Methyl acetoacetate

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 1997 - July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed in accordance with the corresponding OECD-/EU-testing guidelines; original report in Japanese, English translation available

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Methyl acetoacetate
- Analytical purity: 99.4%
- Lot no. 602006
- Supplier: Nippon Synthetic Chemical Industry Co., Ltd.
- Appearance: clear, colorless liquid

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: males/females 8 weeks
- Weight at study initiation: 373-428 g for males and 228-255 g for females
- Housing: 2-3 animals per cage (sexes seperated)
- Diet (e.g. ad libitum): MF, Oriental Yeast Co., Ltd., Japan
- Water (e.g. ad libitum): Water, treated with sodium hypochlorite approx. 2 ppm
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 48-60%
- Air changes (per hr): 13-15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Japanese pharmacopoeia physiological water for injection (Lot No. 6D77, Otsuka Pharmaceutical Factory, Inc.)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of MAA was dissolved in water for injection at the concentrations of 1, 3 and 10 w/v%. Preparation was conducted at a frequency of once a week and the dosing formulations were stored at room temperature until administration. A stability
test of the test mixtures prepared by this method was conducted at this facility, and 0.1 and 20 w/v% solutions were confirmed to remain stable for 8 days under scattered light (in a brown vial) at room temperature. Moreover, the dosing formulations were confirmed to be within the permissible range (nominal concentration ± 5%) by determination of concentration conducted upon initiation of administration (first week of administration) at this facility.

Details on mating procedure:

ESTROUS CYCLE AND FERTILITY:
For females, a vaginal smear was collected at almost the same time every morning for 15 days from the day of the start of administration (day 1 of administration) and the stage of the estrous cycle was examined. The stages of the estrous cycle was classified, the count of estrus and the length of the estrous cycle (average number of days from estrous to the next estrous) during the examination period were calculated. Mating was begun at the age of 12 weeks form about 4:00 P.M. on day 15 of administration. Females were housed overnight on a one-to-one basis with males. Copulation was confirmed by the presence of a vaginal plug or sperm in the
vaginal smear on the following morning, and the day of confirmed copulation was designated as day 0 of gestation. Mating was conducted within the same group for a maximum of 2 weeks. After the end of the mating period, the days required for successful copulation, the copulation index [(number of animals with confirmed copulation/number of animals mated) × 100], and male and female fertility indices [(number of pregnant animals/number of female (male) animals for which copulation was confirmed) × 100] were calculated.

OBSERVATION AT DELIVERY, DURING LACTATION AND OFFSPRING:
All copulated females were allowed natural delivery. The delivery and nursing condition including sign of delivery in the late stages of gestation were observed, and the gestational days and the gestation index [(number of females with live newborns/number of pregnant females) × 100] were calculated. The delivery completed by 12:00 was judged as a delivery on the corresponding day, the animals that completed delivery after 12:00 were observed for lactation the next day. For offspring, the number of litter, stillborns and live newborns were counted and weighed, sexed and examined for external anomalies after delivery. All the live newborns were individually weighed on days 0 and 4 after birth, and the birth index [(number of live newborns/number of implantation) × 100] and the viability on day 4 after birth [(number of live newborns on day 4 after birth/number of live newborns) × 100] were calculated. On day 4 after birth, all the live newborns were sacrificed by exsanguination under ether anesthesia, and macroscopically observed for organs and tissues. Offspring (including stillborn or dead) were fixed and preserved in pure ethanol on a litter basis after necropsy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

Administration period:
- Males, 49 days
- Females, from 14 days before mating to day 3 of lactation

Terminal kill:
- Males, day 50
- Females, day 4 of lactation

Frequency of treatment:

The dosing formulation was administered once daily with a stomach tube from 14 days prior to mating through the mating period up to the day before necropsy (Total: 35 days) in males and 14 days prior to mating through the gestation period up to day 3 of delivery in females.

