Methyl acetoacetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl acetoacetate

EC Number:

203-299-8

EC Name:

Methyl acetoacetate

Cas Number:

105-45-3

Molecular formula:

C5H8O3

IUPAC Name:

methyl 3-oxobutanoate

Details on test material:

- Name of test material (as cited in study report): methyl acetoacetate
- Physical state: transparent and colorless liquid
- Analytical purity: 99.4 %
- Lot/batch No.: Lot No. 602006
- Storage condition of test material: at low temperatures, protected from light

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 Japan, Inc
- Age at study initiation: 10-week old
- Weight at study initiation: 373.2 - 428.2 g (males) and 227.7 - 255.3 g (females)
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): pellets (MF, manufactured by Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): water containing sodium hypochlorite (approx. 2 ppm)
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±2
- Humidity (%): 55±10
- Air changes (per hr): 13 - 15 times
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Using Japanese Pharmacopoeia (JP) water for injection (manufactured by Otsuka Pharmaceutical Factory, Inc., Lot No. 6D77) as a medium, the test article was dissolved therein so that the concentrations were 1, 3 and 10 w/v% to prepare liquids to be administered. The resulting liquids to be administered were preserved at room temperature.
- Incidentally, the concentrations of the liquids to be administered were measured during the week of starting administration to confirm that they were within ±5 % of each preset value.

TREATMENT
- The test article was given through a gastric tube once daily to males for total 49 days (for a 14-day pre-mating period, followed by 35 days including mating period) and females throughout a 14-day pre-mating period, additional mating (max. 14 days) and gestation periods to day 3 of lactation.
- The defined dose level was 10 mL/kg, which was used to obtain individualized doses based on the latest body weights in the case of all males and a part of females (prior to and during mating period), or body weights on day 0 of gestation in the case of females after successful copulation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before starting administration, it was ascertained that 0.1 and 20 w/v%-solutions, which were prepared according to this method, were stable at room temperature under diffused light for at least 8 days.

Duration of treatment / exposure:

males: 49 days;
females: from day 14 prior mating until day 3 of lactation

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Study design:
The test compound was administered orally to male and female rats in a pre-mating period, followed by additional days including mating period in the case of males, or continued administration throughout mating and gestation periods to day 3 postpartum in the case of females, in order to evaluate repeat dose toxicity to parental animals, as well as effects on fertility and development/growth of offspring.

- Dose selection rationale:
Dose levels were defined according to the results of preliminary test. More specifically, as a result of a 2-week repeated administration of the test article at dose levels of 100, 300 and 1000 mg/kg, no changes were seen with regards to general medical conditions, body weights, food consumption, hematology, blood biochemistry, autopsy and organ weights. Hence, for dose levels in this test, a high dose was defined to be 1000 mg/kg, which is a limit dose in conformity with OECD guideline; and similarly, medium and low doses were determined to be 300 and 100 mg/kg, respectively, which were obtained sequentially by dividing the high and medium doses by a common ratio of ca. 3.

- Rationale for animal assignment (if not random):
The stratified continuous random sampling method was adopted for grouping, based on body weights on the day before starting administration

- Post-exposure period: one day

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In all cases of both males and females, observation of general medical conditions and check of viability were conducted more than twice a day.

BODY WEIGHT: Yes
- Time schedule for examinations:
< Males were measured twice a week during administration period.
< Females were weighed twice a week during pre-mating administration and mating periods, on days 0 (the day when gestation was confirmed), 4, 7, 10, 14, 17 and 21 of gestation, and days 0 (parturition day) and 4 of lactation.

FOOD CONSUMPTION
< Food consumption of males was measured twice a week throughout the whole administration period except during mating period.
< As for females, that amount was measured twice a week during pre-mating administration period, on days 1, 4, 7, 10, 14, 17 and 21 of gestation, and on days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- After the completion of administration period, all males were fasted for more than 18 hours, and then subjected to laparotomy under anesthesia by intraperitoneal administration of pentobarbital sodium, in order to collect blood from the abdominal caudal vena cava.
- The collected blood samples were treated with EDTA-2K (EDTA-2K-added blood) to measure
white blood cell count (Electrical resistance detection method),
red blood cell count (Electrical resistance detection method),
hemoglobin content (Oxyhemoglobin method),
hematocrit (Detection method for high-level hemocyte wave pulse) and
platelet count (Electrical resistance detection method),
using an automated multi-channel blood cell counter (SysmexCC-780, manufactured by Toa Medical Electronics); then
mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and
mean corpuscular hemoglobin concentration (MCHC) were obtained from these measured values.

