METHOD FOR PREDICTION ABOUT CARCINOGENICITY OF SUBSTANCE IN RODENT

TECHNICAL FIELD

The present invention relates to a carcinogenicity prediction method for predicting the carcinogenicity of a test substance in a rodent by measuring the expression level of mRNA expressed from given genes after administering the test substance to a rodent such as a rat or a mouse.

BACKGROUND ART

Evaluation of long-term toxicity is one of the evaluation items of the hazard of a chemical substance. In order to evaluate the long-term toxicity of a chemical substance such as carcinogenicity, it is necessary to perform an animal experiment requiring considerable expense and a long testing period.

In an animal experiment for evaluating the carcinogenicity of a chemical substance, the chemical substance is continuously administered to a test animal until cancer is developed in the test animal or until the test animal dies. Since cancer is developed after a long latent period, a long-term animal experiment is needed.

On the other hand, due to a significant advancement of technology associated with genomic information in recent years, the evaluation of the hazard of a chemical substance is starting to be performed at the gene level. For example, Patent Document 1 describes a method for predicting the toxic activity of a chemical substance by detecting a difference in the expression of a gene in a tissue or a cell exposed to the chemical substance.

Also in the evaluation of the carcinogenicity of a chemical substance, it is predicted that there is a gene related to the mechanism of carcinogenesis. It is considered that the carcinogenicity of a chemical substance at the genetic level can be evaluated by detecting a difference in the expression of such a gene. However, the mechanism of carcinogenesis caused by a chemical substance at the genetic level has hardly been elucidated. Accordingly, it is very difficult to predict the carcinogenicity of a chemical substance from a difference in the gene expression.

The present inventors comprehensively obtained the information of the gene expression profiling of rats using a DNA microarray and found a method for predicting the carcinogenicity of a test substance from the gene-expression patterns, which was applied for a patent previously (Patent Document 2).

In this method, carcinogens are divided into three groups in advance according to the similarity of the gene-expression pattern. This method is a prediction method in which the gene-expression pattern common in each of these 3 groups is compared with the gene expression pattern for a test substance, and the carcinogenicity of the test substance is predicted from the degree of consistency of the gene expression pattern. In this method, the gene-expression patterns for a lot of carcinogens are obtained in advance, and the gene-expression patterns are prepared in advance for each group of carcinogens. Subsequently, the degree of consistency between these prepared gene-expression patterns and the gene expression pattern for the test substance is calculated. In order to calculate this degree of consistency, it is necessary to acquire an enormous quantity of data and to perform a computation processing. Therefore, the development of a simpler method for predicting the carcinogenicity of a test substance has been demanded.

- Patent Document 1: JP-A-2003-304888 (Claims)
- Patent Document 2: JP-A-2007-54022 (Claims)

DISCLOSURE OF THE INVENTION
Problems that the Invention is to Solve

An object of the invention is to provide a simple carcinogenicity prediction method which is a method for predicting the carcinogenicity of a test substance in a rodent in a short period by detecting an increase or a decrease in the expression of genes which have relevance to be involved in the mechanism of carcinogenesis at an early stage of development of cancer, and does not require acquisition of an enormous quantity of data or complicated calculation.

Means for Solving the Problems

The present inventors examined in detail the expression level of mRNA expressed from the respective genes for each of the three groups of carcinogens. As a result, the inventors found that a group of genes for which the expression level of mRNA is different between a group with the administration of carcinogens and a group with the administration of non-carcinogens apparently exists for each group of carcinogens. It was confirmed that by using these genes in combination, the carcinogenicity of a test substance can be predicted, and thus, the invention has been completed.

That is, the invention for achieving the above object is as described below.

(1) A method for predicting the carcinogenicity of a test substance in a rodent, comprising the steps of:

administering a solution of the test substance prepared by dissolving or dispersing the test substance in a solvent to a test group and administering the solvent used for the preparation of the solution of the test substance to a control group;

extracting mRNA from each group after a period of administration of the solution of the test substance or the solvent to each group;

measuring the expression level of mRNA expressed from each of genes obtained by selecting one or more genes from each of the following (A) to (C):

(A) genes each comprising a nucleotide sequence depicted in any one of SEQ ID NOs: 1 to 5;

(B) genes each comprising a nucleotide sequence depicted in any one of SEQ ID NOs: 6 to 8; and

determining whether or not a significant difference in the expression level of mRNA expressed from each of the selected genes is observed between the test group and the control group by a significant difference test; and

(2) A method for predicting the carcinogenicity of a test substance in a rodent, comprising the steps of:

extracting mRNA from each group after a period of administration of the solution of the test substance or the solvent to each group;

measuring the expression level of mRNA expressed from each of genes obtained by selecting one or more genes from each of the following (A) to (C):

(A) genes each comprising a nucleotide sequence depicted in any one of SEQ ID NOs: 1 to 5;

(B) genes each comprising a nucleotide sequence depicted in any one of SEQ ID NOs: 6 to 8; and

determining that the test substance has carcinogenicity when a significant difference in the expression level of mRNA expressed from any one of the selected genes is observed between the test group and the control group and the direction of increase or decrease in the expression level of the mRNA in the test group relative to the control group is the same as that previously defined for each gene from which the mRNA is expressed, and determining that the test substance has no carcinogenicity when a significant difference in the expression level of mRNA is not observed between the test group and the control group for all of the selected genes, or even if a significant difference in the expression level of mRNA expressed from any one of the selected genes is observed between the test group and the control group, when the direction of increase or decrease in the expression level of the mRNA expressed from the gene in the test group relative to the control group is not the same as that previously defined for each gene.

(3) The method for predicting the carcinogenicity of a test substance in a rodent according to (1) or (2), wherein the period of administration of the solution of the test substance is from 1 to 90 days.

(4) The method for predicting the carcinogenicity of a test substance in a rodent according to (1) or (2), wherein a test animal in the test group and the control group is a rat, a mouse, a hamster, or a guinea pig.

(5) The method for predicting the carcinogenicity of a test substance in a rodent according to (1) or (2), wherein the gene selected from (A) is a gene comprising a nucleotide sequence depicted in SEQ ID NO: 2, the gene selected from (B) is a gene comprising a nucleotide sequence depicted in SEQ ID NO: 7, and the gene selected from (C) is a gene comprising a nucleotide sequence depicted in SEQ ID NO: 10.

(6) A method for predicting the carcinogenicity of a test substance in a rodent, comprising the steps of:

extracting mRNA from each group after a period of administration of the solution of the test substance or the solvent to each group;

measuring the expression level of mRNA expressed from each of genes obtained by selecting one or more genes from each of the following (A) to (C):

(A) genes each comprising a nucleotide sequence depicted in any one of SEQ ID NOs: 1 to 5;

(B) genes each comprising a nucleotide sequence depicted in any one of SEQ ID NOs: 6 to 8; and

(D) a gene comprising a nucleotide sequence depicted in SEQ ID NO: 33;

determining whether or not a significant difference in the expression level of mRNA expressed from each of the genes is observed between the test group and the control group by a significant difference test; and

determining that the test substance has carcinogenicity when a significant difference in the expression level of mRNA expressed from any one of the genes is observed between the test group and the control group and the direction of increase or decrease in the expression level of the mRNA in the test group relative to the control group is the same as that previously defined for each gene from which the mRNA is expressed.

(7) The method for predicting the carcinogenicity of a test substance in a rodent according to (6), wherein the period of administration of the solution of the test substance is from 1 to 90 days.

(8) The method for predicting the carcinogenicity of a test substance in a rodent according to (6), wherein the gene selected from (A) is a gene comprising a nucleotide sequence depicted in SEQ ID NO: 2, the gene selected from (B) is a gene comprising a nucleotide sequence depicted in SEQ ID NO: 7, and the gene selected from (C) is a gene comprising a nucleotide sequence depicted in SEQ ID NO: 10.

ADVANTAGE OF THE INVENTION

In the invention, by measuring the expression level of mRNA expressed from given genes, the carcinogenicity of a test substance in a rodent such as a rat, a mouse, a hamster, or a guinea pig can be predicted. The calculation necessary for the prediction is calculation of a significant difference in the expression level of mRNA between a test group and a control group. The comparison of the expression level of mRNA may be performed for only about three or four genes, and the method needs only extremely simple data processing.

The administration of the test substance to a rodent is performed for a short period of time ranging from about 1 to 90 days. Therefore, a long-term animal experiment in which, for example, repeated administration of a compound is performed until cancer is developed in a test animal is not needed.

