N,N'-ethylenebis[N-acetylacetamide]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-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 in accordance to the test guidelines in place at the time when the study was performed and GLP. However, as the OECD guideline currently in place contains some parameters which had not to be examined according to the earlier version of the guideline and thus, were not included in the present study, the study is rated as reliable with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 12 May 1981

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): tetra-acetyl-ethylen-diamin

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 Wiga, Sulzfeld
- Age at study initiation (begin of dosing): ca. 6 weeks
- Weight at study initiation (begin of dosing): 141-176 g (males), 118-155 g (females)
- Housing: in groups of 2-3 animals in Makrolon cages Type III
- Diet (e.g. ad libitum): Altromin 1324 DK, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.8
- Humidity (%): 49-56
- Air changes (per hr): not indicated
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 20.01.1986 (1st application) To: 24./25.04.1984 (main groups) and 22.05.1986 (recovery groups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepaired daily immediately before application. The test item was administered as a solution in 1% CMC and 0.5 % Cremophor.

VEHICLE
- Justification for use and choice of vehicle (if other than water): formulation of a homogenous and stable suspension of the test item in the vehicle.
- Concentration in vehicle: 0, 9, 25, and 80 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 90, 250, 800 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex and dose and a recovery control and recovery high dose group consisting of 5 male and 5 female rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses were selected to allow a derivation of a NOAEL
- Rationale for selecting satellite groups: satellite groups were included to determine the reversibility of eventually occurring toxic effects
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

none

Examinations

Observations and examinations performed and frequency:

MORTALITY AND CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


BODY WEIGHT GAIN:
- determined from week 0-6, 6-13 and 0-13


FOOD CONSUMPTION AND WATER CONSUMPTION: Yes
- Food consumption determined as group mean values in g/rat/day
- Water consumption determined as group mean values in ml/rat/day


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at one day before sacrifice
- Dose groups that were examined: control and high dose group


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 6 weeks and at the end of the in life phase
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals per sex and dose
- Parameters examined: erythrocyte count, hematocrit, mean corpuscular volume (MCV), hemoglobin, leukocyte count, platelet count, hemogram: stab cells and segmented cells, lymphocytes, eosinophils, monocytes, basophils, myelocytes, juveniles, diverse


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 6 weeks and at the end of the in life phase
- Animals fasted: No data
- How many animals: 10 animals per sex and dose
- Parameters examined: gamma-glutamyl transferase, alkaline phosphatase, aspartate aminotransaminase, alanine aminotransaminase, sodium, potassium, glucose, urea, protein, calcium, creatinine, cholesterol, chloride, bilirubin


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: brain, testes, heart, liver, spleen, adrenals, kidneys, thymus
HISTOPATHOLOGY: Yes (aorta, bile duct, eyes, duodenum, jejunum, ileum, caecum, colon, rectum, anus, stomach - forestomach, fundus, pylorus, brain - cerebellum and cerebrum, bladder, skin, heart, testes, liver, trachea and lungs, lymph nodes - mesenteric and mandibular, mammary gland, spleen, epididymides, peripheral nerve, kidney, ovary, pancreas, prostate, salivary gland, seminal vesicles, thyroid and parathyroid, oesophagus, skeletal muscle, thymus, uterus, vagina, tongue, vertebrae cervicales, sppinal cord, bone marrow)

Statistics:

T-test (body weight, haematology, clinical chemistry)
U-test (organ weights)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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
Mortality did not occur. Salivation observed in the animals of the high dose group was the only clinical finding.

BODY WEIGHT AND WEIGHT GAIN
Total body weight gain was decreased in male and female rats of all high dose groups.

FOOD AND WATER CONSUMPTION
Water consumption was increased in males of the 250 and 800 mg/kg/d group and in all female test groups,
whereas food consumption was not affected.

OPHTHALMOSCOPIC EXAMINATION
Eye examination revealed no compound related findings.

HAEMATOLOGY
Slightly decreased haematocrit values in all male test groups and an increase in leukocytes in females of the high dose were observed. The changes are not regarded as substance related effects.

