Technical Information

Summary of Toxicological Studies with Chromafenozide
Marketing Department, Agrochemicals Division, Agro & Specialty Chemicals Group
Nippon Kayaku Co., Ltd.* and Crop Protection Company Sankyo Co., Ltd.*(Accepted May 30, 2002)
GENERAL INFORMATION ON CHROMAFENOZIDEChromafenozide (Product name: MATRIC® Flow-able and MATRIC® Dust DL), a
novel insecticide against lepidopteran larvae with a similar mode of action to ecdysone
known as an insect-specific ecdysis hormone, was discovered and developed undercooperative works by Nippon Kayaku Co., Ltd. and Sankyo Co., Ltd.
Chromafenozide is one of the most suitable plant protection products for the
Integrated Pest Management (IPM) to which growing attentions have been attractedwith recent global needs for the sustainable agriculture.
Chromafenozide on the plant is ingested orally by insect larvae to exhibit anecdysis-promoting activity like ecdysone and lead to death.

The activity is selective

only to lepidopteran larvae that damage rice, tea, fruit trees, vegetables or otheragricultural plants.
The chemical structure and physical and chemical properties of chromafenozide areas follows:
Common name: chromafenozide(ISO common name)Chemical name: 2'-tert-butyl-5-methyl-2'-(3,5-xyloyl)-chromane-6-carbohydrate
Chemical structure:
Chemical formula: C24H30N2O3
Molecular weight: 394.51
Physical state: White solid powder
Melting point: 186.4°℃
Solubility (g/l, 20℃): Toluene: 0.32 Dichloromethane: ＞336 Acetone: 186 Ethyl
acetate: 50.6 Ethanol: 173 Diisopropylether: 0.35 Water: 1.12 mg/l
Partition coefficient (n-octanol/water): log Pow＝2.7

ACUTE TOXICITY STUDIES
Summary of acute toxicity studies via various administration routes is presented in
the following table.
IRRITATION STUDIES
1.

Primary Eye Irritation Studies
Primary eye irritation potential of chromafenozide technical, MATRIC® Flowable (5%SC), 1/500 aqueous dilution (the highest field application rate) of 5% SC, and MATRIC®
Dust DL (0.3% D) was assessed with female or male New Zealand White rabbit andevaluated according to the Draize's scoring system. One eye of the rabbit was applied
0.1 g or 0.1 ml of the test substance and assessed irritation appeared on cornea, iris
and/or conjunctiva.

In addition, if irritation was observed, an eye irrigation effect withwater was assessed on the group.
Chromafenozide technical: Mild irritation observed was recovered by 48 hr after
application.

The irrigation was effective at 2 min after application.

(The Institute of Environmental Toxicology, 1995)
5% SC: Mild irritation observed was recovered by 72 hr after application.

The

irrigation was effective at 30 sec after application.
(The Institute of Environmental Toxicology, 1998)
Aqueous dilution (1/500) of 5% SC (the maximum field application rate): No irritation
was observed.
(Safepharm Laboratories, 1998)
0.3% D: Minimum irritation observed was recovered by 24 hr after application.

The

irrigation was effective at 30 sec after application.
(The Institute of Environmental Toxicology, 1998)
2.

Primary Dermal Irritation Studies
Primary dermal irritation potential of chromafenozide technical, 5% SC, and 0.3% D

was assessed with female New Zealand White rabbit and evaluated according to the
Draize's scoring system. The test substance was applied on a 2.5 cm × 2.5 cm area ofthe shaved trunk skin of a rabbit at 0.5 g or 0.5 ml and the appearance of the irritation
responses (erythema, eschar and edema) was assessed over 72 hr.
Chromafenozide technical: No irritation was observed.
(The Institute of Environmental Toxicology, 1995)
5% SC: No irritation was observed.
(The Institute of Environmental Toxicology, 1998)

0.3% D: No irritation was observed.
(The Institute of Environmental Toxicology, 1998)
DERMAL SENSITIZATION STUDIES
Dermal sensitization potential of chromafenozide technical, 5% SC and 0.3% D wasassessed with female Hartly guinea pig.
Chromafenozide technical: Mild sensitization reaction was observed after an
intradermal inductive injection at 5% suspension and a topical inductive application at
25% in white petrolatum followed by a challenge application at 25% in white petrolatum(Maximization test).
(The Institute of Environmental Toxicology, 1995)
5% SC: No sensitization reaction was observed after three topical inductive
applications and a challenge application of the intact test substance (Buehler test).
(The Institute of Environmental Toxicology, 1998)
0.3% D: No sensitization reaction was observed after three topical inductive
applications and a challenge application of 50% aqueous suspension of the test
substance (Buehler test).
(The Institute of Environmental Toxicology, 1998)

SUBCHRONIC TOXICITY STUDIES
1.