According to the invention, the carcinogenicity of a test substance in a rodent can be predicted with high accuracy by a short-term test and simple data processing.

BEST MODE FOR CARRYING OUT THE INVENTION

The method for predicting the carcinogenicity of a test substance of the invention is performed by the following procedure.

First, in order to administer a test substance to a test group, a solution of the test substance is prepared by dissolving or dispersing the test substance in a solvent.

The test substance to be used as a target for the method for predicting the carcinogenicity in the invention is an arbitrary chemical substance. The form of the test substance may be any form of a solid, a powder, a liquid, and a mixture thereof. The test substance is appropriately formed into a solution or a dispersion and is subjected to a test.

The solvent to be used for dissolving the test substance is not particularly limited and any solvent can be used as long as it is a non-carcinogenic vehicle capable of dissolving or dispersing the test substance. For example, a solvent widely used in an animal experiment such as corn oil or purified water can be exemplified. When the test substance is dispersed, a non-carcinogenic dispersant such as a detergent can be used.

The dose of the test substance is preferably a dose which causes a moderate increase or decrease in the expression level of mRNA in a test animal due to stimulation with the test substance. The dose thereof can be determined based on the lethal dose 50% (LD₅₀) value of the test substance in the test animal. The daily dose thereof is preferably from 1/250 to ½ the LD₅₀value, more preferably from 1/50 to ½ the LD₅₀value, furthermore preferably from 1/10 to ½ the LD₅₀value.

The thus prepared solution of the test substance is administered to the test group, and the solvent used for the preparation of the solution of the test substance is administered to the control group. The volume of the solvent to be administered to the control group is set to the same volume as the solution of the test substance administered to the test group. The test animal in the test group and the control group is a rodent such as a rat, a mouse, a hamster, or a guinea pig.

The period of administration to each group is set to 1 to about 90 days. From the viewpoint that the test is performed more rapidly, the administration period is preferably from 1 to 28 days, more preferably from 1 to 14 days. It is preferred that during the administration period, the solution of the test substance or the solvent is repeatedly administered once to several times a day (preferably once a day).

The method for administration of the solution of the test substance or the solvent to the test animal is not particularly limited, and a widely used method such as oral administration, intraperitoneal administration, or intravenous administration can be used.

After completion of the administration period, a tissue is immediately collected from each of the test animals in the test group and the control group. After mRNA is extracted and purified from the tissue of each of the test animals by a known method, the expression level of mRNA is measured.

Examples of the tissue to be collected for measuring the expression level of mRNA include liver, intestine, lung, kidney, stomach, spleen, brain, and blood.

As the method for measuring the expression level of mRNA, a known method such as a method in which fluorescently labeled cDNA or cRNA prepared from the mRNA is hybridized to a DNA microarray or microplate on which cDNA or DNA having a sequence complementary to the mRNA has been immobilized, Northern blotting, quantitative RT-PCR, or an RNase protection assay can be used.

In the case where the expression level of mRNA is measured using a DNA microarray, it is possible to use a commercially available product such as GeneChip (trade name, manufactured by Affymetrix, Inc.) or Rat Oligo Microarray Kit (trade name, manufactured by Agilent Co., Ltd.) as the array.

The mRNA to be measured for the expression level is mRNA expressed from each of genes in the following (A) to (C):

(A) genes each comprising a nucleotide sequence depicted in any one of SEQ ID NOs: 1 to 5;

(B) genes each comprising a nucleotide sequence depicted in any one of SEQ ID NOs: 6 to 8; and

In the invention, one or more genes are selected from each of the above (A) to (C), and the expression level of mRNA expressed from each of the selected genes is measured. The number of genes to be selected from each of the groups (A) to (C) is an arbitrary number of 1 or greater with the proviso that the number of genes belonging to the respective groups is the upper limit. The number of genes to be selected from each of the groups (A) to (C) may be the same or different. From the viewpoint that the operation is simplified while maintaining the accuracy of prediction of carcinogenicity to be high, the number of genes to be selected from (A) to (C) is preferably from 1 to about 3, more preferably 1 or 2, and most preferably 1, respectively.

The expression level of mRNA expressed from each of the selected genes is measured for each of the test group and the control group. Thereafter, the measured expression level of mRNA is compared between the test group and the control group. When the measured expression level of mRNA satisfies both of the following two requirements (1) and (2), the test substance is determined to have carcinogenicity.

(1) A significant difference in the expression level of mRNA expressed from at least any one of the genes measured for the expression level of mRNA is observed between the test group and the control group.

(2) The direction of increase or decrease in the expression level of mRNA expressed from at least any one of the genes for which a significant difference in the expression level of mRNA was observed in (1) in the test group relative to the expression level of mRNA in the control group is the same as that defined for each gene.

The determination as to whether or not a significant difference in the expression level of mRNA is observed between the test group and the control group is performed by a significant difference test. In the significant difference test, a known test method such as a t-test, a U-test, an F-test, a Dunnett method, a Tukey method, a Kruskal-Wallis test, a Wilcoxon test, or a Steel-Dwass method can be adopted.

The direction defined for each gene is a direction which indicates a change, either an increase or a decrease in the expression level of mRNA expressed from each gene when a carcinogen was administered to a rodent. Specifically, it is the direction of increase or decrease shown in the following Table 1.

TABLE 1

Direction of increase (+)

Gene group
SEQ ID NO
or decrease (−)

A
1 to 4
+

5
−

B
6, 7
+

8
−

C
9 to 18
+

19 to 32
−

The present inventors have found that the gene expression patterns obtained when each of a lot of carcinogens was administered to a rodent are divided into three patterns according to the similarity thereof. The groups of the carcinogens divided into three patterns are as follows.

(Group 1)

2,4-diaminotoluene, quinoline, diethylnitrosamine, 2-nitropropane, N-nitrosomorpholine, furan, N-nitrosodimethylamine, N-nitrosopiperidine, 2-acetylaminofluorene, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, methylcarbamate, thioacetoamide, urethane, acetoamide, methapyrilene hydrochloride, 3′-methyl-4-dimethylaminoazobenzene, 1,4-dioxane

(Group 2)

safrole, clofibrate, di(2-ethylhexyl)phthalate, hexachlorobenzene, α-hexachlorocyclohexane, D,L-ethionine, chlorendic acid, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, 4-nitroquinoline-1-oxide, N-ethyl-N-nitrosourea, benzo[a]pyrene, 4-dimethylaminoazobenzene, aldrin, di(2-ethylhexyl)adipate, trichloroethylene, butylated hydroxyanisole, d-limonene, tetrachloroethylene, 1,4-dichlorobenzene, phenyloin sodium salt, trichloroacetic acid

(Group 3)

ethynyl estradiol, chloroform, benz[a]anthracene, pentachloroethane, diethylstilbestrol, phenobarbital

The above-mentioned genes in (A), (B), and (C) are genes for which the expression level of mRNA significantly increases or decreases to the direction as shown in Table 1 when the carcinogens in Group 1, Group 2, and Group 3 were administered to a rodent, respectively. The genes in (A) to (C) are genes for which the expression level of mRNA is not significantly increased or decreased by the administration of the carcinogens in other groups or non-carcinogens. Therefore, when the expression level of mRNA expressed from any one of the genes selected from each of (A) to (C) significantly increases or decreases to the direction as shown in Table 1, the test substance is determined to have carcinogenicity, and in the case where there is no such a gene, the test substance is determined to have no carcinogenicity.

Among the genes in the above (A) to (C), a combination with particularly high prediction accuracy is a combination selected from the genes each comprising a nucleotide sequence depicted in the sequence identification number (SEQ ID NO) shown below.

- (A) SEQ ID NOs: 1 to 4
- (B) SEQ ID NOs: 6 and 7
- (C) SEQ ID NOs: 9, 10, 14, 15, 17, 21, 22, 30, 31, and 32

Among the combinations selected from the above-mentioned genes, combinations shown below are more preferred.

