N,N-dimethylacrylamide

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, near-guideline study, available as published report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethylacrylamide

EC Number:

220-237-5

EC Name:

N,N-dimethylacrylamide

Cas Number:

2680-03-7

Molecular formula:

C5H9NO

IUPAC Name:

N,N-dimethylacrylamide

Details on test material:

- Name of test material (as cited in study report): N,N-dimethylacrylamide
- Batch number: 09430EW
- Physical state: clear, slightly brownish-yellow liquid
- Storage condition of test material: in original container at room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 87-107 g (males), 83-103 g (females)
- Fasting period: 18 hours before blood sample collections, during urine sample collections and prior to terminal sacrifice and necropsy
- Housing: individually in stainless steel cages measuring 24.0 x 17.8 x 17.6 cm, suspended over excrement pans fitted with absorbent cage boards or papers
- Diet: Certified Purina Rodent Chow (Meal) 5002 (PMI Feeds, Inc., St. Louis, MO), ad libitum
- Water: reverse osmosis-purified water, supplied by an automatic watering system, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 30-79
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Remarks:

HPLC grade

Details on exposure:

TEST SITE
- Type of wrap if used: Hill Top Chamber, secured with an elastic band fastened with Velcro
- Time intervals for shavings or clipplings: prior to first test article administration and weekly thereafter

TEST MATERIAL
- Amount applied: 1 ml/kg bw
- Constant volume used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Initially: 10, 200, 500 mg/kg bw/day
Basis:
nominal per unit body weight

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Secondly: 10, 75, 250 mg/kg bw/day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

Sprague Dawley rats (20/sex/group) were exposed 6 hours/day, 7 days/week for 13 weeks to graded concentrations of N,N-Dimetylacrylamide, on the shaved dorsal skin. A vehicle control group of equal size was exposed dermally to the vehicle (distilled water) only. Local skin reaction at the application site was evaluated for all rats, systemic toxicity was evaluated via clinical pathology and organ weights in 10 rats/sex/group, and potential peripheral neuropathy was evaluated via a limited battery of tests and specialized histopathology techniques in the other 10 rats/sex/group.
Rats were initially administered dermal doses of 10, 200 and 500 mg/kg bw/day; however, after six days of administration, it was evident that the highest dose exceeded the maximum tolerated dose since it resulted in overt systemic toxicity (i.e., convulsions, dramatically reduced body weight, body weight gain and food consumption) and two deaths. Therefore, the mid and high dose levels were reduced to 75 and 250 mg/kg bw/day, respectively. The animals that died were replaced with naive animals and the surviving high dose rats were allowed 2 days of rest/recovery prior to administration of the new dose level (i.e., 250 mg/kg/ bwday). Thus, all rats were not dosed for 90 consecutive days.

Positive control:

To aid in neurotoxicity assessment, a positive control group of 10 rats/sex was treated intermittently with intraperitoneal (ip) injections of acrylamide, a known neurotoxicant.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during quarantine period; twice daily on week days and once daily on weekends and holidays during the dosing period.
- Cage side observations included were: morbidity, mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

DERMAL IRRITATION: Yes
- Time schedule for examinations: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: upon receipt of the animals, one week prior to initiation of treatment, one day prior to treatment, weekly during dosing, and at the termination of treatment immediately prior to scheduled sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes (weekly for the first month, and monthly thereafter).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 4, 8 and 12 of treatment
- Anaesthetic used for blood collection: Yes (70% CO2, 30% O2)
- Animals fasted: Yes
- How many animals: all
- Parameters examined were: hematocrit, hemoglobin, total erythrocyte count, total leukocyte count, differential leukocyte count (percent and absolute), mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red blood cell morphology, platelet count and packed cell volume.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: weeks 4, 8 and 12 of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters examined were: albumin, globulin, albumin/globulin ratio, total protein, blood urea nitrogen, glucose, alkaline phosphatase, total bilirubin, aspartate aminotransferase, chloride, alanine aminotransferase, sodium, potassium, creatinine, cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 6 and 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined were: urine volume, refractive index, glucose, urobilinogen, levels of sodium and potassium, color, appearance, specific gravity, microscopic evaluation, and qualitative measurement of pH, protein, ketones, blood, nitrites, leukocytes, and bilirubin.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and after 12 weeks (rotorod); prior to treatment and during weeks 4, 8 and 12 (grip strength and hindlimb foot splay);
- Dose groups that were examined: all dose groups including the positive control groups
- Battery of functions tested: rotorod performance, grip strength, hindlimb foot splay

