o-Acetoacetotoluidide

o-アセトアセトトルイジド


[CAS No. 93-68-5]

2-Acetoacetylaminotoluene

2-アセトアセチルアミノトルエン

Molecular formula: C11H13NO2　Molecular weight: 191.23

ABSTRACT

o-Acetoacetotoluidide was studied for oral toxicity in rats in a single dose oral toxicity test at doses of 0, 819, 1024, 1280, 1600, 2000 and 2500 mg/kg. The LD50 values (95 % confidence limits) were estimated to be 1854 (1549 - 2298) mg/kg for males and 1945 (1654 - 2318) mg/kg for females.

o-Acetoacetotoluidide was studied for oral toxicity in rats in an OECD combined repeat dose and reproductive/developmental toxicity screening test at doses of 0, 8, 25, 80 and 250 mg/kg/day.

With regard to repeat dose toxicity in males, hematological examinations revealed a decrease in erythrocyte counts, and an increase in MCV in the 80 and 250 mg/kg groups. Further, decreases in hemoglobin concentration and hematocrit values, increases in MCH and reticulocyte counts, a tendency for increase in methemoglobin concentration, and appearance of Heinz-bodies in erythrocytes were observed in the 250 mg/kg group. Blood chemical examinations revealed an increase in bilirubin in the 80 and 250 mg/kg groups. Further, an increase in potassium was noted in the 250 mg/kg group. At necropsy, blackening of the spleen was observed in the 80 and 250 mg/kg groups. Increases in absolute and relative weights of the spleen and pituitary were observed in the 250 mg/kg group. Histopathologically, hemosiderin deposits in the liver and increased in severity of hemosiderin deposits in the spleen were observed in the 80 and 250 mg/kg groups. Increased extramedullary hematopoiesis and congestion in the spleen, and an increased incidence of eosinophilic bodies in the proximal tubular epithelial cells of the kidneys were also observed in the 250 mg/kg group. No histopathological changes attributable to the compound were detected in the pituitary. In females, similar pathological changes in the spleen and liver to those observed in males were detected in the 80 and 250 mg/kg groups. The NOELs for repeat dose toxicity are considered to be 25 mg/kg/day for males and females. In terms of reproductive and developmental toxicity, the test substance showed no effects on any reproductive parameters of the parental animals or developmental parameters of the offspring. The NOELs for reproductive/developmental toxicity are considered to be 250 mg/kg/day or more for reproductive performance of parental animals and offspring development.

o-Acetoacetotoluidide was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA with or without an exogeneous metabolic activation system.

o-Acetoacetotoluidide induced structural chromosomal aberrations in CHL cells without an exogeneous metabolic activation system. Polyploidy was not induced with or without an exogenous metabolic activation system.

SUMMARIZED DATA FROM THE STUDIES

1. Single Dose Oral Toxicity 1)

Purity:99.93 %
Test species/strain:Rat/Crj:CD(SD)
Test method:OECD Test Guideline 401
　Route:Oral (gavage)
　Dosage:0 (vehicle), 819, 1024, 1280, 1600, 2000, 2500 mg/kg
　Number of animals/group:Males, 5; females, 5
　Vehicle:1 % methylcellulose solution
GLP:Yes

　Test results:

Deaths occurred in males of the 1280 mg/kg or more groups and in females of the 1600 mg/kg or more groups. Clinical signs such as decreased locomotor activity, adoption of a prone position, hypotonia, ptosis, deep respiration, piloerection, hypothermia, lacrimation and pale skin were observed in treated groups. Body weights in treated groups were dose-dependently lower than those of the control group on the day after treatment. At necropsy, bloody material in the stomach and intestine, petechiae in the glandular stomach and distension of the urinary bladder were detected in the animals that died.

The LD50 values (95 % confidence limits) were estimated to be 1854 (1549 - 2298) mg/kg for males and 1945 (1654-2318) mg/kg for females.

2. Repeat Dose and Reproductive/Developmental Toxicity 1)

Purity:99.93 %
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), 8, 25, 80, 250 mg/kg/day
　Number of animals/group:Males, 10; females, 10
　Vehicle:1 % methylcellulose solution
　Administration period:Males, 44 days
Females, from 14 days before mating to day 3 of lactation
　Terminal kill:Males, day 45
Females, day 4 of lactation
GLP:Yes

　Test results:

<Repeat dose toxicity>

In males, hematological examination revealed a decrease in erythrocyte counts, and an increase in MCV in the 80 and 250 mg/kg groups. Further, decreases in hemoglobin concentration and hematocrit values, increases in MCH and reticulocyte counts, a tendency for increase in methemoglobin concentration, and the appearance of Heinz-bodies in erythrocytes were observed in the 250 mg/kg group. Blood chemical examination revealed an increase in bilirubin in the 80 and 250 mg/kg groups. Further, an increase in potassium was noted in the 250 mg/kg group. At necropsy, blackening of the spleen was observed in the 80 and 250 mg/kg groups. Increases in absolute and relative weights of the spleen and pituitary were observed in the 250 mg/kg group. Histopathologically, hemosiderin deposits in the liver and increased severity of hemosiderin deposits in the spleen were observed in the 80 and 250 mg/kg groups. Increased extramedullary hematopoiesis and congestion in the spleen, and an increased incidence of eosinophilic bodies in the proximal tubular epithelial cells of the kidneys were also observed in the 250 mg/kg group. No histopathological changes attributable to the compound were detected in the pituitary.