- (A) SEQ ID NO: 1, (B) SEQ ID NO: 7, (C) SEQ ID NO: 10
- (A) SEQ ID NO: 3, (B) SEQ ID NO: 7, (C) SEQ ID NO: 10
- (A) SEQ ID NO: 4, (B) SEQ ID NO: 7, (C) SEQ ID NO: 10
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 9
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 10
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 14
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 15
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 17
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 21
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 22
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 30
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 31
- (A) SEQ ID NO: 2, (B) SEQ ID NO: 7, (C) SEQ ID NO: 32

Further, among the above-mentioned combinations, the most preferred combination is a combination of (A) a gene having a nucleotide sequence depicted in SEQ ID NO: 2, (B) a gene having a nucleotide sequence depicted in SEQ ID NO: 7, and (C) a gene having a nucleotide sequence depicted in SEQ ID NO: 10.

As another prediction method of the invention, there is a method in which the expression level of mRNA expressed from a gene comprising a nucleotide sequence depicted in SEQ ID NO: 33 as a gene (D) is also measured in addition to the genes in the above (A) to (C).

The gene (D) is basically a gene for which the expression level of mRNA decreases when a carcinogen in Group 2 was administered to a rodent.

The carcinogens in Group 2 have a tendency that a gene for which a significant difference in the expression level of mRNA is observed is different between liver carcinogens and the other carcinogens. In the case of the genes in the above (B), a significant difference in the expression level of mRNA is sometimes not observed by the administration of carcinogens other than liver carcinogens. Therefore, by using the gene in (B) and the gene (D) in combination, the accuracy of the prediction for the carcinogens in Group 2 is improved. Therefore, by measuring the expression level of mRNA expressed from each of the genes in the above (A) to (C) and the gene (D), the occurrence of cases where a carcinogen in Group 2 is erroneously determined to be a non-carcinogen is decreased, and the accuracy of the prediction of carcinogenicity is further increased.

EXAMPLES
Examination Example 1

A test solution was prepared by dissolving each of the carcinogens and the non-carcinogens shown in Tables 2 to 6 in the corresponding solvent shown in Tables 2 to 6. Each of the thus prepared test solutions and the solvents used for the preparation was administered by oral gavage to rats in each group. As the rats, male rats (F344 SPF rats) at 5 weeks of age obtained from Charles River Laboratories Japan, Inc. were used. The animals were divided into groups, each containing four individuals. The volume of the solvent administered to the vehicle control group was set to the same volume as that of the test solution administered to the carcinogen administration group or the non-carcinogen administration group. The administration of the test solution or the solvent was performed once daily for 28 days. Apiece was excised from the liver of each of the rats after a lapse of 28 days from the initiation of the administration, and the total RNA was extracted and purified. By using an in-house oligo-DNA microarray (number of UniGene ID based on UniGene database released on 2007/05/16: 6,689, length of gene per probe: 60 mer), the amount of each mRNA contained in the total RNA was measured.

The substance numbers (Substance No.) of the used carcinogens and non-carcinogens, the solvents used for the preparation of the test solutions, and the doses administered to the rats are shown in Tables 2 to 6.

TABLE 2

Substance

Dose

Chemical substance
No.
Solvent
(mg/kg/day)

Clofibrate
TS001
Corn oil
250

Di(2-ethylhexyl)phthalate
TS002
Corn oil
300

Carbon tetrachloride
TS003
Corn oil
50

2,4-Diaminotoluene
TS004
Water
10

2,6-Diaminotoluene
TS005
Water
10

Quinoline
TS006
Corn oil
25

8-Hydroxyquinoline
TS007
Corn oil
25

Phenobarbital
TS008
Water
100

D-Mannitol
TS009
Water
1000

L-Ascorbic acid
TS010
Water
1000

Diethylnitrosamine
TS011
Water
20

2-Nitro-p-phenylenediamine
TS012
1 w/v %
100

CMCNa

2-Nitropropane
TS013
Corn oil
40

N-Nitrosomorpholine
TS014
Water
10

Aldrin
TS015
Corn oil
0.3

Dichloro-diphenyl-trichloroethane
TS016
Corn oil
25

Dieldrin
TS017
Corn oil
0.3

TABLE 3

Substance

Dose

Chemical substance
No.
Solvent
(mg/kg/day)

Di(2-ethylhexyl)adipate
TS018
Corn oil
1000

Ethinylestradiol
TS019
Corn oil
0.5

Hexachlorobenzene
TS020
Corn oil
5

α-Hexachlorocyclohexane
TS021
Corn oil
20

Trichloroethylene
TS022
Corn oil
700

Butylated hydroxyanisole
TS023
Corn oil
750

D-Limonene
TS024
Corn oil
1000

Safrole
TS025
Corn oil
300

1,4-Dichlorobenzene
TS026
Corn oil
300

1,4-Dioxane
TS027
Water
1000

Furan
TS028
Corn oil
10

Methyl carbamate
TS029
Water
500

Thioacetoamide
TS030
Water
20

Acetaminophen
TS031
1% CMC-
700

0.1% Tween 80

2-Chloroethanol
TS032
Water
40

TABLE 4

Substance

Dose

Chemical substance
No.
Solvent
(mg/kg/day)

2-Chloromethylpyridine HCl
TS033
Water
150

DL-Menthol
TS034
Corn oil
1000

4-Nitro-o-phenylenediamine
TS035
1 w/v % CMCNa
250

1-Nitropropane
TS036
Corn oil
80

Quercetin
TS037
1 w/v % CMCNa
200

Benzoin
TS038
5.0 w/v % aqueous
500

solution of gum arabic

Iodoform
TS039
Corn oil
200

Lithocholic acid
TS040
5.0 w/v % aqueous
1000

solution of gum arabic

N-Nitrosodimethylamine
TS041
Water
0.2

N-Nitrosopiperidine
TS042
Water
10

2-Acetylaminofluorene
TS043
Corn oil
6

2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline
TS044
1 w/v % CMCNa
20

2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine
TS045
1 w/v % CMCNa
5

Benz[a]anthracene
TS046
Corn oil
50

7,12-Dimethylbenz [a]anthracene
TS047
Corn oil
1

3-Methylcholanthrene
TS048
Corn oil
2

4-Nitroquinoline-1-oxide
TS049
Corn oil
2

N-Ethyl-N-nitrosourea
TS050
Water
3

TABLE 5

Substance

Dose

Chemical substance
No.
Solvent
(mg/kg/day)

Trichloroacetic acid
TS051
Water
300

Tannic acid
TS052
Water
1000

Methapyrilene HCl
TS053
Water
50

Urethane
TS054
Water
80

Pentachloroethane
TS055
Corn oil
200

Chloroform
TS056
Corn oil
90

Lindane
TS057
Corn oil
10

2-Chloro-p-phenylenediamine SO₄
TS058
1 w/v %
100

CMCNa

p-Phenylenediamine 2HCl
TS059
Water
60

2,5-Toluenediamine SO₄
TS060
1 w/v %
50

CMCNa

4-Acetylaminofluorene
TS061
Corn oil
40

α-Tocopherol (Vitamin E)
TS062
Corn oil
1000

Benzo[a]pyrene
TS063
Corn oil
15

1-Nitropyrene
TS064
Corn oil
5

Tetrachloroethylene
TS066
Corn oil
100

Acetamide
TS067
Water
1180

Diethylstilbestrol
TS068
Corn oil
10

Phenytoin Na
TS069
Water
160

D,L-Ethionine
TS070
Corn oil
30

Aspirin
TS071
Corn oil
27

TABLE 6

Substance

Dose

Chemical substance
No.
Solvent
(mg/kg/day)

4-(Chloroacetyl)acetanilide
TS072
Corn oil
250

Phthalamide
TS073
Corn oil
1000

Caprolactam
TS074
Water
375

4-Aminoazobenzene
TS075
Corn oil
50

3′-Methyl-4-
TS076
Corn oil
50

dimethylaminoazobenzene

4-Dimethylaminoazobenzene
TS077
Corn oil
50

Chlorendic acid
TS078
Water
100

1-Chloro-2-propanol
TS079
Water
100

3-Chloro-p-toluidine
TS080
Corn oil
300

Glutaraldehyde
TS081
Water
50

4-Nitroanthranilic acid
TS082
Corn oil
1,000

1-Nitronaphthalene
TS083
Corn oil
100

Sodium benzoate
TS084
Water
1000

Indomethacin
TS085
Corn oil
5

Methyleugenol
TS086
0.5 w/v % MC
40

o-Nitrotoluene
TS087
Corn oil
300

Tris(2-chloroethyl)phosphate
TS088
Corn oil
88

The expression level of mRNA was compared between each of the chemical substance administration groups and the corresponding vehicle control group, and genes that satisfy the requirements described in (A) to (D) were selected, respectively.