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Parameters examined were: eyes, brain, pituitary, lung, thymus, heart, aorta, thyroid/parathyroid, esophagus, trachea, liver, kidneys, adrenal glands, spleen, pancreas, salivary glands, submandibular lymph nodes, skeletal muscle, bone marrow smear (femur), bone/bone marrow (sternebrae), tests, epididymis, seminal vesicle, prostate, ovaries, uterus, stomach, duodenum, jejunum, ileum, cecum, colon, urinary bladder, mesenteric lymph nodes, spinal cord (thoracic), spinal cord (lumbar), mammary gland, sciatic nerve, skin (application site & untreated), tumor/mass, gross lesions, ear tag.

Statistics:

All data were statistically analyzed by two-factor, fixed-effects repeated-measures analysis of variance and/or analysis of variance (ANOVA) followed, where appropriate, by Dunnett's post hoc test for comparing multiple treatment groups to a single control. Hematology data were analyzed using the LABCAT-provided statistical tools. All other data (i.e., F.O.B. parameters, body weights, body weight gains, food consumption, clinical chemistry, urine sodium and potassium, and absolute and relative organ-to-body weights) were statistically analyzed using SYSTAT software (SPSS, Inc., Chicago, IL, version 5.0) on a PlusData 486-DX2 personal computer. It should be noted that the positive control animal data were excluded from the comparisons of the treated groups with the vehicle control for body weight, body weight gain and food consumption data. For those parameters, the positive controls were compared independently by ANOVA with the first ten animals/sex (Cohort I) in the vehicle control group. Furthermore, with regard to rotorod data, some groups had values that were all 60 seconds and a lack of variance precluded statistical comparison. In those groups, variance was introduced into the data by setting the value for the first animal in the group to 61 seconds. This data adjustment in no way affected the overall outcome of the analysis. A minimum significance level of p ≤ 0.05 was used in all comparisons.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
Three categories of clinical signs were observed: observations for dermal irritation at the application site, adverse systemic toxic effects, and incidental, sporadic or non-treatment related. The latter category included such observations as red material around nose/eyes, swollen cars, enlarged face, sore/swollen hindlimbs, broken/infected toenail, malocclusion and eye damage resulting from the trauma during orbital sinus bleeding. Clinical observations of the skin included sores, scabs, and scaly and/or red skin; these signs were not considered test article-related since they were 1) present around, and not on, the application site, 2) observed in the vehicle control and all dermally exposed dose groups, and 3) not observed in a dose-related incidence. Therefore, they were considered as having resulted from the adhesive of the Hill Top Chamber.
Salient clinical signs associated with the high dose level, especially females, consisted of hypothermia, (after the first dose only), hyperexcitability, piloerection, hunched posture, emaciation, as well as convulsions and death of two females (later replaced). After the high dose was lowered to 250 mg/kg/day, these signs (with the exception of hyperexcitability) subsided by week 4. However, during weeks 8 through 12 these signs (tremors, convulsions, emaciation, hunched posture and piloerection) returned and/or the incidence was increased in only the high dose females which had been fasted, anesthetized with a 70% CO2 30% 02 mixture and bled. The fact that these signs reappeared in only those animals exposed to orbital sinus bleeding is suggestive of an interaction between test article-treatment and repeated fasting, anesthesia and orbital bleeding. This phenomenon is most likely associated with fasting. Since fasting results in significant body weight loss and the rats were dosed based on non-fasted body weights, the overall dose on a mg/kg bw basis was higher than 250 mg/kg bw/day on the days the rats were fasted. Adverse signs attributed to treatment with acrylamide included hyperexcitability, piloerection, rough coat, excessive urination, wet/discolored inguinal fur, tremors, labored breathing, coldness to the touch, emaciation, hunched posture, ptosis and, especially, ataxia and dragging of hindlimbs/possible hindlimb paralysis.
Eight animals died during the study. Two high dose females died during the first week of dosing; these animals were replaced and, as a result, the high and mid dose levels were lowered. Two postive control males died during weeks 2 and 3, and one positive control female was sacrified in extremis during week 11. One low dose male and one vehicle control female died accidentally from suffocation due to wrapping/restraint during dosing at weeks 1 and 2, respectively. One mid dose male died spontaneously during week 2.