13-Week Oral Subchronic Toxicity Study in Rats
Chromafenozide technical mixed with a rodent diet at 0, 300, 1000, 3000, 10,000 or

20,000 ppm was administered to 12 male and 12 female Fischer rats per group for 13
weeks.
Decreased body weight, anemia, increased creatine phosphokinase, increased weightsof the liver and spleen, periportal hepatocellular swelling, and increased incidence ofdark in color and brown pigment deposition of the spleen were noted in males andfemales at 20,000 ppm, the highest dose level.

Decreased total cholesterol andtriglyceride in males and increased blood urea nitrogen in females were also observed at
20,000 ppm.

No test substance-related findings were observed in males at 1000 ppm or

below and in females at 3000 ppm or below.
Based on these findings, under the conditions of this study, the no observed adverse
effect levels (NOAELs) of chromafenozide were determined to be 1000 ppm (64.4
mg/kg/day) for males and 3000 ppm (207.7 mg/kg/day) for females.
Institute of Environmental Toxicology, 1996)

(The

2.

13-Week Oral Subchronic Toxicity Study in Mice
Chromafenozide technical mixed with a rodent diet at 0, 310, 1250, 5000, 20,000 or

30,000 ppm was administered to 12 male and 12 female ICR mice per group for 13
weeks.
Darkening in color and brown pigment deposition of the spleen in males andfemales and centrilobular hepatocellular swelling in males were noted at 20,000 and
30,000 ppm.

Increased spleen weight and extramedullary hematopoiesis in the spleen

in males and females and decreased erythrocyte count and increased relative weight of
the liver in males were observed at 30,000 ppm, the highest dose level.
Based on these findings, under the conditions of this study, the NOAEL ofchromafenozide was determined to be 5000 ppm for both males and females (624.7
mg/kg/day for males and 723.1 mg/kg/day for females).
(The Institute of Environmental Toxicology, 1996)
3.

13-Week Oral Subchronic Toxicity Study in Dogs
Chromafenozide technical mixed with a diet at 0, 2000, 10,000 or 30,000 ppm wasadministered to 4 male and 4 female beagle dogs per group for 13 weeks.
At 30,000 ppm, increased platelet count, darkening in color of the spleen andcongestion in the spleen in both sexes, mild anemia, increased mean corpuscularvolume, increased relative weight of the liver, increased absolute and relative weight ofthe spleen in males, increased hematopoiesis in the bone marrow and increased
triglyceride in females were observed. Darkening in color of the spleen and congestionin the spleen in both sexes, increased platelet count in males and increased total
bilirubin and increased absolute and relative weight of the spleen in females were noted
at 10,000 ppm.
Based on these findings, under the conditions of this study, the NOAEL ofchromafenozide was determined to be 2000 ppm for both sexes (52.2 mg/kg/day formales and 52.4 mg/kg/day for females).
(The Institute of Environmental Toxicology, 1996)
CHRONIC TOXICITY AND ONCOGENICITY STUDIES
1.

24-Month Oral Chronic Toxicity and Oncogenicity Study in Rats
Chromafenozide technical mixed with a rodent diet at 0, 250, 1000, 4000 or 15,000

ppm was administered to 85 male and 85 female Fischer rats per group for 24 months.
Lower body weight gain at 15,000 ppm, mild anemia and periportal hepatocellularhypertrophy, increased liver weight, and increased weight, darkening in color, andbrown pigment deposition to be considered hemosiderin in the spleen at 4000 and
15,000 ppm were noted in both sexes.
Based on these findings, under the conditions of this study, the NOAEL ofchromafenozide was determined to be 1000 ppm for both males and females (35.8mg/kg/day for males and 44.0 mg/kg/day for females).
no tumorigenic potential in the test substance.

It was considered that there was(The Institute ofEnvironmental Toxicology, 1998)
2.