(A) Genes for which the number of substances causing a significant change in the expression level among the carcinogens (17 substances) in Group 1 shown in Table 7 is 14 or more and the number of substances causing a significant change in the expression level among the non-carcinogens (26 substances) is 8 or less.

(B) Genes for which the number of substances causing a significant change in the expression level among the carcinogens (23 substances) in Group 2 shown in Table 7 is 13 or more and the number of substances causing a significant change in the expression level among the non-carcinogens (26 substances) is 8 or less.

(C) Genes for which the number of substances causing a significant change in the expression level among the carcinogens (6 substances) in Group 3 shown in Table 7 is 5 or more and the number of substances causing a significant change in the expression level among the non-carcinogens (26 substances) is 8 or less.

(D) Genes for which the expression level is significantly changed by a carcinogen by which the expression level for the gene in (B) is not changed among the carcinogens in Group 2 shown in Table 7.

TABLE 7

Substance No.

Carcin-
Group 1
TS004, TS006, TS011, TS013, TS014, TS027, TS028

ogen

TS029, TS030, TS041, TS042, TS043, TS044, TS053,

TS054, TS067, TS076

Group 2
TS001, TS002, TS015, TS018, TS020, TS021, TS022,

TS023, TS024, TS025, TS026, TS045, TS047, TS048,

TS049, TS050, TS051, TS063, TS066, TS069, TS070,

TS077, TS078

Group 3
TS008, TS019, TS046, TS055, TS056, TS068

Non-carcinogen
TS005, TS007, TS009, TS010, TS032, TS033, TS034,

TS035, TS038, TS039, TS040, TS057, TS058, TS059,

TS060, TS062, TS071, TS072, TS073, TS074, TS079,

TS080, TS082, TS083, TS084, TS085

The determination as to whether or not a significant difference in the expression level of mRNA is observed between the carcinogen administration group or the non-carcinogen administration group and the vehicle control group was performed as follows. The case where the expression level of mRNA in the carcinogen administration group or the non-carcinogen administration group is 1.5 times or more or 1/1.5 or less of the expression level of the corresponding mRNA expressed in the control group was determined to be significant. In this connection, the determination as to whether or not the change is significant was performed by taking into consideration the direction of increase or decrease in the expression level. In the case where the direction of increase or decrease is different as compared with the case where another carcinogen was administered, the change was determined to be not significant.

The gene numbers (Gene No.) of the genes selected by the above-mentioned method, the sequence identification numbers (SEQ ID NO) depicting the nucleotide sequences of the probes used in the detection of mRNA expressed from the genes, Unigene numbers (Unigene No.) of the genes, the sequence identification numbers (SEQ ID NO) depicting the nucleotide sequences of the genes listed in the Unigene database, the direction of the change in the expression level when the carcinogens were administered, and the number of substances which caused a change in the expression level are shown in Table 8.

TABLE 8

Nucleotide

Nucleotide

Number of substances causing

sequence of

sequence of

change in the expression level

probe

Unigene
Direction of
Carcinogens in
Non-

Gene No.
(SEQ ID NO)
Unigene No.
(SEQ ID NO)
change
Groups 1 to 3
carcinogens

A
4218
1
Rn.167075
34
+
14
2

4846
2
Rn.144554
35
+
17
2

5600
3
Rn.9836
36
+
14
2

6182
4
Rn.5834
37
+
15
6

2026
5
Rn.19133
38
−
14
6

B
2045
6
Rn.19329
39
+
14
6

2203
7
Rn.21240
40
+
14
4

1494
8
Rn.1430
41
−
13
8

C
533
9
Rn.106184
42
+
5
8

813
10
Rn.201760
43
+
5
3

893
11
Rn.164817
44
+
6
6

1119
12
Rn.9757
45
+
6
5

1163
13
Rn.11766
46
+
5
5

1334
14
Rn.120914
47
+
5
4

2494
15
Rn.23969
48
+
5
1

3929
16
Rn.4620
49
+
5
5

6620
17
Rn.120914
50
+
5
4

6742
18
Rn.9779
51
+
5
6

154
19
Rn.185941
52
−
6
8

725
20
Rn.108075
53
−
5
5

1131
21
Rn.144946
54
−
5
6

1562
22
Rn.14744
55
−
5
3

1741
23
Rn.1647
56
−
6
6

1993
24
Rn.18728
57
−
6
5

2546
25
Rn.24561
58
−
6
7

3776
26
Rn.43232
59
−
5
5

4095
27
Rn.119024
60
−
6
6

4973
28
Rn.118529
61
−
6
8

5634
29
Rn.9862
62
−
6
8

5878
30
Rn.143213
63
−
5
0

6026
31
Rn.167685
64
−
5
3

6303
32
Rn.123063
65
−
5
7

D
5726
33
Rn.9939
66
−

The following Tables 9 to 14 show the behavior of each gene selected by the requirements (A) to (D) when a chemical substance was administered. The case of “+” indicates that a significant difference in the expression level of mRNA was observed between the chemical substance administration group and the vehicle control group by the administration of the chemical substance and the expression level of mRNA was changed in the direction defined for the gene. The case of “−” indicates that a significant difference in the expression level of mRNA was not observed between the chemical substance administration group and the vehicle control group or the direction of the change was not the same as that previously defined.

