Acetoacetanilide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed OECD and GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acetoacetanilide

EC Number:

202-996-4

EC Name:

Acetoacetanilide

Cas Number:

102-01-2

Molecular formula:

C10H11NO2

IUPAC Name:

3-oxo-N-phenylbutanamide

Details on test material:

- Name of test material (as cited in study report): Acetoacetanilide

Test animals

Species:

rat

Strain:

other: Charles River Crl: CD(R) SD BR VAF/Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Total number 68 (34 male and 34 female)
- Age: 28 days +/- 1 day
- Weight range: 63 - 85 g
- Free access to tap water and Biosure LAD 1 diet.

- Temperature: 21.5 - 21.9 °C
- Rel. humidity: 47.9 - 53.2 %
- Artificial light: 12 hrs per 24 hrs period
- Acclimation: 23 days

- Housing: in groups of five
- Identification: by cage number, ear punch and tattoed tail mark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous methylcellulose

Details on oral exposure:

- Dosage volume: 10 ml/kg/day
- Test substance in 1% aqueous methylcellulose at concentrations of: 8.5, 1.0, 0.12 % (w/v)
- Prior to dosing, the test substance formulations were mixed by inversion (x20). Subsequent mixing using a magnetic stirrer was carried out for a period of at least 10 minutes before dosing commences. Dosing was completed within one hour of the commencement of stirring.
- Dosage levels: 0, 12, 100, 850 mg/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration analyses of formulations prepared for administration on Days 1 and 8 were conducted.

Duration of treatment / exposure:

- once daily for 28 days

Frequency of treatment:

- once daily for 28 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
12 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
850 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1: Control Group: 10 male & 10 female
Group 2: Dosage level 12 mg/kg/day: 5 male & 5 female
Group 3: Dosage level 100 mg/kg/day: 5 male & 5 female
Group 4: Dosage level 850 mg/kg/day: 10 male & 10 female

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observation during experimental period: Twice daily (Early each work day and late afternoon; during public holidays and weekends last daily check be mid-day)


BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing and subsequently at weekly intervals


FOOD CONSUMPTION: Yes
- The quantity of food consumed in each cage was measures at weekly intervals


WATER CONSUMPTION: Yes
- Daily monitoring by visual appraisal.
- Following several days of dosing an apparent increase in water consumption was noted for animals in the high dosage group in comparison with the controls. Gravimetric measurement of water consumption was therefore initiated during week 3 of tratment and also during the recovery period.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: - Blood was withdrawn prior to termination (week 4). Additional investigations where carried out during week 6 for recovery animals
- Anaesthetic used for blood collection: Yes (identity: ether)
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see previous section
- Animals fasted: Yes

Sacrifice and pathology:

- Terminal studies: After 28 days of treatment (day 29) five male and five female rats selected from group 1 and 4 and all remaining animals from groups 2 and 3 were killed by carbon dioxide asphyxiation and a complete autopsy untertaken.
- Microscopic examination: In the first instance, microscopic examinations were carried out for all rats of group 1 and group 4 killed on day 29. In view of changes seen at this initial examination, microscopic pathology was extended to include the kidneys, liver and spleen from all animals in the intermediate and low dosage groups and recovery system.
In view of changes seen in hematological parameters during the fourth week of treatment, bone marrow smears were prepared from each animal killed on day 29. Slides prepared from rats in the control and high dosage groups were examined in the first instance and bone myelograms conducted. Changes in the erythroid series of cells were noted and examinations were therefore extended to rats of the lower dosage groups. Bone marrow smears from recovery animals killed on day 43 were also prepared and examined

Statistics:

- All statistical analyses were carried out separately for males and females.
- Bodyweight data were analysed using weight gains.

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):

effects observed, treatment-related

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- No mortalities
- For male and female rats treated at 850mg/kg/day pilo-erection, hunched posture, pallor of the extremities, abnormal gait (waddling) and darkened eyes, often accompanied by lethargy, ptosis and increased salivation, were noted throughout the treatment period. The cage tray paper was noted to be wetter than normal during week 3 and 4. These signs ameliorated over the recovery period, with only pilo-erection and hunched posture being evident at termination (day 43). The cage tray paper was still wetter than normal during the 14d recovery period
- Pilo erection, often accompanied by increased salivation and hunched posture was noted during the treatment period for male and female rats treated at 100mg/kg/day. Abnormal gait (waddling) was noted during weeks 3 and 4.
- No clinical signs were recorded for rats treated at 12mg/kg/day


BODY WEIGHT AND WEIGHT GAIN
- Lower body weight gains were recorded for rats receiving 850mg/kg/day (males: significant during weeks 1 to 5 females: significant during 3 to 5 inclusive). By week 6 body weight gain was back to normal in the high-dosage group
- 100mg/kg/day and 12mg/kg/day were comparable to control