18-Month Oral Oncogenicity Study in Mice
Chromafenozide technical mixed with a rodent diet at 0, 800, 5000 or 30,000 ppm wasadministered to 50 male and 50 female ICR mice per group for 18 months.
Lower body weight gain in females and increased incidence of brown pigment
deposition in the spleen in both sexes were noted at 30,000 ppm
Based on these findings, under the conditions of this study, the NOAEL ofchromafenozide was determined to be 5000 ppm for both males and females (553.3
mg/kg/day for males and 484.8 mg/kg/day for females).

It was considered that there

was no tumorigenic potential in the test substance.

(The Institute ofEnvironmental Toxicology, 1998)
3.

12-Month Oral Chronic Toxicity Study in Dogs
Chromafenozide technical mixed with a diet at 0, 1200, 6000 or 30,000 ppm wasadministered to 4 male and 4 female beagle dogs per group for 12 months.
Decreased erythrocyte count, increasing tendency of reticulocyte count and increased
platelet count in females, and increased total bilirubin in plasma in both sexes wereobserved at 30,000 ppm. In addition, increased organ weight, swelling, darkening incolor, congestion and brown pigment (hemosiderin) deposition of the spleen, increased
hemosiderin deposition in the Kupffer cells of the liver, and increased hematopoiesis in
the bone marrow were also observed at the same dose level. Increased total bilirubinin plasma in males, swelling, darkening in color, and congestion of the spleen, increased
hemosiderin deposition in the Kupffer cells of the liver, and increased hematopoiesis in
the bone marrow were noted at 6000 ppm.
Based on these findings, under the conditions of this study, the NOAEL ofchromafenozide was determined to be 1200 ppm for both males and females (27.9mg/kg/day for males and 27.2 mg/kg/day for females).
(The Institute of Environmental Toxicology, 1998)

REPRODUCTION STUDY IN RATS
Chromafenozide technical mixed with a rodent diet at 0, 200, 2000 or 20,000 ppm wasadministered to 24 male and 24 female SD rats per group to assess potential toxicity
and effects on the reproduction performance over successive 2 generations.
Decreased body weight gains in F1 male parental animals were observed at 20,000
ppm.

Darkening in color and brown pigment deposition of the spleen and periportal

hepato-cellular hypertrophy in both sexes, increased relative weight of the liver inmales, increased absolute and relative weight of the liver in females and increased
absolute and/or relative weight of the spleen in females were also observed in 20,000
ppm F0 and F1 parental animals. No test substance-related adverse effects werenoted in any of the parameters tested for reproductive performance of F0 and F1
parental animals up to the highest dose level. Temporary decreased body weight gains
observed in F1 and F2 offsprings at 20,000 ppm were not observed at 2000 ppm and 200
ppm.
Based on these findings, under the conditions of this study, the NOAEL ofchromafenozide was determined to be 2000 ppm (128‐152 mg/kg/day for males and 144‐165 mg/kg/day for females) for the general toxicity to the parental animals andoffsprings.

The NOAEL for the reproductive performance was determined to be 20,000

ppm (1284‐1549 mg/kg/day for males and 1416‐1666 mg/kg/day for females).
(The Institute of Environmental Toxicology, 1997)
TERATOLOGY STUDIES
1.

Teratogenicity Study in Rats
Chromafenozide technical suspended in 1% CMC aqueous solution was orally

administered daily at 0, 100, 300 and 1000 mg/kg to 24 copulated female SD rats pergroup for 10 days from 6 to 15 of gestation.
No test substance-related effects on the maternal rats were noted and any inductionof the fetal malformation or variation was caused at dose levels up to 1000 mg/kg.
Based on these findings, under the conditions of this study, the NOAEL ofchromafenozide was determined to be 1000 mg/kg for the maternal rats and the fetuses.
Therefore, the teratogenic potential of chromafenozide was determined to be negative.
(The Institute of Environmental Toxicology, 1997)
2.

Teratogenicity Study in Rabbits

Chromafenozide technical suspended in 1% CMC aqueous solution was orally
administered daily at 0, 100, 300 and 1000 mg/kg to 18 copulated female Kbl: JW
rabbits per group for 13 days from 6 to 18 of gestation.
No test substance-related effects on the maternal rabbits were noted and any
induction of the fetal malformation or variation was caused at dose levels up to 1000
mg/kg.
Based on these findings, under the conditions of this study, the NOAEL ofchromafenozide was determined to be 1000 mg/kg for the maternal rabbits and thefetuses.