TABLE 9

Substance No.
TS004
TS006
TS011
TS013
TS014
TS027
TS028
TS029

Liver carcinogenicity
Cr+
Cr+
Cr+
Cr+
Cr+
Cr+
Cr+
Cr+

Carcinogenicity
C+
C+
C+
C+
C+
C+
C+
C+

Mutagenicity by Ames test
M+
M+
M+
M+
M+
M−
M+
M−

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
+
−
+
+
+
+
+
+

4846
Rn.144554
+
+
+
+
+
+
+
+

5600
Rn.9836
+
−
+
+
+
+
+
+

6182
Rn.5834
+
−
+
+
+
+
+
+

2026
Rn.167075
+
+
+
+
+
+
+
+

(B)
2045
Rn.19329
−
−
−
−
−
−
−
−

2203
Rn.21240
−
−
−
−
−
−
−
−

1494
Rn.1430
+
+
+
+
+
+
+
+

(D)
5726
Rn.9939
−
−
−
−
−
−
−
−

(C)
533
Rn.106184
−
−
+
+
−
+
+
+

813
Rn.201760
−
+
−
−
+
+
+
−

893
Rn.164817
−
−
+
−
−
−
−
−

1119
Rn.9757
−
−
−
−
−
+
+
−

1163
Rn.11766
−
−
−
−
+
−
−
−

1334
Rn.120914
−
−
−
−
−
−
−
−

2494
Rn.23969
−
−
+
−
−
−
−
−

3929
Rn.4620
−
−
−
−
−
−
+
−

6620
Rn.120914
−
−
−
−
−
−
−
−

6742
Rn.9779
−
−
−
−
−
+
+
−

154
Rn.185941
−
−
+
+
−
+
+
+

725
Rn.108075
+
−
−
+
−
−
−
−

1131
Rn.144946
−
−
−
−
−
−
−
−

1562
Rn.14744
−
−
+
+
−
+
+
+

1741
Rn.1647
−
−
+
−
−
+
+
+

1993
Rn.18728
−
−
+
+
+
+
+
+

2546
Rn.24561
−
−
+
−
−
−
−
−

3776
Rn.43232
−
−
−
−
−
−
+
+

4095
Rn.119024
−
−
+
−
−
−
+
−

4973
Rn.118529
−
−
+
+
−
−
+
−

5634
Rn.9862
−
−
−
−
−
−
+
−

5878
Rn.143213
−
−
−
−
−
−
+
−

6026
Rn.167685
−
−
+
−
−
−
−
−

6303
Rn.123063
−
−
+
+
−
−
+
+

Substance No.
TS030
TS041
TS042
TS043
TS044
TS053
TS054
TS067

Liver carcinogenicity
Cr+
Cr+
Cr+
Cr+
Cr+
Cr+
Cr+
Cr+

Carcinogenicity
C+
C+
C+
C+
C+
C+
C+
C+

Mutagenicity by Ames test
M−
M+
M+
M+
M+
M−
M−
M−

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
+
+
+
+
+
+
−
−

4846
Rn.144554
+
+
+
+
+
+
+
+

5600
Rn.9836
+
+
+
+
+
+
−
−

6182
Rn.5834
+
+
+
+
+
+
+
−

2026
Rn.167075
+
−
+
+
−
+
−
+

(B)
2045
Rn.19329
−
−
−
−
−
−
−
−

2203
Rn.21240
−
−
−
−
−
−
−
−

1494
Rn.1430
+
−
+
−
−
+
−
−

(D)
5726
Rn.9939
+
−
+
−
−
−
+
−

(C)
533
Rn.106184
+
−
+
−
−
+
−
+

813
Rn.201760
+
−
+
−
−
−
−
−

893
Rn.164817
+
−
−
+
−
+
−
−

1119
Rn.9757
−
−
−
−
−
+
−
−

1163
Rn.11766
−
−
−
+
+
−
−
−

1334
Rn.120914
−
−
−
−
−
−
−
−

2494
Rn.23969
+
−
−
−
−
+
−
−

3929
Rn.4620
−
−
−
−
−
−
−
−

6620
Rn.120914
−
−
−
−
−
−
−
−

6742
Rn.9779
−
+
−
−
−
−
−
−

154
Rn.185941
+
−
−
−
−
+
−
+

725
Rn.108075
+
−
−
+
+
−
−
−

1131
Rn.144946
−
−
+
−
−
−
−
−

1562
Rn.14744
+
−
+
+
−
+
−
−

1741
Rn.1647
+
−
−
−
−
+
−
−

1993
Rn.18728
+
−
+
+
−
+
−
−

2546
Rn.24561
−
−
−
−
−
+
−
−

3776
Rn.43232
+
−
−
−
−
+
−
−

4095
Rn.119024
+
−
−
+
−
+
−
−

4973
Rn.118529
+
−
−
+
−
+
−
−

5634
Rn.9862
−
−
−
−
−
+
−
−

5878
Rn.143213
−
−
−
−
−
+
−
−

6026
Rn.167685
−
−
−
−
+
+
−
−

6303
Rn.123063
+
−
+
+
−
+
−
+

TABLE 10

Substance No.
TS076
TS001
TS002
TS015
TS018
TS020
TS021
TS022

Liver carcinogenicity
Cr+
Cr+
Cr+
Cr−
Cr−
Cr+
Cr+
Cr−

Carcinogenicity
C+
C+
C+
C+
C+
C+
C+
C+

Mutagenicity by Ames test
M+
M−
M−
M−
M−
M−
M−
M−

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
+
−
−
−
−
−
−
−

4846
Rn.144554
+
−
−
−
−
−
−
+

5600
Rn.9836
+
−
−
−
−
−
−
−

6182
Rn.5834
+
+
−
+
+
+
+
+

2026
Rn.167075
+
+
+
−
−
−
−
−

(B)
2045
Rn.19329
+
+
+
+
+
+
+
+

2203
Rn.21240
−
+
+
+
+
+
+
+

1494
Rn.1430
+
+
+
−
+
+
+
+

(D)
5726
Rn.9939
−
−
−
−
−
−
−
−

(C)
533
Rn.106184
+
+
+
−
+
+
+
−

813
Rn.201760
−
+
+
−
−
−
+
−

893
Rn.164817
−
+
−
−
−
−
−
−

1119
Rn.9757
−
−
−
−
−
−
−
−

1163
Rn.11766
−
+
−
−
−
−
−
−

1334
Rn.120914
−
−
−
−
−
−
−
−

2494
Rn.23969
−
−
−
+
−
−
−
−

3929
Rn.4620
−
−
−
−
−
−
−
−

6620
Rn.120914
−
−
−
−
−
−
−
−

6742
Rn.9779
−
+
+
−
−
−
+
+

154
Rn.185941
+
−
−
−
−
−
−
−

725
Rn.108075
+
+
+
−
−
−
−
−

1131
Rn.144946
−
−
+
−
−
−
−
−

1562
Rn.14744
+
−
−
−
−
−
−
−

1741
Rn.1647
+
−
−
−
−
−
+
−

1993
Rn.18728
+
−
−
−
−
−
−
−

2546
Rn.24561
−
−
−
−
−
−
−
−

3776
Rn.43232
−
−
−
−
−
−
−
−

4095
Rn.119024
+
+
+
−
+
−
+
−

4973
Rn.118529
+
+
+
−
+
−
+
−

5634
Rn.9862
−
−
−
−
−
−
+
−

5878
Rn.143213
−
−
−
−
−
−
−
−

6026
Rn.167685
−
−
−
−
−
−
−
−

6303
Rn.123063
+
−
−
−
−
−
+
−

Substance No.
TS023
TS024
TS025
TS026
TS045
TS047
TS048
TS049

Liver carcinogenicity
Cr−
Cr−
Cr+
Cr−
Cr−
Cr−
Cr−
Cr−

Carcinogenicity
C+
C+
C+
C+
C+
C+
C+
C+

Mutagenicity by Ames test
M−
M−
M−
M−
M+
M+
M+
M+

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical su bstance

(A)
4218
Rn.167075
−
−
+
−
−
−
−
−

4846
Rn.144554
−
−
+
−
−
−
−
−

5600
Rn.9836
−
−
+
−
−
−
−
−

6182
Rn.5834
−
−
+
−
−
−
−
−

2026
Rn.167075
−
−
−
−
−
−
+
−

(B)
2045
Rn.19329
−
−
+
−
+
−
−
−

2203
Rn.21240
+
−
+
−
+
−
−
−

1494
Rn.1430
−
+
+
+
−
−
−
−

(D)
5726
Rn.9939
−
−
−
+
+
+
+
+

(C)
533
Rn.106184
−
+
+
+
−
−
−
−

813
Rn.201760
−
−
+
−
−
−
−
−

893
Rn.164817
−
−
−
−
−
−
−
−

1119
Rn.9757
−
−
−
−
−
−
−
−

1163
Rn.11766
+
−
−
−
−
+
−
−

1334
Rn.120914
−
−
−
−
−
−
−
−

2494
Rn.23969
−
−
−
−
+
−
−
−

3929
Rn.4620
−
−
−
−
−
−
−
−

6620
Rn.120914
−
−
−
−
−
−
−
−

6742
Rn.9779
−
−
+
−
−
−
−
−

154
Rn.185941
+
−
+
−
−
−
−
−

725
Rn.108075
+
−
+
−
−
+
−
−

1131
Rn.144946
−
−
−
−
−
−
−
−

1562
Rn.14744
−
−
−
−
−
−
−
−

1741
Rn.1647
−
−
+
−
−
−
−
−

1993
Rn.18728
−
−
−
−
−
−
−
−

2546
Rn.24561
−
−
−
−
−
−
−
−

3776
Rn.43232
−
−
+
−
−
−
−
−

4095
Rn.119024
+
−
+
+
−
−
−
−

4973
Rn.118529
+
−
+
−
−
−
−
−

5634
Rn.9862
−
−
+
−
−
−
+
−

5878
Rn.143213
−
−
−
−
−
−
−
−

6026
Rn.167685
−
−
−