BODY WEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION
Body weights were significantly decreased in the high dose animals following the first week of treatment and for the duration of the study. Cumulative body weight gains were also significantly reduced, while reductions in weekly body weight gains were noted only during weeks 1, 5 through 8 and 10 for high dose males and at week 1 for high dose females. Reductions in food consumption were noted in the high dose males and mid and high dose females following the first week of treatment, but, with reduction of mid and high dose levels, subsequent mean weekly food consumption was unaffected by treatment, except during week 8 when high dose male mean food consumption was once again decreased. Since an intermittent dosing regimen was used for the positive control rats, body weight, body weight gain and food consumption parameters fluctuated between significant reductions or increases, with reductions being observed following dosing initiation and increases subsequent to cessation of treatment.

HAEMATOLOGY
No statistically significant effects on hematology parameters were observed in male test article-treated rats, whereas mean platelet counts were consistently significantly increased in mid and high dose females compared to vehicle control females at all timepoints, with the increases appearing dose-related. Also observed in high dose females were significant decreases in mean red blood cell counts at Weeks 0 and 12 (mean high dose female red blood cell count was decreased at Week 4 but the decrease was not found to be statistically significant), with concomitant increases in mean corpuscular volume and increased mean corpuscular hemoglobin (Week 8 only). Treatment-related alterations in erythrocyte morphology were also observed and consisted of polychromasia, poikilocytosis and anisocytosis with an increased severity of 2+ to 3+. The high dose animals also exhibited macrocytosis 1+ to 3+, Howell-Jolly bodies and platelet counts increased and occasionally abnormally formed. These observations were noted in both sexes in each of the test article treatment groups, albeit not in all animals and not in a consistently dose-related fashion. Polychromasia, poikilocytosis, anisocytosis and target cells of minimal severity, ;+, were seen in the vehicle controls; a 1+ severity for these observations are commonly seen in rats and are considered normal variations. With regard to differential white blood cell counts, mid dose males exhibited significantly increased mean absolute monocyte counts, but not relative counts, at Week 4. On Week 8, both absolute and relative mature neutrophil counts were increased in high dose males and females and mean absolute and relative lymphocyte counts were significantly decreased in high dose females. On Week 12, relative lymphocyte counts were decreased, while relative mature neutrophil counts were increased in the high dose females.

CLINICAL CHEMISTRY
Sporadic but statistically significant differences between treated and vehicle control rats were noted in alanine aminotransferase (males), chloride, creatinine, total protein, globulin, albumin, blood urea nitrogen and glucose at one or more of the sampling time points (weeks 4, 8, 12). The changes were slight and generally fell within the range of values found in the vehicle control group and were therefore considered of minimal clinical significance. Clinically relevant elevations in alkaline phosphatase, alanine aminotransfersse and aspartate aminotransferase levels were observed in the high dose females at one or more of the sampling time points.

URINALYSIS
Mean urinary sodium was significantly increased in mid dose females and potassium was significantly decreased in high dose female rats at the 6-week sampling timepoint. No other significant effects on these parameters were observed in male rats or in any rats at the 13-week timepoint. There were no remarkable findings with regard to individual urological observations.

NEUROBEHAVIOUR
Rotorod performance, forelimb and hindlimb grip strength and hindlimb foot splay were not affected by dermal exposure to N,N-Dimethylacrylamide. The positive control male and female rats, on the other hand, displayed statistically significant decreases in mean grip strength (forelimb at week 12 and hindlimb at weeks 4 and 12) and increases in mean hindlimb foot splay (males at weeks 4, 8 and 12 and females at week 8).

ORGAN WEIGHTS
Absolute brain weights in high dose female rats were significantly decreased compared to those of the vehicle control females; no other significant differences with regard to absolute organ weight were observed. With regard to organ-to-body weight ratios, high dose male and female rats had generally increased mean relative organ weights, with statistically significant increases observed in mean relative brain, kidney, liver, testes weights in high dose male rats compared to vehicle control males and in mean relative adrenal, heart, and kidney weights in high dose female rats compared to those of female vehicle control rats. In addition, relative spleen weights were also significantly increased in the mid dose male rats compared to vehicle controls. Fasted body weights were significantly decreased in the high dose group compared to their vehicle control counterparts. Thus, the increases in relative organ weights can be attributed, at least in part, to the decreased fasted body weights.