CLINICAL CHEMISTRY: Yes
- Following the hematological examination, the collected blood samples were left standing at room temperature for approximately 60 minutes, then underwent centrifugation at 3000 rpm for 10 minutes;
- the serum samples thus obtained were measured for
total protein (Biuret method),
albumin (BCG method),
A/G ratio (obtained from total protein and albumin),
total billirubin (Alkaline azo-billirubin method),
GOT (Karmen method),
GPT (Wróblewski-La Due method),
γ-glutamyl trans-peptidase (γ-GTP, L-γ-glutamyl-DBHA substrate method),
alkaline phosphatase (p-nitrophenylphosphate substrate method),
total cholesterol (COD-DAOS method),
triglyceride (GPO-DAOS and glycerin elimination methods),
phospholipid (enzymatic and DAOS chromogenic methods),
glucose (Glucokinase/G-6-PDH method),
urea nitrogen (Urease-GIDH method),
creatinine (Jaffé method),
inorganic phosphorus (Molybdic acid direct method) and
calcium (OCPC method), using an automated analyzer (736-10, manufactured by Hitachi, Ltd.).
The serum samples were also measured for Na (Electrode method),
K (Electrode method) and
Cl (Coulometric titration method), using an electrolyte analyzer (PVA-α III, manufactured by Analytical Instruments).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
see chapter 7.8.1 of this document

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Following collection of blood samples after the completion of administration period in the case of males, or on day 4 of lactation in the case of females, rats were killed by exsanguination via transection of the external iliac artery under anesthesia with ether.
- Subsequently, they were dissected for gross observation of various organs and tissues; in addition, females were examined for numbers of corpus lutea and implantation sites.
- Following the autopsy, brain, heart, lungs (including bronchi), thymus, liver, spleen, kidneys, adrenal glands, testes, epididymes and ovaries were removed, in order to measure organ weights (absolute weights) and obtain the ratios of respective organs to body weight (relative weights) based on body weights on the day of autopsy.
- In addition to the weighed organs, gross abnormal sites were removed and fixed in a 10 %-neutral buffered formalin solution (testes and epididymes were prefixed in Bouin fluid).

HISTOPATHOLOGY: Yes
- As for control and 300 mg/kg groups, paraffin sections were prepared from brain, heart, lungs (including bronchi), thymus, liver, spleen, kidneys, adrenal glands, testes, epididymes and ovaries in the usual manner, in order to stain with hematoxylin and eosin (HE) and observe under a light microscope. - Additionally, one case with gross splenic hypertrophy in 100 mg/kg group was suspected of myelogenic leukemia; accordingly thigh bones (marrow), liver and mesenteric lymph nodes were also removed to conduct the same examination.
- Moreover, liver, spleen and thigh bones were stained with esterase. In the cases of death, kidneys and lungs were stained with PTAH.
- All gross abnormal sites (excluding inversions of intra-thoracic and intraperitoneal organs) underwent histopathological examination.

Other examinations:

Other examinations were performed for reproductive toxicity and are included in chapter 7.8.1 of this document.

Statistics:

For each group, mean value and standard deviation of body weights, food consumption, hematology, blood biochemistry, organ weights were obtained. Then the uniformity of dispersion was tested first by using the Bartlett method between control group and each of other administration groups which were exposed to the test article. The Dunnett’s multiple comparison test (when the dispersion was uniform) and the Steel’s multiple comparison test (when the dispersion was not uniform) were used respectively to perform a comparison with control group. The Mann-Whitney U test for pathology was used to perform a comparison between control group and each of other administration groups. In each case, the levels of significance were set to 1 and 5 %.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
- With regard to males, no death occurred, and no changes in general medical conditions were seen in each group.
As for females, one case in 300 mg/kg group exhibited decreased activity, bradypnea, lowered body temperature and prone position from prior to administration on day 2 of lactation, and died on day 3 of lactation.
- In this case, no lactation activities were seen from day 2 of lactation when these symptoms developed, and histopathological examination revealed stress-related changes such as forestomach ulcer, glandular stomach erosion, thymic atrophy, and hypertrophy of adrenal zona glomerulosa and zona fasciculata; accordingly it was assumed that the female died because of puerperium. Moreover, in this case, focal necrosis of liver, pulmonary thrombus, epithelial cell necrosis of renal proximal tubule and hemorrhage into renal tubule were also seen. However, these changes were sometimes found in animals which died due to puerperal disease (NARAMA Isao, “Course in Toxicity Screening Test 5, Toxicologic Pathology”, edited by MAEKAWA Akihiko and HAYASHI Yuzo, Chijin Shokan Co., Ltd., Tokyo, 1992, p. 66) and thus considered to be related to puerperium. Hence, these changes were not considered to be related to administration of the test article.
- In 100 and 1000 mg/kg groups, no death occurred, and no changes in general medical conditions were seen.
- Therefore no effects of test article administration were assumed with regard to general medical conditions.