Details on study schedule:

See above

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:
nominal conc.
vehicle control

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

Number of animals/group:
- Males = 12
- Females = 12

Control animals:

yes, concurrent no treatment

Details on study design:

Dose levels were determined from the results of the previous preliminary study. In the previously conducted 2-week repeated oral dose study (dose levels: 100, 300, and 1000 mg/kg), no effects of administration of test article were observed on clinical signs, body weight, food consumption, hematological or blood chemical examination, necropsy or organ weight. Therefore, the high dose in this study was set at 1000 mg/kg, which was the dose of maximum limit stated in the OECD Guideline, and the middle and low dose levels were set at 300 and 100 mg/kg, respectively, in a common ratio of about 3.

Positive control:

No details available.

Examinations

Parental animals: Observations and examinations:

1) Clinical observation, body weight and food consumption:
All the male and female rats were observed at least twice daily for clinical signs and mortality.
The body weights of all males were measured twice a week throughout administration period. The body weights of all females were measured twice a week from prior to mating through the mating period, on days 0, 4, 7, 10, 14, 17 and 21 of gestation during the gestation period and on days 0 and 4 of lactation during the lactation period.
Food consumption of all males was measured twice a week during the administration period except during the mating period. Food consumption of all females was measured twice a week from prior to mating through the mating period, on days 1, 4, 7, 10, 14, 17 and 21 of gestationduring the gestation period and on days 1 and 4 of lactation during the lactation period.

Oestrous cyclicity (parental animals):

Estrous cycle and fertility:
For females, a vaginal smear was collected at almost the same time every morning for 15 days from the day of the start of administration (day 1 of administration) and the stage of the estrous cycle was examined. The stages of the estrous cycle was classified, the count of estrus and the length of the estrous cycle (average number of days from estrous to the next estrous) during the examination period were calculated. Mating was begun at the age of 12 weeks form about 4:00 P.M. on day 15 of administration. Females were housed overnight on a one-to-one basis with males. Copulation was confirmed by the presence of a vaginal plug or sperm in the vaginal smear on the following morning, and the day of confirmed copulation was designated as day 0 of gestation. Mating was conducted within the same group for a maximum of 2 weeks.
After the end of the mating period, the days required for successful copulation, the copulation index [(number of animals with confirmed copulation/number of animals mated) × 100], and male and female fertility indices [(number of pregnant animals/number of female (male) animals
for which copulation was confirmed) × 100] were calculated.

Sperm parameters (parental animals):

See above

Litter observations:

Observation at delivery, during the lactation period (up to day 4 of lactation) and offspring):
All copulated females were allowed natural delivery. The delivery and nursing conditionincluding sign of delivery in the late stages of gestation were observed, and the gestational days and the gestation index [(number of females with live newborns/number of pregnant females) × 100] were calculated. The delivery completed by 12:00 was judged as a delivery on the corresponding day, the animals that completed delivery after 12:00 were observed for lactation the next day. For offspring, the number of litter, stillborns and live newborns were counted and weighed, sexed and examined for external anomalies after delivery. All the live newborns were individually weighed on days 0 and 4 after birth, and the birth index [(number of live newborns/number of implantation) × 100] and the viability on day 4 after birth [(number of live newborns on day 4 after birth/number of live newborns) × 100] were calculated. On day 4 after birth, all the live newborns were sacrificed by exsanguination under ether anesthesia, and macroscopically observed for organs and tissues. Offspring (including stillborn or dead) were fixed and preserved in pure ethanol on a litter basis after necropsy.