+
−
−
−
−

6303
Rn.123063
−
−
+
+
−
−
−
−

TABLE 11

Substance No.
TS050
TS051
TS063
TS066
TS069
TS070
TS077
TS078

Liver carcinogenicity
Cr−
Cr−
Cr−
Cr−
Cr−
Cr+
Cr+
Cr+

Carcinogenicity
C+
C+
C+
C+
C+
C+
C+
C+

Mutagenicity by Ames test
M+
M−
M+
M−
M−
M−
M+
M−

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
−
−
−
−
−
−
−
−

4846
Rn.144554
−
−
−
−
−
−
−
−

5600
Rn.9836
−
−
−
−
−
−
−
−

6182
Rn.5834
+
−
+
+
+
−
+
−

2026
Rn.167075
−
−
+
−
−
−
−
+

(B)
2045
Rn.19329
−
−
+
+
+
−
+
+

2203
Rn.21240
−
−
+
−
+
+
−
+

1494
Rn.1430
−
−
−
−
+
+
+
+

(D)
5726
Rn.9939
+
+
−
−
−
−
+
−

(C)
533
Rn.106184
−
−
−
+
−
−
−
−

813
Rn.201760
−
−
−
−
−
−
−
−

893
Rn.164817
−
−
−
−
−
−
−
−

1119
Rn.9757
−
−
−
−
−
−
−
−

1163
Rn.11766
−
−
−
−
−
−
−
+

1334
Rn.120914
−
−
−
−
−
−
−
−

2494
Rn.23969
−
−
−
−
−
−
−
−

3929
Rn.4620
−
−
−
−
+
−
−
−

6620
Rn.120914
−
−
−
−
−
−
−
−

6742
Rn.9779
−
−
−
−
−
−
−
−

154
Rn.185941
−
−
−
−
+
−
−
−

725
Rn.108075
−
+
−
−
+
+
−
−

1131
Rn.144946
−
−
−
−
−
−
−
−

1562
Rn.14744
−
−
−
−
−
−
−
−

1741
Rn.1647
−
−
−
−
+
−
−
−

1993
Rn.18728
−
−
−
−
−
−
−
−

2546
Rn.24561
−
−
−
−
−
−
−
−

3776
Rn.43232
−
−
−
−
−
−
−
−

4095
Rn.119024
−
+
−
−
+
−
−
−

4973
Rn.118529
−
+
−
−
+
−
−
+

5634
Rn.9862
−
−
+
−
+
−
−
+

5878
Rn.143213
−
−
−
−
−
−
−
−

6026
Rn.167685
−
−
−
−
−
−
+
−

6303
Rn.123063
+
−
−
−
−
−
+
−

Substance No.
TS008
TS019
TS046
TS055
TS056
TS068
TS005
TS007

Liver carcinogenicity
Cr+
Cr+
Cr−
Cr−
Cr+
Cr−
Cr−
Cr−

Carcinogenicity
C+
C+
C+
C+
C+
C+
C−
C−

Mutagenicity by Ames test
M−
M−
M+
M−
M−
M−
M+
M+

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
−
−
−
−
−
+
−
−

4846
Rn.144554
+
+
−
−
−
+
−
−

5600
Rn.9836
−
−
−
−
−
+
−
−

6182
Rn.5834
−
+
+
−
−
+
−
−

2026
Rn.167075
−
−
+
−
+
−
−
−

(B)
2045
Rn.19329
−
−
−
−
−
−
−
−

2203
Rn.21240
−
+
+
−
−
−
−
−

1494
Rn.1430
+
−
−
+
+
−
−
−

(D)
5726
Rn.9939
−
−
+
+
−
−
−
+

(C)
533
Rn.106184
+
+
−
+
+
+
−
−

813
Rn.201760
+
−
+
+
+
+
−
−

893
Rn.164817
+
+
+
+
+
+
−
−

1119
Rn.9757
+
+
+
+
+
+
−
−

1163
Rn.11766
−
+
+
+
+
+
−
−

1334
Rn.120914
+
+
−
+
+
+
−
−

2494
Rn.23969
+
+
−
+
+
+
−
−

3929
Rn.4620
+
+
−
+
+
+
−
−

6620
Rn.120914
+
+
−
+
+
+
−
−

6742
Rn.9779
+
−
+
+
+
+
−
−

154
Rn.185941
+
+
+
+
+
+
−
−

725
Rn.108075
+
+
+
−
+
+
−
−

1131
Rn.144946
−
+
+
+
+
+
+
−

1562
Rn.14744
+
+
−
+
+
+
−
−

1741
Rn.1647
+
+
+
+
+
+
−
−

1993
Rn.18728
+
+
+
+
+
+
−
−

2546
Rn.24561
+
+
+
+
+
+
−
−

3776
Rn.43232
−
+
+
+
+
+
−
−

4095
Rn.119024
+
+
+
+
+
+
−
−

4973
Rn.118529
+
+
+
+
+
+
−
−

5634
Rn.9862
+
+
+
+
+
+
−
−

5878
Rn.143213
−
+
+
+
+
+
−
−

6026
Rn.167685
+
+
−
+
+
+
−
−

6303
Rn.123063
+
+
−
+
+
+
−
−

TABLE 12

Substance No.
TS009
TS010
TS032
TS033
TS034
TS035
TS038
TS039

Liver carcinogenicity
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−

Carcinogenicity
C−
C−
C−
C−
C−
C−
C−
C−

Mutagenicity by Ames test
M−
M−
M+
M+
M−
M+
M−
M+

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
−
−
−
−
−
−
−
−

4846
Rn.144554
−
−
−
−
−
−
−
−

5600
Rn.9836
−
−
−
−
−
−
−
−

6182
Rn.5834
−
−
−
−
−
−
−
−

2026
Rn.167075
−
−
−
−
+
+
+
−

(B)
2045
Rn.19329
−
−
−
−
+
−
−
−

2203
Rn.21240
−
−
−
−
+
−
−
−

1494
Rn.1430
−
−
−
−
+
+
−
+

(D)
5726
Rn.9939
−
−
−
−
−
−
−
−

(C)
533
Rn.106184
−
−
−
−
+
+
−
−

813
Rn.201760
−
−
−
−
−
+
−
−

893
Rn.164817
−
−
−
−
−
−
−
+

1119
Rn.9757
−
−
−
−
−
−
+
−

1163
Rn.11766
−
−
−
−
−
+
−
−

1334
Rn.120914
−
−
−
−
−
+
−
+

2494
Rn.23969
−
−
−
−
−
−
−
−

3929
Rn.4620
−
−
−
−
−
−
−
−

6620
Rn.120914
−
−
−
−
−
+
−
+

6742
Rn.9779
−
−
−
−
−
−
−
+

154
Rn.185941
−
−
−
−
+
−
+
+

725
Rn.108075
−
−
−
−
−
+
−
−

1131
Rn.144946
−
+
−
−
−
−
−
−

1562
Rn.14744
−
−
−
−
−
−
−
−

1741
Rn.1647
−
−
−
−
−
+
−
+

1993
Rn.18728
−
−
−
−
−
+
+
−

2546
Rn.24561
−
−
−
−
−
+
+
−

3776
Rn.43232
−
−
−
−
−
−
−
−

4095
Rn.119024
−
−
−
−
−
−
+
−

4973
Rn.118529
−
−
−
−
−
−
+
−

5634
Rn.9862
−
−
−
−
−
+
+
+

5878
Rn.143213
−
−
−
−
−
−
−
−

6026
Rn.167685
−
−
−
−
−
−
+
−

6303
Rn.123063
−
−
−
−
+
+
+
+

Substance No.
TS040
TS057
TS058
TS059
TS060
TS062
TS071
TS072

Liver carcinogenicity
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−

Carcinogenicity
C−
C−
C−
C−
C−
C−
C−
C−

Mutagenicity by Ames test
M−
M−
M+
M+
M+
M−
M−
M+

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
−
−
−
−
−
−
−
+

4846
Rn.144554
−
−
−
−
−
−
−
+

5600
Rn.9836
−
−
−
−
−
−
−
+

6182
Rn.5834
−
−
−
−
−
−
−
+

2026
Rn.167075
−
−
−
−
−
−
−
+

(B)
2045
Rn.19329
−
−
+
−
−
−
−
+

2203
Rn.21240
−
−
−
−
−
−
−
+

1494
Rn.1430
−
−
−
−
−
−
−
+

(D)
5726
Rn.9939
−
−
−
+
−
−
−
−

(C)
533
Rn.106184
−
+
−
−
−
−
−
+

813
Rn.201760
−
−
−
−
−
−
−
−

893
Rn.164817
−
−
−
+
+
−
−
−

1119
Rn.9757
−
−
+
+
−
−
−
−

1163
Rn.11766
−
−
+
−
+
+
−
+

1334
Rn.120914
−
−
−
+
−
−
−
−

2494
Rn.23969
−
−
+
−
−
−
−
−

3929
Rn.4620
−
−
−
+
+
−
−
−

6620
Rn.120914
−
−
−
+
−
−
−
−

6742
Rn.9779
+
−
−
−
−
+
−
−

154
Rn.185941
−
−
+
+
−
−
−
−

725
Rn.108075
−
−
−
−
−
−
−
+

1131
Rn.144946
−
+
−
−
+
−
+
−

1562
Rn.14744
−
−
−
+
−
−
−
−

1741
Rn.1647
−
−
−
+
−
−
−
−

1993
Rn.18728
−
−
−
+
−
−
−
−

2546
Rn.24561
−
−
+
+
−
−
−
−

3776
Rn.43232
−
−
−
+
−
−
−
+

4095
Rn.119024
−
−
−
+
−
−
−
−

4973
Rn.118529
−
−
−
+
−
−
−
+

5634
Rn.9862
−
−
+
+
+
−
−