BODY WEIGHT AND WEIGHT GAIN
- As for males and females in each group, no differences were seen in comparison with control group throughout administration period.

FOOD CONSUMPTION
- No effects of test article administration were seen with regard to general medical conditions, body weights, food consumption, hematology, blood biochemistry and pathology.

OPHTHALMOSCOPIC EXAMINATION
- no data

HAEMATOLOGY
- In each group, no differences were seen in comparison with control group.

CLINICAL CHEMISTRY
- Decreases in total cholesterol (61 ± 5 mg/dL [100 mg/kg bw] compared to 69 ± 9 mg/dL [control]) and γ-GTP (0.0 ± 0.0 IU/L [300 mg/kg bw] compared to 0.1 ± 0.1 IU/L [control]) were seen respectively in 100 and 300 mg/kg groups compared to the control level.
However, these changes were not found in 1000 mg/kg group. Hence, these changes were not considered to be related to administration of the test article.

URINALYSIS
- no data

NEUROBEHAVIOUR
- no data

ORGAN WEIGHTS
- As for males, increases in absolute and relative weights of spleen were seen in 300 mg/kg group compared to control:
absolute spleen weight: 0.90 ± 0.07 g compared to 0.81 ± 0.08 g
relative spleen weight: 0.17 ± 0.01 g compared to 0.15 ± 0.02 g
- However, the same change was not seen in any females and other males in 1000 mg/kg group.
- Consequently, this change was considered to be incidental.

GROSS PATHOLOGY
- At autopsy in males after the completion of administration period, one case in 100 mg/kg group showed splenic hypertrophy and was histo-pathologically diagnosed with myelogenic leukemia. However, this kind of change was not seen in more than 300 mg/kg groups, and, while relatively rare, a similar change was reported as a spontaneous lesion in rats of the same type and the same-week old (C. B. Richter, Laboratory Investigation, 26, 419, 1972). Accordingly, this change was not considered to be related to administration of the test article.
- In addition, one case in 1000 mg/kg group showed a lymphocytic infiltrate beneath the endocardium of the left ventricle. However, its expression frequency was extremely low, and this kind of change was also reported as a spontaneous lesion in rats (NARAMA Isao, “Course in Toxicity Screening Test 5, Toxicologic Pathology”, edited by MAEKAWA Akihiko and HAYASHI Yuzo, Chijin Shokan Co., Ltd., Tokyo, 1992, p. 59). Therefore, this change was not considered to be related to administration of the test article.
- One case in 100 mg/kg group showed inversions of intra-thoracic and intraperitoneal organs.
- At autopsy in females on day 4 of lactation, no anomalies were seen in each group. Incidentally, one case in 300 mg/kg group, which died on day 3 of lactation, showed dark-red renal papilla, dark-red urine in bladder, blood pooling in vagina, dark-red spots in forestomach and glandular stomach mucosa. However, these changes were considered to be related to puerperium and not considered to be related to administration of the test article.
Therefore, for both, males and females no effects of test article administration were assumed with regard to pathology.

HISTOPATHOLOGY: NON-NEOPLASTIC
Males
- One case in 1000 mg/kg group showed a lymphocytic infiltrate beneath the endocardium of the left ventricle.
- Additionally, one case with gross splenic hypertrophy in 100 mg/kg group was diagnosed with myelogenic leukemia, by reason of the following histological findings.
- More specifically, in this case, femoral bone marrow was dominated by tumor cells with a relatively light, round or oval nucleus which had very little cytoplasm; at the same time, similar tumor cells were found around the hepatic portal vein and showed diffuse growth in lobular sinusoid.
- Moreover, in spleen, tumor cells showed infiltrative growth over the entire region to replace the normal structure of spleen. No neoplastic changes were seen in mesenteric lymph nodes.
Females
- No organ changes were seen in each group.
- Incidentally, one case of death in 300 mg/kg group showed focal necrosis of liver, pulmonary thrombus, epithelial cell necrosis of renal proximal tubule and hemorrhage into renal tubule. In addition, forestomach ulcer, glandular stomach erosion, thymic atrophy, and hypertrophy of adrenal zona glomerulosa and zona fasciculata were found.

OTHER FINDINGS
- Other findings see chapter 7.8.1 for reproductive toxicity.

The authors assumed that the no-observed effect level for repeat dose toxicity under the conditions of this test was more than 1000 mg/kg/day in both males and females.

Effect levels

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed.

Target system / organ toxicity

Critical effects observed:

not specified