Therefore, the teratogenic potential of chromafenozide was determined to be

negative.
(The Institute of Environmental Toxicology, 1997)
MUTAGENICITY STUDIES
1.

Reverse Mutation Assay
Gene mutation potential of chromafenozide technical treated to histidine auxotrophic

strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and atryptophan auxotrophic strain of Escherichia coli (WP2 uvrA) with or without S-9 Mix
at 0, 313, 1250 and 5000 μg/plate was assessed using the reverse mutation assay(Ames test).
No increased revertant colonies were noted on the treated plates up to 5000 μg/plate,the highest concentration, comparing with control plates regardless with or without S-9
Mix.

Reverse mutation potential of chromafenozide was considered to be negative.

(BML, 1994)
2.

In Vitro Cytogenetics Test
Chromosome aberration potential of chromafenozide was assessed using cultured

Chinese hamster lung (CHL) cells by the direct method and the metabolic activation
method.

Dose levels of chromafenozide technical were 0, 78, 156, 313 and 625 μg/mL

for the 24 hr-treatment of the direct method and the metabolic activation method withand without S-9 Mix, and 0, 39, 78, 156 and 313 μg/ml for the 48 hr-treatment of thedirect method.
No increased incidence of abnormal cells was observed in treated cultures comparing
with control cultures.

Consequently, chromosome aberration potential ofchromafenozide was determined to be negative.
(The Environmental Toxicology Institute, 1995)

3.

DNA Repair Test (Rec-Assay)
DNA-damaging activity of chromafenozide was assessed with the DNA repair test

(rec-assay) using the recombination-proficient strain (H17, rec＋) and therecombination-deficient strain (M45, recE−) of Bacillus subtilis at 0, 313, 1250, 2500,
5000 and 10,000 μg/disc.
Comparable growth inhibitory zones were not observed in both strains of B. subtilis
up to the highest dose, 10,000 μg/disc, indicating a negative DNA-damaging activity ofchromafenozide.
(The Environmental Toxicology Institute, 1995)

GENERAL PHARMACOLOGICAL STUDIES
1.

Effects on Central Nervous System
1.1 Clinical signs and body weight in mice
Behavior of 3 male and 3 female ICR mice per group injected intraperitoneally withchromafenozide technical in 1% Tween 80 aqueous solution at 0, 320, 800, 2000 or 5000
mg/kg was examined according to the Irwin's method.
Slightly reduced body muscle tone was observed in both sexes received 5000 mg/kg at
1 hr after treatment, and decreased body weight in males received 5000 mg/kg on day 1of the study.
1.2 Toxicological signs and body weight in rats
Observation for acute toxicological signs after oral administration of chromafenozide
technical suspended in 1% Tween 80 aqueous solution at 0, 2000 and 5000 mg/kg was
performed with 6 male SD rats.
No abnormal findings and body weight changes were observed up to 5000 mg/kg, thehighest dose level.
1.3 Effects on the hexobarbital sleep in mice
Effects of chromafenozide technical on the sleeping time induced by subcutaneous
injection of hexobarbital at 100 mg/kg were assessed with 8 male ICR mice per groupwhich were received intraperitoneal injection of the test substance suspended in 1%
Tween 80 aqueous solution at 0, 128, 320, 800, 2000 and 5000 mg/kg at 1 hr before thehexobarbital treatment.
Prolonged sleeping time was noted at dose levels of 2000 and 5000 mg/kg.

1.4 Effects on body temperature of rats
Body temperature was measured after oral administration of chromafenozide
technical suspended in 1% Tween 80 aqueous solution at 0, 2000 and 5000 mg/kg to 6
male SD rats per group.
No treatment-related changes were observed.
2.

Effects on Respiratory and Cardiovascular Systems
Maximum blood pressure and heart rates were measured after oral administration ofchromafenozide technical suspended in 1% Tween 80 aqueous solution at 0, 2000 and5000 mg/kg to 6 male SD rats per group.
No treatment-related changes were observed.
3.

Effects on Autonomic Nervous System
Pupillometry was performed after oral administration of chromafenozide technicalsuspended in 1% Tween 80 aqueous solution at 0, 2000 and 5000 mg/kg to 6 male SD
rats per group.
No treatment-related changes were observed.
4.

