N,N'-ethylenebis[N-acetylacetamide]

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: The test was done according to the respective OPPTS Guideline and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethylenebis[N-acetylacetamide]

EC Number:

234-123-8

EC Name:

N,N'-ethylenebis[N-acetylacetamide]

Cas Number:

10543-57-4

Molecular formula:

C10H16N2O4

IUPAC Name:

N,N'-ethylenebis[N-acetylacetamide]

Details on test material:

- Name of test material (as cited in study report): Tetraacetylethylenediamine, TAED
- The test material was recieved from the sponsor on March 3, 2000
- Storage condition of test material: 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 ., Raleigh, North Carolina
- Age at study initiation: approximately eight-week-old
- Weight at study initiation:
- Fasting period before study: none
- Housing: Upon receipt, animals were housed 2 per cage by sex in polycarbonate cages measuring 9 x 8.5 x 8 inches (length x width x height)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: seven days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): between 66 and 77°F
- Humidity (%): between 30 and 70%
- Air changes (per hr): 10 to 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The vehicle, 1 % carboxymethylcellulose (CMC), was prepared in deionized water, and refrigerated . Prior to use, the vehicle formulation was equilibrated to room temperature and stirred well. The required amount of test article for each animal was weighed on an appropriate scale using a weigh boat and a spatula. The weighed test article was mixed with the CMC and stirred to form a paste. The weigh boat was emptied by wiping with a 2 in x 2 in gauze pad which was placed on a plastic wrap ensuring that the compound was exposed. The gauze pad with the test article formulation was placed on the dorsal surface covering the shaved area, and secured with vet wrap which was held in position by taping the ends . Each animal was exposed daily for six hours. At the end of the exposure, the animals were unwrapped, and the exposure area was wiped with gauze moistened with 1% CMC. If necessary, Elizabethan collars were used. The rats were given the appropriate dosing formulation via the dermal route once daily for at least 90 days; and dosing continued to the day prior to necropsy. The control group (0 mg/kg/day) received the vehicle only.
Approximately 24 hours prior to the first dose, an area approximately 10% of the body surface starting at the scapular and extending to the ileum was shaved for each animal. Additional shaving was done weekly or as needed for each animal . Control animals were dosed first, followed by each successive higher dose-group. Individual dose amount was based on the most recently recorded body weight. The test article was administered dermally because this is the expected route of human exposure. Centrilobular hypertrophy in the high-dose animals served as evidence of absorption.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 200, 2000 mg/kg bw/d
Basis:
nominal per unit body weight

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose were selcted based on the existing toxicity data

Positive control:

none

Examinations

Observations and examinations performed and frequency:

OBSERVATIONS AND RECORDS
All of the rats were observed for mortality, moribundity and clinical signs of toxicity twice daily by cageside observation, at least six hours apart each day. Animals were observed weekly for erythema and edema. Animals were observed for clinical signs at the end of each daily exposure. Rats were removed from their cages and detailed physical examinations were performed at randomization and weekly thereafter. Individual body weights were recorded at randomization, prior to dosing, and weekly thereafter . Food consumption was measured weekly. Ophthalmic examinations were performed on all animals prior to dosing and on all animals in Groups 1 and 4 prior to termination, and included examination of the anterior portion, optic media, and ocular fundus .

CLINICAL PATHOLOGY
Hematology and serum chemistry samples (approximately 1 mL each) and coagulation samples (approximately 2 mL) were obtained from all animals via puncture of the orbital sinus with 70 %CO2/30 % O2 as anesthesia prior to termination. All animals were food fasted overnight with water available . EDTA and sodium citrate were used as anticoagulants for the hematology and coagulation samples, respectively . Samples were sent to Lab Corp (Chevy Chase, Maryland), (PAI) for analysis.
Hematology determinations were conducted on a BioChem Immunosystems 9010 analyzer. Serum chemistry was conducted on a Boehringer Manheim Corporation (BMC) Hitachi 717. Coagulation parameters were conducted on an ACL 100 Coagulation Analyzer. The following parameters were evaluated:

