Methyl acetoacetate was studied for oral toxicity in Crj:CD(SD) rats in a single dose toxicity test at doses of 0 and 2000 mg/kg for both sexes. The single dose oral toxicity test revealed a lethal dose value of more than 2000 mg/kg for both sexes.
Methyl acetoacetate was studied for oral toxicity in Crj:CD(SD) rats in an OECD combined repeat dose and reproductive/developmental toxicity screening test at doses of 0, 100, 300 and 1000 mg/kg.
With regard to repeat dose toxicity, no effects related to the test article were observed on clinical observation, body weights, food consumption and hematological, blood chemical and histopathological data. The NOEL for repeat dose toxicity is considered to be more than 1000 mg/kg/day for males and females. In terms of reproductive/developmental toxicity, there were no effects related to the test article on reproductive performance of parents and development of the next generation. The NOEL is considered to be more than 1000 mg/kg/day for reproductive performance of parents and for development of offspring.
Methyl acetoacetate was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.
Genotoxicity of methyl acetoacetate was studied by the chromosomal aberration test in cultured Chinese hamster lung(CHL/IU) cells.
Methyl acetoacetate induced structural chromosomal aberrations and polyploidy at 1.2 mg/mL(10 mM, high concentration) on short-term treatment with an exogenous metabolic activation system and on continuous treatment. However, no chromosomal aberrations were observed under pH-adjusted conditions in the confirmation test.
Therefore, it was suggested that the chromosome aberrations induced were caused by lowering of the pH with methyl acetoacetate in the medium.
| Purity | : | 99.4 % |
| Test species/strain | : | Rat/Crj:CD(SD) |
| Test method | : | OECD Test Guideline 401 |
| Route | : | Oral(gavage) |
| Doses | : | 0(Vehicle), 2000 mg/kg/day |
| Number of animals/group | : | Males, 5; females, 5 |
| Vehicle | : | Water for injection |
| GLP | : | Yes |
Test results:
Lethal dose: Male, > 2000 mg/kg; female, > 2000 mg/kg
| Purity | : | 99.4 % |
| Test species/strain | : | Rat/Crj:CD(SD) |
| Test method | : | OECD Combined Repeat Dose and Reproductive/ Developmental Toxicity Screening Test |
| Route | : | Oral(gavage) |
| Dosage | : | 0(Vehicle), 100, 300, 1000 mg/kg/day |
| Number of animals/group | : | Males, 12; females, 12 |
| Vehicle | : | Water for injection |
| 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 |
| GLP | : | Yes |
Test results:
No effects related to the test article were observed on clinical observation, body weights, food consumption and hematological and blood chemical data. On pathological examination, there were no effects related to the test article in necropsy findings, organ weights and histopathology. The NOEL is considered to be more than 1000 mg/kg/day for males and females.
<Reproductive and developmental toxicity>
As for reproductive performance, no effects related to the test article were observed on the estrous cycle, numbers of corpora lutea and implantations, copulation index or fertility indices for males or females. Examination at delivery and during the lactation period revealed no effects related to the test article on gestational days, litter and live born numbers, gestation index, stillborn index, birth index, sex ratio, body weights of offspring at birth and at day 4 after birth, or viability index on day 4. No external anomalies were observed. The NOEL is considered to be more than 1000 mg/kg/day for reproductive performance of parents and for development of offspring.
| Purity | : | 99.4 % |
| Test species/strains | : | Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA |
| Test method | : | Guidelines for Screening Toxicity Testing of Chemicals (Japan) and OECD Guidelines No. 471 and 472 |
| Procedures | : | Pre-incubation method |
| Solvent | : | Distilled water |
| Positive controls | : | -S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide(TA100, TA98, WP2 uvrA), Sodium azide(TA1535)and 9-Aminoacridine(TA1537) +S9 mix; 2-Aminoanthracene(five strains) |
| Doses | : | -S9 mix; 0, 313, 625, 1250, 2500, 5000 μg/plate(five strains) +S9 mix; 0, 313 - 5000 μg/plate(five strains) |
| S9 | : | Rat liver, induced with phenobarbital and 5,6-benzoflavone |
| Plates/test | : | 3 |
| Number of replicates | : | 2 and 3(WP2 uvrA without S9 mix) |
| GLP | : | Yes |
Test results:
Genetic effects:
Salmonella typhimurium TA100, TA1535, TA98, TA1537
| + | ? | - | |
| Without metabolic activation: | [ ] | [ ] | [*] |
| With metabolic activation: | [ ] | [ ] | [*] |
Escherichia coli WP2 uvrA
| + | ? | - | |
| Without metabolic activation: | [ ] | [ ] | [*] |
| With metabolic activation: | [ ] | [ ] | [*] |
| Purity | : | 99.4 % |
| Type of cell used | : | Chinese hamster lung(CHL/IU)cells |
| Test method | : | Guidelines for Screening Mutagenicity Testing of Chemicals(Japan)and OECD Guideline No. 473 |
| Solvent | : | Distilled water |
| Positive controls | : | -S9 mix, Mitomycin C +S9 mix, Cyclophosphamide |
| Doses | : | -S9 mix(continuous treatment): 0, 0.30, 0.60, 1.2 mg/mL -S9 mix(short-term treatment): 0, 0.30, 0.60, 1.2 mg/mL +S9 mix(short-term treatment): 0, 0.30, 0.60, 1.2 mg/mL +S9 mix(short-term treatment with pH-adjustement): 0, 1.2 mg/mL |
| S9 | : | Rat liver, induced with phenobarbital and 5,6-benzoflavone |
| Plates/test | : | 2 |
| GLP | : | Yes |
Test results:
Structural chromosomal aberrations(including gaps) and polyploidy were significant on short-term treatment with an exogenous metabolic activation system at 1.2 mg/mL(10 mM, high concentration, 11.0 % and 2.75 %, respectively). A confirmation test was conducted, because methyl acetoacetate decreased the pH of the medium at 1.2 mg/mL on short-term treatment with an exogenous metabolic activation system. After pH adjustment, no significant structural chromosomal aberrations or polyploidy were observed. Therefore, it was suggested that the chromosome aberrations induced were caused by lowering of the pH with methyl acetoacetate in the medium.
Genotoxic effects:
| clastogenicity | polyploidy | |||||
| + | ? | - | + | ? | - | |
| Without metabolic activation: | [ ] | [ ] | [*] | [ ] | [ ] | [*] |
| With metabolic activation: | [ ] | [ ] | [*] | [ ] | [ ] | [*] |
| 1) | The tests were performed by the Safety Assessment Laboratory, Panapharm Laboratories Co., Ltd. 1285 Kurisaki-machi, Uto-shi, Kumamoto, 869-0425, Japan Tel +81-964-23-5111 Fax +81-964-23-2282 |
| 2) | The tests were performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257-0025, Japan. Tel +81-463-82-4751 Fax +81-463-82-9627 |