FOOD CONSUMPTION
- Male rats, 850mg/kg/day: lower food consumption; females of the same group: slightly reduced
- during recovery: food consumption back to normal

WATER CONSUMPTION
- Male and female rats, 850mg/kg/day: increased water consumption starting at week 3
- Still increased water consumption in this group at week 6

HAEMATOLOGY
- 850 mg/kg/day: At week 4 rats showed low packed cell volume (PCV), low haemoglobin concentration (Hb), low red blood cell count (RBC), high mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), incidences of polychromasia, anisocytosis and nucleated red blood cells, raised methaemoglobin concentrations and a high neutrophil count.
- 100 mg/kg/day: At week 4 rats showed low PCV, low Hb, low RBC, raised methaemoglobin (males only) and high neutrophil count (males only) were recorded at Week 4.
- After the recovery period all parameters improved. However, statistically significant differences comprising low PCV (males only), low RBC, high MCHC, high MCV and a high neutrophil count (females only) were again recorded for rats from the high dosage group (850 mg/kg/day). The lymphocyte count was also raised for female rats of this group. The methaemoglobin concentration for male and female rats from the high dosage group was similar to that for control animals.

CLINICAL CHEMISTRY
- Bilirubin elevated in males and females of the high dosagae group (significant)
- slightly elevated in mid dosage group
- potassium was dose-dependent elevated, significant in females of mid and high dosage groups
- creatinine signif. raised for male rats of the mid and high dosage groups
- albumin was signif. raised for females of the high dosage
- after recovery, all parameters improved

BONE MYELOGRAMS (WEEK 5)
- Signif. higher values for the erythroid series for male and female rats of the high dosage group
- Some of parameters of the erythroid series were still evaluated for female rats of both lower dosage groups
- Female rats of all dosage groups: myeloid:erythroid ratio was dose related decreased
- For female rats at 12mg/kg/day there was no clear indication of a treatment related effect as the values were similar to those obtained two weeks later in control females of the recovery group
- Lymphocytes were decreased for male and female rats of the high dosage group and neutrophil metamyelocytes of the female high dosage group


ORGAN WEIGHTS
- Spleen (signif. higher for high dose male and female; mid dose male)
- Liver (higher for high dose male, signif. higher in female rats)
- Kidney (signif. higher for males of high and mid dose)
- Adrenal weights signif. lower in high and mid dose (was not found microscopically, hence not regarded as treatment related)
- After recovery: general improvement, yet still partly significant higher weights. Ovaries weighed signif. more after recovery, not at end of exposure

GROSS PATHOLOGY
- High dose: enlarged spleen
- Mid dose: darkened spleen
- after recovery partly improvement

HISTOPATHOLOGY: NON-NEOPLASTIC
- Treatment-related effects were noted in the livers (centrilobular hepatocyte enlargement, pigmented Kupffer cells and extramedullary haemopoiesis), spleens (haemosiderosis) and kidneys (brownish pigment and, in males only, eosinophilic droplets in the cortical tubules) of rats treated at 850 mg/kg/day.
- Brownish pigment in the cortical tubules of the kidneys was also noted in the majority of female rats treated at 100 mg/kg/day, and haemosiderosis was noted in the spleens of the majority of rats of this intermediate dosage group.
- Following the 2-week recovery period, moderate to minimal treatment-related changes were seen in the spleen and kidneys of rats from the high dosage group.

Effect levels

Dose descriptor:

NOEL

Effect level:

12 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Group/ dosage (mg/kg/day)	Hb (g/dl)	Met Hb (%Hb)
Control male	15.3	2.36
12 male	15.2	2.42
100 male	14.2	3.50
850 male	12.6	6.64
Control female	14.7	2.36
12 female	14.2	1.86
100 female	13.3	2.02
850 female	12.7	4.28

Applicant's summary and conclusion

Conclusions:

The no effect level of the test substance was found to be 12 mg/kg/day.

Executive summary:

Acetoacetanilide was formulated daily as 8.5%, 1.0% and 0.12% w/v suspensions in 1% aqueous methylcellulose (MC) and administered to rats by oral gavage at dosage levels of 12, 100 and 850 mg/kg/day. Treatment was carried out for twenty-eight consecutive days. Control animals similarly received 1% MC alone. Animals were killed following the 28-day treatment period or, for additional animals of the control and high dosage groups, following a further 2-week post treatment recovery period.

Acetoacetanilide caused typical symptoms of haematotoxicity including changes of haematological parameters and morphological effects in liver, spleen and kidneys. The most prominent histopathological finding was hemosiderin deposition in the kidneys of male rats in the mid and high dose group.

The no effect level of Acetoacetanilide was found to be 12 mg/kg/day.

Following the 2 -week recovery period most of the effects seen in the high dose group disppeared or were less pronounced. But moderate to minimal treatment-related changes were still seen in the spleen and kidneys.