Postmortem examinations (parental animals):

Necropsy, organ weight measurement and histopathological examination:
The animals were sacrificed by exsanguination from the lateral iliac artery under ether anesthesia,and all the organs and tissues were examined macroscopically after blood sampling at thecompletion of administration period for males and on day 4 of lactation for females and the numbers of corpora lutea and implantations were counted for females. After necropsy, the brain, heart, lungs (with bronchi), thymus, liver, spleen, kidneys, adrenals, testes, epididymides and ovaries were weighed (absolute organ weight) and the ratio of organ weight to body weight was calculated based on the body weight on the day of necropsy (relative organ weight).
Above weighed organs and organs with macroscopic lesions at necropsy were removed and fixed in 10% neutral buffered formalin solution. The testes and epididymides were prefixed in Bouin’s solution. The brain, heart, lungs (with bronchi), thymus, liver, spleen, kidneys, adrenals, testes, epididymides, ovaries and ovaries which copulation was unsuccessful from the control and 300 mg/kg groups were embedded in paraffin according to the established method, sectioned and stained with hematoxylin and eosin (H.E. stain), and examined microscopically. As 1 animal in the 100 mg/kg group, in which hypertrophy of the spleen was noted macroscopically, was suspected to have developed myeloid leukemia, the femur (bone marrow), liver, and mesenteric lymph nodes were also collected and similarly examined. Moreover, the, liver, pancreas, and femur were esterase stained for a further examination. For animals that died, kidneys and lung were PTAH stained. All the organs with macroscopic lesions from all the groups were performed histopathological examination (not performed for inversion of thoracic or abdominal cavity).

Postmortem examinations (offspring):

See above.

Statistics:

As regards body weight, food consumption, hematological data, blood chemical data, number of days required for successful copulation, estrous cycle data (count of estrus, estrus cycle), organ weights, gestational days, numbers of corpora lutea, numbers of implantation, litter and live newborns and body weight of live newborns, the mean and standard deviations were calculated for every group, and the homogeneity of variance was tested by Bartlett’s method. Comparison of the treated groups with the control group was conducted by Dunnett’s method when the variance was observed to be homogeneous and by Steel’s method when the variance was not homogeneous. The copulation, male or female fertility and gestation indices and sex ratios of live newborns were analyzed by the χ2 test, the stillbirth and birth indices and viability index on day 4 after birth were analyzed by Wilcoxon’s rank sum test, histopathological data was analyzed by the Mann-Whitney’s U-test, and the test article group was compared with the control group. Levels of p<0.05 were considered to be significant in all cases. The values for offspring were recorded with each litter treated as a unit.

Reproductive indices:

See above.

Offspring viability indices:

See above.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See text field "any other information on results"

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

no effects

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

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:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P1)

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

not examined

Effect levels (F1)

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

REPEATED DOSE TOXICITY:

1) Clinical signs:

No death occurred in any group and no abnormal clinical signs were observed in males. One of female in the 300 mg/kg group showed hypoactivity, bradypnea, hypothermia and prone position from before administration on day 2 of lactation, and died on day 3 of lactation. No death occurred in any female nor were there any abnormal clinical signs observed in the 100 and 1000 mg/kg groups.

2) Body weight:

No differences were seen any groups as compared to the control group throughout the administration period for males or females.

3) Food consumption:

No differences were seen in any group as compared to the control group throughout the administration period for males or females.

4) Hematology:

No differences were seen in any group as compared to the control group for males or females.

5) Blood chemistry:

A decrease in T. protein was observed in males of 100 mg/kg group and a decrease in γ-GTP was observed in males in the 300 mg/kg group.

6) Necropsy:

At completion of the administration, enlargement in the spleen was observed in 1 male in 100 mg/kg group. Moreover, inversion of thoracic and abdominal cavity was observed in 1 male in 100 mg/kg group. In females on the day 4 of lactation, no abnormalities were observed in any group. In addition, blackish red coloration in the renal papillae, retention of blackish red urine in the urinary bladder, retention of blood in the vagina and blackish red spot in the fore stomach and glandular mucosa were observed one female that died on day 3 of lactation.

7) Organ weight:

An increase in absolute and relative spleen weights was observed in males in the 300 mg/kg group. No changes were seen any groups in females.