CLINICAL CHEMISTRY
Changes in biochemical parameters considered to be compound-related comprised increased protein values in male and female rats of the high dose group and increased cholesterol values in female animals treated with 800 mg/kg.

ORGAN WEIGHTS
At the dose level of 250 mg/kg relative liver and testes weights were significantly increased in male rats. At 800 mg/kg absolute and relative liver weights of both sexes and relative adrenal and testes weights in male rats were statistically significantly increased.

GROSS PATHOLOGY
No compound related macroscopically visible findings were present at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination revealed centrilobular hypertrophy of hepatocytes in all high dose animals. This effect reversed completely within
the 28 day recovery period. In the animals of the low and mid dose group centrilobular hypertrophy was borderline in some rats and not considered a clear substance related effect, since this finding was also present in some control animals of the recovery group.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)


HISTORICAL CONTROL DATA (if applicable)


OTHER FINDINGS
none

Effect levels

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

At the dose of 800 mg/kg bw/d the liver was identified as target organ.

Increased liver weights were correlated with histopathological findings. 

The histopathological changes of the liver were fully reversible.

The increased values for leucocytes, protein and cholesterol were also regarded as substance related effects. In addition, the adrenal and gonadal weights were increased and body weight gain was reduced. At 250 mg/kg bw/d relative gonadal and liver weights were increased. Also, a retarded (statistically not significant) body weight gain was observed. This dose level was regarded as the boarderline of systemic tolerance. At 90 mg/kg bw/d no toxicologically relevant systemic effects occurred. It cannot be totally excluded that the observed minimal centrolobular hypertrophy of hepatocytes at this dose level was related to treatment with the substance. However this finding was not substantiated by any other changes and minimal centrolobular hypertrophy was also seen in some recovery control animals parameter. Thus, 90 mg/kg bw/d was considered as the dose without relevant toxicological effect.

Applicant's summary and conclusion

Conclusions:

Clear substance related adverse effects occurred at the dose level of 800 mg/kg BW/d.
Changes in organ weights were present at 250 mg/kg BW/d.
No adverse effects were observed at 90 mg/kg BW/day.
A “no-adverse-effect-level” of 90 mg/kg BW/day was established.

Executive summary:

Subchronic oral toxicity was evaluated in groups of 10 male and 10 female Sprague-Dawley rats receiving TAED by gavage at daily dose levels of 0, 90, 250 and 800 mg/kg BW for 90 days (Henkel, 1987). A recovery control and high dose group consisting of 5 male and 5 female rats was terminated 28 days after the 90 day treatment period.
Mortality did not occur. Salivation observed in the animals of the high dose group was the only clinical finding. Water consumption was increased in males of the 250 and 800 mg/kg BW/d group and in all female test groups, whereas food consumption was not affected. Total body weight gain was decreased in male and female rats of all high dose groups. Slightly decreased haematocrit values in all male test groups and an increase in leukocytes in females of the high dose were observed. Changes in biochemical parameters considered to be compound-related comprised increased protein values in male and female rats of the high dose group and increased cholesterol values in female animals treated with 800 mg/kg BW. Eye examination revealed no compound related findings. At the dose level of 250 mg/kg BW relative liver and testes weights were significantly increased in male rats. At 800 mg/kg BW absolute and relative liver weights of both sexes and relative adrenal and testes weights in male rats were statistically significantly increased. No compound related macroscopically visible findings were present at necropsy. Histopathological examination revealed centrilobular hypertrophy of hepatocytes in all high dose animals. This effect reversed completely within the 28 day recovery period. In the animals of the low and mid dose group centrilobular hypertrophy was borderline in some rats and not considered a clear substance related effect, since this finding was also present in some control animals of the recovery group.
Clear substance related adverse effects occurred at the dose level of 800 mg/kg BW/d. Changes in organ weights were present at 250 mg/kg BW/d. No adverse effects were observed at 90 mg/kg BW/day. Hence, a “no-adverse-effect-level” of 90 mg/kg BW/day can be deduced.