−

5878
Rn.143213
−
−
−
−
−
−
−
−

6026
Rn.167685
−
+
−
−
−
−
−
−

6303
Rn.123063
−
+
−
−
−
−
−
−

TABLE 13

Substance No.
TS073
TS074
TS079
TS080
TS082
TS083
TS084
TS085

Liver carcinogenicity
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−
Cr−

Carcinogenicity
C−
C−
C−
C−
C−
C−
C−
C−

Mutagenicity by Ames test
M−
M−
M+
M−
M+
M+
M−
M−

Training
Training
Training
Training
Training
Training
Training
Training

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
−
−
−
+
−
−
−
−

4846
Rn.144554
−
−
−
+
−
−
−
−

5600
Rn.9836
−
−
−
+
−
−
−
−

6182
Rn.5834
−
+
+
+
+
−
+
−

2026
Rn.167075
−
−
−
+
−
+
−
−

(B)
2045
Rn.19329
+
+
+
−
−
−
−
−

2203
Rn.21240
−
−
+
+
−
−
−
−

1494
Rn.1430
−
−
−
+
+
+
+
−

(D)
5726
Rn.9939
−
−
+
+
−
−
−
−

(C)
533
Rn.106184
−
−
−
+
+
+
+
−

813
Rn.201760
−
−
−
+
−
+
−
−

893
Rn.164817
−
−
−
+
+
+
−
−

1119
Rn.9757
−
−
−
−
+
+
−
−

1163
Rn.11766
−
−
−
−
−
−
−
−

1334
Rn.120914
−
−
−
−
−
+
−
−

2494
Rn.23969
−
−
−
−
−
−
−
−

3929
Rn.4620
−
+
−
−
+
+
−
−

6620
Rn.120914
−
−
−
−
−
+
−
−

6742
Rn.9779
−
−
−
−
+
+
+
−

154
Rn.185941
−
−
−
+
+
+
−
−

725
Rn.108075
−
+
−
+
−
+
−
−

1131
Rn.144946
−
−
−
−
+
−
−
−

1562
Rn.14744
−
−
−
−
+
+
−
−

1741
Rn.1647
−
+
−
−
+
+
−
−

1993
Rn.18728
−
−
−
−
+
+
−
−

2546
Rn.24561
−
−
−
+
+
+
−
−

3776
Rn.43232
−
+
−
−
+
+
−
−

4095
Rn.119024
−
+
−
+
+
+
−
−

4973
Rn.118529
−
+
−
+
+
+
+
−

5634
Rn.9862
−
+
−
−
+
−
−
−

5878
Rn.143213
−
−
−
−
−
−
−
−

6026
Rn.167685
−
−
−
−
−
+
−
−

6303
Rn.123063
−
−
−
−
+
+
−
−

Substance No.
TS003
TS012
TS016
TS017
TS031
TS036
TS037
TS052

Liver carcinogenicity
Cr+
Cr−
Cr+
Cr−
Cr−
Cr−
Cr−
Cr+

Carcinogenicity
C+
C+
C+
C+
C−
C−
C−
C+

Mutagenicity by Ames test
M−
M+
M−
M−
M−
M−
M+
M−

Validate
Validate
Validate
Validate
Validate
Validate
Validate
Validate

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
+
−
−
−
−
−
−
−

4846
Rn.144554
+
−
−
−
−
−
−
−

5600
Rn.9836
+
−
−
−
−
−
−
−

6182
Rn.5834
+
−
+
+
−
−
−
−

2026
Rn.167075
+
+
−
−
+
−
−
−

(B)
2045
Rn.19329
+
−
+
+
−
−
−
−

2203
Rn.21240
+
−
+
+
−
−
−
+

1494
Rn.1430
+
+
+
+
+
−
−
−

(D)
5726
Rn.9939
−
−
−
−
−
−
−
−

(C)
533
Rn.106184
+
+
+
+
+
−
−
−

813
Rn.201760
−
+
−
−
−
−
−
−

893
Rn.164817
−
−
−
−
−
+
−
−

1119
Rn.9757
−
−
−
−
−
+
−
−

1163
Rn.11766
+
−
−
−
−
−
−
+

1334
Rn.120914
−
−
−
−
−
−
−
−

2494
Rn.23969
−
−
+
−
−
−
−
−

3929
Rn.4620
−
−
−
−
−
+
−
−

6620
Rn.120914
−
−
−
−
−
−
−
−

6742
Rn.9779
−
−
+
−
+
−
−
−

154
Rn.185941
+
−
−
−
−
+
−
−

725
Rn.108075
−
+
−
−
−
+
−
−

1131
Rn.144946
−
+
−
−
−
+
−
−

1562
Rn.14744
−
−
−
−
−
+
−
−

1741
Rn.1647
+
+
+
−
+
+
−
−

1993
Rn.18728
−
−
−
−
−
+
−
−

2546
Rn.24561
−
−
−
−
+
+
−
−

3776
Rn.43232
−
−
−
−
−
+
−
−

4095
Rn.119024
−
−
+
−
−
+
−
−

4973
Rn.118529
+
−
−
−
−
+
−
−

5634
Rn.9862
−
−
+
−
−
−
−
−

5878
Rn.143213
−
+
−
−
−
+
−
−

6026
Rn.167685
−
−
−
−
−
−
−
−

6303
Rn.123063
−
+
−
−
−
−
−
−

TABLE 14

Substance No.
TS061
TS064
TS075
TS081
TS086
TS087
TS088

Liver carcinogenicity
Cr−
Cr−
Cr+
Cr−
Cr+
Cr+
Cr−

Carcinogenicity
C−
C+
C+
C−
C+
C+
C+

Mutagenicity by Ames test
M+
M+
M+
M+
M−
M−
M−

Validate
Validate
Validate
Validate
Ext Validate
Ext Validate
Ext Validate

Gene No.
Unigene No.
Behavior of gene caused by administration of chemical substance

(A)
4218
Rn.167075
−
−
−
−
−
+
−

4846
Rn.144554
+
−
−
−
+
+
−

5600
Rn.9836
−
−
−
−
+
+
−

6182
Rn.5834
+
−
−
−
+
+
−

2026
Rn.167075
+
−
−
−
−
+
−

(B)
2045
Rn.19329
−
−
−
−
−
−
−

2203
Rn.21240
−
+
+
−
−
−
−

1494
Rn.1430
−
−
−
−
−
+
+

(D)
5726
Rn.9939
−
−
−
−
−
−
−

(C)
533
Rn.106184
−
−
−
+
+
+
−

813
Rn.201760
−
−
−
−
−
−
−

893
Rn.164817
−
−
+
−
−
−
−

1119
Rn.9757
−
−
−
−
−
−
−

1163
Rn.11766
+
−
+
−
−
−
−

1334
Rn.120914
−
−
−
−
−
−
−

2494
Rn.23969
−
−
−
−
−
−
−

3929
Rn.4620
−
−
+
−
−
−
−

6620
Rn.120914
−
−
−
−
−
−
−

6742
Rn.9779
−
−
−
+
−
−
−

154
Rn.185941
−
−
−
−
−
−
−

725
Rn.108075
−
−
−
−
−
+
−

1131
Rn.144946
−
−
−
−
−
−
+

1562
Rn.14744
−
−
−
−
−
−
−

1741
Rn.1647
−
−
−
−
−
−
−

1993
Rn.18728
−
−
+
−
−
−
−

2546
Rn.24561
−
−
+
−
−
−
−

3776
Rn.43232
−
−
−
−
−
−
−

4095
Rn.119024
−
−
+
−
−
−
−

4973
Rn.118529
−
−
+
+
−
+
−

5634
Rn.9862
−
−
−
−
−
−
−

5878
Rn.143213
−
−
−
−
−
−
−

6026
Rn.167685
−
−
−
−
−
−
−

6303
Rn.123063
−
−
−
−
−
+
−

Example 1

By using the genes in (A) to (C) selected in Examination Example 1, prediction of the carcinogenicity of each of chemical substances (training substances) which were used for the selection of the genes and chemical substances (validation substances) which were not used for the selection of the genes was performed. The prediction was performed by the following procedure.

One gene was selected from each of (A) to (C), and the expression level of mRNA for each of the selected genes was compared between a chemical substance administration group and a vehicle control group. The chemical substance was determined to have carcinogenicity when the expression level of mRNA for any one of the genes significantly increased or decreased as compared with the vehicle control group and the direction of increase or decrease was the same as that shown in Table 1, and in the other cases, the chemical substance was determined to have no carcinogenicity.

