAC levels in air differed for the high- and unspecified exposure groups (mean DMAC in air levels of 1.9 and 1.3 ppm, 12-hour time-weighted average, respectively) as well as mean urinary NMAC values (26.7 vs. 13.5 mg/g creatinine). No DMAC exposure related trends in hepatic serum clinical chemistry results were detected. None (0 of 21) of the high exposure group individuals had an abnormal serum biochemistry result during the study period. Comment: dermal exposure was not quantified but might contribute significantly to high DMAC and NMAC concentrations in urine.

Conclusion: There was no liver injury in workers exposed to DMAC excreting >60 mg N-methylacetamide/g creatinine (trigger value) via the urine (2-fold BEI value).

 

In a clinical study with 8 male volunteers no toxic effects were reported after repeated inhalation exposure to 10 ppm (36 mg/m³). Eight male volunteers were exposed to 10 ppm at 6 h per day for 5 consecutive days. Behaviour was recorded during and after the exposure period. Blood samples were collected prior and after the exposure period for hematology and clinical chemistry. Urine was collected during and after the exposure period for urinalysis and porphyrin excretion. No treatment related effects were detected (1974, RL2; see IUCLID Section 7.10.3).

There was some indication for increased hepatic injury in workers employed in departments of elastane fibre plants with DMAC exposure but there is not sufficient evidence for the dose dependency of these effects (Lee et al., 2006; RL2, see IUCLID Section 7.10.2).

Some indications for DMAC-induced hepatic injuries in 38 out of 1045 monitored workers were reported but no sufficient quantitative data on exposure were given (Jung et al., 2007; RL2, IUCLID Section 7.10.1).

Two cases of toxic hepatitis after repeated dermal exposure to DMAC were reported by Baum et al. (1997; see IUCLID Section 7.10.3). Two female workers of an acrylic-fiber production plant were repeatedly exposed to DMAC via the skin by splashes of this substance to the clothes above the protection gloves. Clinical data suggested toxic hepatitis.

The odour threshold in the trained panel of four staff members of the Food and Flavour Section was 47 ppm (170 mg/m³); odour description: amine, burnt, oily (see IUCLID Section 7.12; Leonardos et al., 1969, RL2).

 

Parenteral application in humans

Hallucinogenic effects accompanied by altered EEG were found in cancer patients after repeated i.v. injection at a dose level of >=400 mg/kg bw/day for 3 days; there was also evidence for hepatotoxic effects in some patients (Weiss et al., 1962; RL2, see Section 7.10.3). Supporting evidence for hallucinogenic effects were also given after acute dermal and inhalation exposure (Marino et al., 1994; RL2, case report in IUCLID Section 7.10.3; study limited by co-exposure).

 

Summary of short-term repeated dose toxicity most relevant for the derivation of acute/short term DNELs

In a two-week inhalation toxicity study groups of 10 male mice were whole body exposed to DMAC concentrations of 0, 30, 100, 310, 490 or 700 ppm (5 days/week) (Valentine et al., 1997). The NOAEC was 100 ppm (360 mg/m³). In a further two-week inhalation toxicity study with recovery groups, 10 male and 10 female rats were whole body exposed to DMAC concentrations of 0, 100, 288 or 622 ppm (5 days/week) (Kelly et al., 1984). The concentration of 100 ppm was derived as LOAEC by the study authors due to local effects in the nasal cavity and changes in some isolated clinical chemistry/hematology parameters. All of the effects were of questionable relevance. Thus, 100 ppm (360 mg/m³) can be taken as the NOAEC with respect to acute/short term effects.

 

In a sub-acute oral toxicity study (1975) groups of 10 rats per sex were treated once daily, 5 days/week for 4 weeks at 0, 290, 590, 1170, 2350 mg/kg bw/day by gavage. The dose of 290 mg/kg bw/day was derived as LOAEL on the basis of changes in organ weights (heart in males and adrenal in females), atrophy of uterus and some changes in clinical chemistry parameters. These effects can be attributed to the repeated administration of the test substance and are not expected to occur after an acute exposure. Signs of toxicity observed soon after onset of exposure of a repeated dose study can be used to establish a NOAEL for acute effects from a repeated dose study (ECHA, 2012). Consequently, the NOAEL of 590 mg/kg bw/day can be derived from the study for acute toxicity based on clinical signs and body weight reduction observed already during the first week of treatment at 1170 mg/kg bw/day.

 

From a one-generation reproductive toxicity study (1973a, study performed at IBL; see IUCLID section 7.8.1) in rats with dermal application of DMAC (28-day treatment at 0, 250, 500 and 1000 mg/kg bw/day, determination of clinical signs and body weight, necropsy/histopathology on day 164) a NOAEL for acute/short term effects of 500 mg/kg bw/day can be derived. At 1000 mg/kg bw/day, reduced body weights in males were noted (starting at test day 7).

In a feeding study 6 rats per sex received 0 or 60 mg/kg bw/day for 93-94 days (Kennedy and Sherman, 1986). Clinical signs, mortality, body weight gain, food consumption, hematology, and clinical chemistry were assessed. At termination macroscopic and microscopic pathology was performed of the main organs. No treatment related adverse effects were seen at 60 mg/kg bw/d.

 

Two dogs per dose level received DMAC at 0, 0.1, 0.32, 1.0, or 4.0 mL/kg bw/day (0, 94, 300, 940, 3760 mg/kg bw/day) to the clipped skin (open; 5 days/weeks; washing after 5 h exposure) for 6 weeks at the two higher dose levels (lethal effects) and for 6 months at the two lower dose levels (Horn, 1961). At a dose level of 0.32 mL/kg bw/day systemic toxicity consisted of decrease in body weight, increased activity of alkaline phosphatase, and altered BSP retention. Fatty degeneration of the liver was detected in histopathology at ≥1.0 mL/kg bw/day (940 mg/kg bw/day). The reliability of the derived NOAEL of 94 mg/kg bw/d is restricted due to the low number of animals per dose.

 

Reference

ECHA (2012). Guidance on information requirements and chemical safety assessment, Chapter R.8: Characterisation of dose [concentration]-response for human health; Version 2.1.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.