Hematology: Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Platelet count (PLT), Leukocyte differential, Mean Corpuscular Hemoglobin Concentration (MCHC), Mean corpuscular volume (MCV), Mean platelet volume (MPV), Mean corpuscular hemoglobin (MCH), Leukocyte count (WBC), Cellular Morphology

Blood Chemistry: Sodium (NA), Potassium (K), Chloride (CL), Total Protein (TPROT), Albumin (ALB), Calcium (CA), Phosphorus (P04), Total bilirubin (TBIL), Urea nitrogen (BUN), creatinine (CREAT), SGOT/AST, SGPT/ALT, Globulin (GLOB), Alkaline Phosphatase (ALP), Cholesterol (CHOL), Triglycerides (TRIG), Albumin/Globulin Ratio (A/G), Glucose (GLU)

Coagulation: Activated Partial Thromboplastin time (APTT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

TERMINATION
Following at least 90 days of treatment, all animals were weighed, euthanized by carbon dioxide asphyxiation, and exsanguinated. The animals were necropsied as close as possible to the time of sacrifice. Necropsies were conducted on each animal by trained personnel and included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents.

ORGAN WEIGHTS
The following organs were weighed wet from all animals :
Liver, Adrenals, Epididymides/ovaries, Spleen, Heart, Kidneys, Testes/Uterus, Thymus, Brain

TISSUE PRESERVATION
The following tissues from each necropsied animal were preserved in 10% neutral buffered formalin:
Adrenals Aorta, Bone marrow (sternum) Bone, Brain (3 levels), Cecum, Colon Duodenum, Epididymides, Esophagus, Eyes with optic nerve, Gross lesions, Ileum, Heart, Kidneys, Jejunum, Larnyx, Lungs, Liver, Mammary glands, Lymph nodes (mesenteric and mandibular), Nerve (sciatic), nasal cavity, Ovaries and fallopian tubes, pancreas, Pharynx, Pituitary, Parathyroid/thyroid, Prostate, Rectum, Salivary glands, Seminal vesicles, Skeletal muscle (gastrocnemius), Skin, Spinal cord (3 levels), Spleen, Sternum, Stomach, Testes, Thymus, Vagina, Trachea, Urinary bladder, Uterus, Thyroid

HISTOPATHOLOGY
All preserved tissues from animals in Groups 1 and 4 were embedded in paraffin, stained with hematoxylin and eosin, and examined microscopically by the board-certified pathologist at Experimental Pathology Laboratories, Inc .

Statistics:

Mean body weight, body weight gain, food consumption, clinical pathology, and organ weight data were analyzed using tests for homogeneity of variances, ANOVA and Dunnett's TTest, if applicable, at the 5.0% one-tailed probability level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were four unscheduled deaths during the study (one control male, two 200mg/kg females and one 2000 mg/kg female) . The cause of death for each of these rats could not be attributed to administration of TAED.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights for the animals at 20, 200 and 2000 mg/kg/day were generally comparable (with no statistical differences) to the respective control group mean value, and exhibited normal growth and development patterns .
Mean body weight gains were significantly increased for the 200 mg/kg/day males, and decreased for the 200 and 2000 mg/kg/day females at the Week 1 to 2 body weight change interval compared to the respective control mean value . The noted decreases did not appear to be the result of TAED administration. The remaining body weight gain values were generally comparable between the treated and control groups for each sex.
FOOD CONSUMPTION
Mean food consumption values for the animals at 20, 200 and 2000 mg/kg/day groups were generally comparable (with no significant differences) to the respective control mean value during the course of the study.

OPHTHALMOSCOPIC EXAMINATION
All animals examined exhibited no visible ophthalmoscopic lesions .