GROSS PATHOLOGY
Gross findings at necropsy were incidental, sporadic and not attributable to the test article treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Minimal nephropathy was observed in 6/10 high dose males (mean severity = 0.70 of 4.0 maximum possible), in 5/10 mid dose males (mean severity = 0.50 of 4.0 maximum possible) and in 3/10 low dose males (mean severity = 0.33 of 4.0 maximum possible) compared to 1/10 vehicle control males (mean severity = 0.10 of 4.0 of maximum possible). Minimal dilatation of the renal pelvis, also considered indicative of renal toxicity, was observed in 2/10 high dose males (mean severity = 0.30 of maximum 4.0) and in 4/10 mid dose males (mean severity = 0.60 of maximum 4.0). No treatment-related microscopic lesions were observed at the application site skin nor were any other significant treatment-related microscopic lesions observed in any other tissue examined. Microscopic examination of sciatic nerve tissue from positive control animals revealed multifocal axon degeneration in 7/7 male rats and in 9/10 female rats. These positive neuropathology responses were considered indicative of the adequacy of the assay to detect neuropathology. No such evidence of neuropathology was observed in any of the N,N-Dimethylacrylamide-treated rats.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

LOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: minimal renal toxicity in males, alterations in clinical pathology parameters in females

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Sprague-Dawley rats were initially administered dermal doses of 10, 200 and 500 mg/kg bw/day; however, after six days of administration, it was evident that the highest dose exceeded the maximum tolerated dose since it resulted in overt systemic toxicity (i.e., convulsions, dramatically reduced body weight, body weight gain and food consumption) and two deaths. Therefore, the mid and high dose levels were reduced to 75 and 250 mg/kg bw/day, respectively.

Salient clinical signs associated with the 500 mg/kg bw/day dose level, especially females, consisted of hypothermia (after the first dose only), hyperexcitability, piloerection, hunched posture, emaciation, as well as convulsions and death of two females (later replaced). After the high dose was lowered to 250 mg/kg bw/day, these signs (with the exception of hyperexcitability) subsided by week 4. However, during weeks 8 through 12 these signs (tremors, convulsions, emaciation, hunched posture and piloerection) returned and/or the incidence was increased only in the high dose females which had been repeatedly fasted, anesthetized with a 70% CO2 30 % O2 mixture and bled. The fact that these signs reappeared in only those animals exposed to orbital sinus bleeding is suggestive of an interaction between test article-treatment and repeated fasting, anesthesia and retro orbital bleeding. This phenomenon is most likely associated with fasting because fasting resulted in significant body weight loss and the rats were dosed on a non-fasted body weight bases. Thus, the overall dose on a mg/kg bw basis was higher than 250 mg/kg bw/day on the days the rats were fasted.

Treatment-related increases in neutrophil counts and concomitant decreases in lymphocyte counts were observed; generally, for dermal studies these changes are associated with an inflammatory response resulting from skin irritation. Although no treatment-related skin irritation was grossly or microscopically observed at the application site skin, these alterations in the white blood cell population are most likely associated with very minimal skin irritation. Alterations in red blood cell morphology indicative of anemia and associated reductions in red cell count with increased mean corpuscular volume and mean corpuscular hemoglobin and increased platelet counts were observed in high dose females.

Several clinical chemistry parameters were altered; however, these changes were slight and generally fell within the range of values found in the vehicle control group and were therefore considered of minimal clinical significance. Clinically relevant elevations in alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase levels were observed in the high dose females at one or more of the sampling time points; these changes may be indicative of early liver dysfunction. No histopathological alterations in any of the tissues examined could account for the changes in clinical pathology parameters. Transient alterations in urinary sodium (elevated in mid dose females) and potassium (reduced in high dose females) levels were noted at the week 6 time point; the clinical significance of this is unclear.

No significant gross lesions were observed at necropsy. Absolute brain weights were reduced in the high dose females at necropsy and were most likely caused by the early body weight depression (growth suppression) induced by the 500 mg/kg bw/day dose level and later maintained at the lower dose of 250 mg/kg bw/day. Organ-to-body weight ratios were generally increased in the high dose rats and fasted body weights were significantly decreased; thus, the increased in organ-to-body weight ratios can be attributed, at least part, to the decreased fasted-body weight.

Test article-related histopathological changes were confined to the kidneys of mid and high dose males and consisted of minimal nephropathy and renal pelvis dilatation. As expected, positive control rats exhibited multifocal axon degeneration in the sciatic nerve. No test article-related changes were observed in the tests (grip strength, foot splay and rotorod performance) used to evaluate peripheral neuropathy and no histopathological evidence of neurotoxicity was observed in the test article-treated rats. Peripheral neuropathy was detected by the battery of tests in the positive control rats and clinical signs of toxicity consisting of ataxia, dragging of hindlimbs, hyperexcitability and piloerection were noted. In conclusion based on the results of this study, the no effect level for dermal application of N,N-Dimethylacrylamide is 10 mg/kg bw/day.