In females, similar pathological changes in the spleen and liver to those observed in males were detected in the 80 and 250 mg/kg groups.

The NOELs for repeat dose toxicity are considered to be 25 mg/kg/day for males and females.

<Reproductive and developmental toxicity>

The parental animals exhibited no alteration in reproductive parameters. There were no significant differences in offspring parameters.

The NOELs for reproductive/developmental toxicity are considered to be 250 mg/kg/day or more for reproductive performance and offspring development.

3. Genetic Toxicity

3-1. Bacterial test 2)

Purity:99.93 %
Test species/strains:Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
Test method:Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Test Guidelines 471 and 472
　Procedures:Pre-incubation method
　Solvent:DMSO
　Positive controls:-S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98 and WP2 uvrA), Sodium azide (TA1535), 9-Aminoacridine hydrochloride (TA1537)
+S9 mix; 2-Aminoanthracene (all strains)
　Doses:-S9 mix; 156, 313, 625, 1250, 2500 and 5000 μg/plate
+S9 mix; 156, 313, 625, 1250, 2500 and 5000 μg/plate
　S9:Rat liver, induced with phenobarbital and 5,6-benzoflavone
　Plates/test:3
　Number of replicates:2
GLP:Yes

　Test results:

This chemical did not induce gene mutations in the S. typhimurium and E. coli strains. No toxicity was observed up to a concentration of 5000 μg/plate, with or without metabolic activation.

Genetic effects:
Salmonella typhimurium TA100, TA1535, TA98, TA1537
+?-
Without metabolic activation:[ ][ ][*]
With metabolic activation:[ ][ ][*]

Escherichia coli WP2 uvrA
+?-
Without metabolic activation:[ ][ ][*]
With metabolic activation:[ ][ ][*]

3-2. Non-bacterial in vitro test (chromosomal aberration test) 2)

Purity:99.93 %
Type of cell used:Chinese hamster lung (CHL) cells
Test method:Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Test Guideline 473
　Solvent:DMSO
　Positive controls:-S9 mix (24 and 48 hr continuous treatment); Mitomycin C
-S9 mix (short-term treatment); Cyclophosphamide
+S9 mix (short-term treatment); Cyclophosphamide
　Doses:-S9 mix (24 hr continuous treatment); 0, 625, 1250, 2500, 5000 μg/mL
-S9 mix (48 hr continuous treatment); 0, 450, 900, 1800, 3600 μg/mL
-S9 mix (short-term treatment); 0, 1250, 2500, 5000 μg/mL
+S9 mix (short-term treatment); 0, 1250, 2500, 5000 μg/mL
-S9 mix (continuous treatment, confirmative test); 0, 1500, 2000, 2500, 3000, 3500 μg/mL
　S9:Rat liver, induced with phenobarbital and 5,6-benzoflavone
　Plates/test:2
GLP:Yes
　Test results:
Structural chromosomal aberrations (including gaps) were induced under the following conditions: 24 hr continuous treatment (2.5 mg/mL, 10.0 %); 48 hr continuous treatment (1.8 mg/mL, 5.0 %); short-term treatment without S9 mix (5 mg/mL, 9.0 %); short-term treatment with S9 mix (5 mg/mL, 5.0 %). The confirmative examination was conducted with 24 hr continuous treatment, because structural chromosomal aberrations were only induced at the dosage of 2.5 mg/mL. As a result, structural chromosomal aberrations were induced dose-dependently. Polyploidy was not induced under any test conditions.

Cytotoxicity was observed at 3.5 mg/mL with 24 hr continuous treatment and at 3.6 mg/mL with 48 hr continuous treatment.

Lowest concentration producing cytogenetic effects in vitro:
　Without metabolic activation (continuous treatment): 2.5 mg/mL (clastogenicity)

Genotoxic effects:
clastogenicitypolyploidy
+?-+?-
　Without metabolic activation:[*][ ][ ][ ][ ][*]
　With metabolic activation:[ ][*][ ][ ][ ][*]

1)The tests were performanced by the Research Institute for Animal Science in Biochemistry and Toxicology, 3-7-11 Hashimotodai, Sagamihara-shi, Kanagawa, 229-1132, Japan. Tel +81-42-762-2775 Fax +81-42-762-7979
2)The tests were performed by the Biosafety Research Center, Foods, Drugs and Pesticides (An-pyo Center), 582-2 Shioshinden Arahama, Fukude-cho, Iwata-gun, Shizuoka, 437-1213, Japan. Tel +81-538-58-1266 Fax +81-538-58-1393