8) Histopathology:

In males, lymphocytic infiltration was noted in the left ventricular endocardium of 1 animal in the 1000 mg/kg group. Moreover, the effect seen in one animal in the 100 mg/kg group, in which hypertrophy of the spleen was noted macroscopically, was judged to be myeloid leukemia based on the following histopathological findings: there was a depletion of bone marrow cells with normal cytoplasm and filled dominantly with tumor cells with relatively light and circular to elliptic nuclei, and similar tumor cells were also observed in the periportal region and diffusely proliferated in sinusoid in the liver. In the spleen, infiltrative hyperplasia of the tumor cells displaced the normal structure entirely. No tumorous change was noted in the mesenteric lymph nodes. No changes were seen in organs of females in any group. In addition, focal necrosis in the liver, thrombus in the lung, necrosis in the proximal tubular epithelium, hemorrhage in the renal tubule, ulcer in the fore stomach, erosion in the glandular stomach, atrophy in the thymus and hypertrophy in the zona glomerulosa and zona fasciculata were observed in the one animal that died. Furthermore, pulmonary thrombosis was positive by the PTAH staining

2. REPRODUCTIVE/DEVELOPMENTAL TOXICITY

1) Estrus cycle and fertility:

No differences were seen in the count of estrous or estrous cycle in any group as compared to the control group. In the fertility, copulation was confirmed in all pairs except for 1 pair of the 100 mg/kg group and fertility was confirmed in all pairs. Accordingly, the copulation index was 100%, 91.67%, 100% and 100% in the control, 100, 300 and 1000 mg/kg groups, respectively, no differences were seen in any group as compared to the control group. No differences in the days required for successful copulation were seen in any group as compared to the control group, respectively. Moreover, no abnormality was seen at the necropsy of female which not copulated or of the ovary at the histopathology.

2) Observation on delivery, lactation (up to day 4 of lactation) and offspring:

At delivery, the number of newborn females was higher than males, indicating female-biased sex ratio in the 100 mg/kg group, and increase in numbers of implantation, newborns and live newborns was observed in the 300 mg/kg group. However, no differences were seen in any group as compared to the control group in the gestation period, number of corpora lutea, birth and stillbirth indices, numbers of live newborns and body weight of live newborns, and no abnormality was seen by external examination of live newborns in any group.

Applicant's summary and conclusion

Conclusions:

The NOEL for repeat dose toxicity, reproduction performance of parents and development of offspring was found to be 1000 mg/kg/day.

Executive summary:

The study was performed in Japan according to OECD-guideline no. 422 as GLP-study and summarized in the JETOC Information sheet No. 43 of April 2000 - Special Issue no. 6. MAA at dosages of 0 (vehicle control), 100, 300 and 1000 mg/kg/day was orally administered to Crj:CD(SD) male and female rats (12 animals/sex/group) from 14 days before mating through the mating period and up to the day before necropsy (Total: 35 days) in males and 14 days prior to mating through the gestation period up to day 3 of delivery in females to investigate the effect on the repeat dose and reproductive toxicity for the parent animals and the development for the offspring,

and the following results were obtained.

1. Repeat dose toxicity:

No effects of administration of test article were observed on the clinical signs, body weight, food consumption, hematological, blood chemistry and histopathological examination.

2. Reproductive/developmental toxicity:

As for reproductive performance in parent animal, no effects of administration of test article were observed on the estrus cycle, numbers of corpora lutea or implantations, copulation (mating performance) or fertility indices. Examination at delivery and during the lactation period revealed, no effects of administration of test article were observed on the gestational days, number of litter or live newborns, gestation, birth or stillbirth indices, sex ratio, body weight at birth or on day 4 after birth, viability index on day 4 after birth. No external malformations in live newborns were observed.

As described above, the no observed effect level (NOEL) under this study conditions was assumed to be 1000 mg/kg/day for repeat dose toxicity in both males and females, and also to be 1000 mg/kg/day for reproductive/developmental toxicity in parent animals and offspring.