HAEMATOLOGY and CLINICAL CHEMISTRY
There were no treatment-related findings in the clinical pathology data. There was a significant increase in the mean MPV value for the 2000 mg/kg/day males which may have occurred as a result of an increase in platelet size, and not as a result'of TAED administration. A significant decrease was noted for the mean triglyceride concentration values for the 20 mg/kg/day females which was mild and not related to administration of TAED.

ORGAN WEIGHTS
Mean fasted terminal body weights were generally comparable between the control and treated groups for the male and females. Mean absolute organ weights, organ-to-body weight percentages, and organ-to-body weight ratios for the animals at 20, 200 and 2000 mg/kg/day were generally comparable (with no statistical differences) to the respective control group mean value .

GROSS PATHOLOGY
The gross pathology findings noted were sporadic in nature and are of the type commonly seen in rats this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
A test article-related lesion in the liver was observed in 8/10 males and 4/10 females at 2000 mg/kg/day. The lesion was centrilobular hypertrophy (cytomegaly) and was considered minimal in degree of severity . Minimal centrilobular hypertrophy was not noted in the lower dosed rats that died on study or had gross lesions associated with the liver. Hemorrhage, necrosis, chronic inflammation in the capsule and subcapsular area were diagnosed in several animals including controls . These lesions were the result of the gauze wrapping and unrelated to the test-article exposure.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: minimal centrilobular hypertrophy at 2000 mg/kg

2

Dose descriptor:

LOEL

Effect level:

2 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: minimal centrilobular hypertrophy in 8/10 males and 4/10 females

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Tetraacetylethyldiamine (TAED) when administered to Sprague-Dawley rats once daily for six hours to the skin by dermal application for 90 days at dose levels of 0, 20, 200, and 2000 mg/kg/day resulted in minimal centrilobular hypertrophy (cytomegaly) in 8/10 and 4/10 males and females, respectively at the high-dose only. No other treatment-related findings were noted. Based on the effects in this study the no-effect-level is equal to or greater than 200 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

The only effect observed in the present study was characterized by centrilobular hypertrophy and was graded minimal in degree of severity. No increase in liver weight was found. This type of effect is a physiological response to the need for increased metabolic activity and represents an adaptive effect.
As the centrilobular hypertrophy was only of minimal degree with no change of liver weight, this effect is regarded as non-adverse.
Thus, A NOAEL of 2000 mg/kg bw can be derived from the study.

Executive summary:

Subchronic dermal toxicity was studied in groups of 10 female and 10 male Sprague-Dawley rats for 90 days according to OPPTS Guideline No. 870.3250. TAED pasted in 1% carboxymethylcellulose (CMC) was applied dermally once daily for 6 hours at 0, 20, 200, and 2000 mg/kg BW/d. A gauze pad with the test article formulation was placed on the dorsal surface covering the shaved area of approximately 10% of the body surface and secured with vet wrap held in position by taping the ends. At the end of the exposure, the animals were unwrapped and the exposure area was wiped with gauze moistened with 1% CMC. Following the treatment period, all animals were weighed then euthanised by carbon dioxide asphyxation and ensanguined and subsequently necropsied. One control male, two 200 mg/kg BW/d females, and one 2000 mg/kg BW/d female did not survive to termination. These deaths were considered to be the result of the wrapping procedure and not the effect of TAED since no clinical signs suggestive of TAED involvement were noted prior to death. No treatment related findings were noted at 0, 20, or 200 mg/kg BW/d in clinical observation, body weight and food consumption data, ophthalmologic findings, clinical pathology findings, gross necropsy, and organ weights. The only treatment-related finding was at the high dose, 2000 mg/kg BW/d, which was hypertrophy of centrilobular hepatocytes in 8/10 and 4/10 females and males, respectively, and graded minimal in degree of severity.

Based on the effects in this study the NOEL would be equal or greater than 200 mg/kg BW/d in both males and females. The NOEL of 200 mg/kg BW/d is a result of the large factor of 10 between dose levels. Based on the minimal effects observed in this study an actual NOEL much closer to 2000 mg/kg BW/d can be assumed and a NOAEL of 2000 mg/kg BW/d can be derived..