Effects on Dygestive System
Transportability of charcoal meal administered orally at 10 ml/kg through the small

intestine was assessed with 8 male ICI mice per group into which chromafenozide
technical suspension had been injected intraperitoneally in 1% Tween 80 aqueous
solution at 0, 128, 320, 800, 2000 and 5000 mg/kg at 1 hr before the charcoal meal
administration.
Suppressed transportability of charcoal meal was noted at dose levels of 320 mg/kg or
more.
5.

Effects on Skeletal Muscle
Forelimb grip strength was measured after oral administration of chromafenozide

technical suspended in 1% Tween 80 aqueous solution at 0, 2000 and 5000 mg/kg to 6
male SD rats per group.
No treatment-related changes were observed.
6.

Effects on Blood
6.1 Effects on in vivo hemolysis and hemocoagulation
Prothrombin time, activated partial thromboplastin time and hemoglobin

concentration were measured with blood samples collected after oral administration ofchromafenozide technical suspension in 1% Tween 80 aqueous solution at 0, 2000 and5000 mg/kg to 6 male SD rats per group.
No treatment-related changes were observed.
6.2 Effects on in vitro hemolysis and hemocoagulation
Prothrombin time, activated partial thromboplastin time and hemoglobin
concentration were measured by adding chromafenozide technical solved in DMSO anddiluted with 1% Tween 80 saline solution at 0, 10−７, 10−６ and 10−５ g/ml toplasma and 10% erythrocyte suspension.
No treatment-related changes were observed.
(The Environmental Toxicology Institute, 1998)

SUMMARY
Results of various toxicology studies conducted for characterization of effects onhuman health demonstrate that chromafenozide is a pesticide with extremely low risk
properties.
The acute toxicity of chromafenozide to rats, mice and rabbits corresponds to the low
toxic substance group specified in the Japanese Toxic Substance Regulation Act. In
irritation studies, mild eye irritation induced by the technical substance, 5% SC and
0.3% D can be remedied by eye irrigation. No eye irritation was occurred with 5% SC
at field application rate (1/500 dilution).

Neither dermal irritation nor dermal

sensitization was observed.
Subchronic toxicity, chronic toxicity and oncogenicity studies with chromafenozide inrats, mice and dogs were revealed decreased body weight gain, mild anemia,
hepatocellular hypertrophy, increased weight of the liver and spleen, and darkening incolor and brown pigment deposition of the spleen without any tumorigenesis in animals
received the high dose of the test substance. In the reproduction study in rats and theteratology studies in rats and rabbits, no test substance-related effects were noted.

In

the three mutagenicity studies chromafenozide was demonstrated to be negative. In
general pharmacology studies, prolonged hexobarbital sleeping time and suppressedtransportability of charcoal meal in the small intestine were characteristic ofchromafenozide.
MATRIC® Flowable (5% SC) and MATRIC® Dust DL (0.3% D) were registered as
insecticides containing chromafenozide as the active ingredient by Japanese Ministry ofAgriculture, Forestry and Fisheries on December 27, 1999.

Contact
Marketing Department, Agrochemicals Division, Agro & Specialty Chemicals Group,
Nippon Kayaku Co., Ltd.
11‐2, Fujimi 1-chome, Chiyoda-ku, Tokyo 102‐8172
andCrop Protection Company, Sankyo Co., Ltd.
7‐12, Ginza 2-chome, Chuo-ku, Tokyo 104‐8113

Table

Acute toxity studies.

__________________________________________________________________________
Chroma-Speciesfenozide

Dosing

LD50 (mg/kg)

route

Male

FemaleLaboratory(year)__________________________________________________________________________
Technical

Rat

Oral

>5000

>5000

Mouse

Oral

>5000

>5000

IETd

RabittDermal

>2000

>2000

(1995)

Rat

Inhalation

>4.68

>4.68

(mg/l)(mg/l)__________________________________________________________________________
Oral>5000c>5000cDermal>2000c>2000c

Mouse

Oral

>5000c>5000cRat

Inhalation>5.4c*>5.4c*(mg/l)(mg/l)

Rat
5% SCaIETd__________________________________________________________ (1998)
Rat
0.3% Db
Mouse

Oral

>5000c>5000cDermal>2000c>2000cOral>5000c>5000c__________________________________________________________________________
aMATRIC®

Flowable;bMATRIC®

Environmental Toxicology; *LC50.

Dust DL;cValueas product;dThe

Institute of