The determination as to whether the expression level of mRNA in the chemical substance administration group significantly increased or decreased as compared with the vehicle control group was performed based on whether or not the expression level of mRNA in the chemical substance administration group was 1.5 times or more or 1/1.5 or less of the expression level of mRNA in the control group.

It was difficult to examine the combinations of all of the genes, however, the following results were obtained. The combination of the genes which gave the highest predictive value was 4846/2203/813. Also in the case of other combinations of the genes, a predictive value of 60% or more was obtained for the validation substances.

The gene numbers (Gene No.) of the genes used for the prediction, the number of substances for which a correct prediction was made, the predictive value calculated from the number of substances for which a correct prediction was made are shown in Tables 15 and 16.

TABLE 15

Training substances
Validation substances

Total of
Total of

Total of
Total of

Grp 1
Grp 2
Grp 3
C+
C−
Total
C+
C−
Total

Number of substances

Gene No.
17
23
6
46
26
72
10
5
15

4846/2203/813
Number of substances for which
17
14
6
37
20
57
9
4
13

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
76.9
79.2
90.0
80.0
86.7

4846 gene was replaced by another gene

4218/2203/813
Number of substances for which
15
14
6
35
20
55
8
5
13

correct prediction was made

Predictive value (%)
88.2
60.9
100.0
76.1
76.9
76.4
80.0
100.0
86.7

5600/2203/813
Number of substances for which
15
14
6
35
20
55
9
5
14

correct prediction was made

Predictive value (%)
88.2
60.9
100.0
76.1
76.9
76.4
90.0
100.0
93.3

6182/2203/813
Number of substances for which
16
17
6
39
17
56
9
4
13

correct prediction was made

Predictive value (%)
94.1
73.9
100.0
84.8
65.4
77.8
90.0
80.0
86.7

2026/2203/813
Number of substances for which
14
15
6
35
19
54
8
3
11

correct prediction was made

Predictive value (%)
82.4
65.2
100.0
76.1
73.1
75.0
80.0
60.0
73.3

2203 gene was replaced by another gene

4846/2045/813
Number of substances for which
17
14
6
37
17
54
6
4
10

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
60.0
80.0
66.7

4846/1494/813
Number of substances for which
17
13
6
36
18
54
7
3
10

correct prediction was made

Predictive value (%)
100.0
56.5
100.0
78.3
69.2
75.0
70.0
60.0
66.7

813 gene was replaced by another gene

4846/2203/533
Number of substances for which
17
17
6
40
17
57
9
2
11

correct prediction was made

Predictive value (%)
100.0
73.9
100.0
87.0
65.4
79.2
90.0
40.0
73.3

4846/2203/893
Number of substances for which
17
14
6
37
17
54
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
80.0
60.0
73.3

4846/2203/1119
Number of substances for which
17
14
6
37
17
54
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
80.0
60.0
73.3

4846/2203/1163
Number of substances for which
17
15
6
38
18
56
8
4
12

correct prediction was made

Predictive value (%)
100.0
65.2
100.0
82.6
69.2
77.8
80.0
80.0
80.0

4846/2203/1334
Number of substances for which
17
14
6
37
18
55
8
4
12

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
69.2
76.4
80.0
80.0
80.0

4846/2203/2494
Number of substances for which
17
14
6
37
21
58
8
4
12

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
80.8
80.6
80.0
80.0
80.0

4846/2203/3929
Number of substances for which
17
14
6
37
17
54
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
80.0
60.0
73.3

TABLE 16

Training substances
Validation substances

Total
Total

Total
Total

Grp 1
Grp 2
Grp 3
of C+
of C−
Total
of C+
of C−
Total

Number of substances

Gene No.
17
23
6
46
26
72
10
5
15

4846/2203/6620
Number of substances for which
17
14
6
37
18
55
8
4
12

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
69.2
76.4
80.0
80.0
80.0

4846/2203/6742
Number of substances for which
17
14
6
37
16
53
8
2
10

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
80.0
40.0
66.7

4846/2203/154
Number of substances for which
17
14
6
37
16
53
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
80.0
60.0
73.3

4846/2203/725
Number of substances for which
17
16
5
38
19
57
9
3
12

correct prediction was made

Predictive value (%)
100.0
69.6
83.3
82.6
73.1
79.2
90.0
60.0
80.0

4846/2203/1131
Number of substances for which
17
14
6
37
16
53
10
3
13

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
100.0
60.0
86.7

4846/2203/1562
Number of substances for which
17
14
6
37
19
56
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
73.1
77.8
80.0
60.0
73.3

4846/2203/1741
Number of substances for which
17
14
6
37
16
53
9
2
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
90.0
40.0
73.3

4846/2203/1993
Number of substances for which
17
14
6
37
17
54
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
80.0
60.0
73.3

4846/2203/2546
Number of substances for which
17
14
6
37
16
53
8
2
10

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
80.0
40.0
66.7

4846/2203/3776
Number of substances for which
17
14
6
37
18
55
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
69.2
76.4
80.0
60.0
73.3

4846/2203/4095
Number of substances for which
17
16
6
39
17
56
8
3
11

correct prediction was made

Predictive value (%)
100.0
69.6
100.0
84.8
65.4
77.8
80.0
60.0
73.3

4846/2203/4973
Number of substances for which
17
15
6
38
16
54
8
2
10

correct prediction was made

Predictive value (%)
100.0
65.2
100.0
82.6
61.5
75.0
80.0
40.0
66.7

4846/2203/5634
Number of substances for which
17
15
6
38
14
52
8
4
12

correct prediction was made

Predictive value (%)
100.0
65.2
100.0
82.6
53.8
72.2
80.0
80.0
80.0

4846/2203/5878
Number of substances for which
17
14
6
37
22
59
9
3
12

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
84.6
81.9
90.0
60.0
80.0

4846/2203/6026
Number of substances for which
17
16
6
39
19
58
8
4
12

correct prediction was made

Predictive value (%)
100.0
69.6
100.0
84.8
73.1
80.6
80.0
80.0
80.0

4846/2203/6303
Number of substances for which
17
17
6
40
16
56
9
4
13

correct prediction was made

Predictive value (%)
100.0
73.9
100.0
87.0
61.5
77.8
90.0
80.0
86.7

Example 2

By using the genes obtained by selecting one gene from each of (A) to (C) and the gene (D) shown in Table 7, the carcinogenicity of a chemical substance was predicted in the same manner as in Example 1. It was difficult to examine all of the combinations, however, the following results were obtained. A predictive value of 60% or more was obtained for the validation substances. The genes used for the prediction, the number of substances for which a correct prediction was made, the predictive value calculated from the number of substances for which a correct prediction was made are shown in Tables 17 and 18.

TABLE 17

Training substances
Validation substances

Total
Total
Degree of
Total
Total
Degree of

Grp 1
Grp 2
Grp 3
of C+
of C−
consistency
of C+
of C−
consistency

Number of substances

17
23
6
46
26
72
10
5
15

4846/2203/5726/813
Number of substances for which
17
21
6
44
18
62
9
4
13

correct prediction was made

Predictive value (%)
100.0
91.3
100.0
95.7
69.2
86.1
90.0
80.0
86.7

4846 gene was replaced by another gene

4218/2203/5726/813
Number of substances for which
15
14
6
35
20
55
8
5
13

correct prediction was made

Predictive value (%)
88.2
60.9
100.0
76.1
76.9
76.4
80.0
100.0
86.7

5600/2203/5726/813
Number of substances for which
15
14
6
35
20
55
9
5
14

correct prediction was made

Predictive value (%)
88.2
60.9
100.0
76.1
76.9
76.4
90.0
100.0
93.3

6182/2203/5726/813
Number of substances for which
16
17
6
39
17
56
9
4
13

correct prediction was made

Predictive value (%)
94.1
73.9
100.0
84.8
65.4
77.8
90.0
80.0
86.7

2026/2203/5726/813
Number of substances for which
14
15
6
35
19
54
8
3
11

correct prediction was made

Predictive value (%)
82.4
65.2
100.0
76.1
73.1
75.0
80.0
60.0
73.3

2203 gene was replaced by another gene

4846/2045/5726/813
Number of substances for which
17
14
6
37
17
54
6
4
10

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
60.0
80.0
66.7

4846/1494/5726/813
Number of substances for which
17
13
6
36
18
54
7
3
10

correct prediction was made

Predictive value (%)
100.0
56.5
100.0
78.3
69.2
75.0
70.0
60.0
66.7

813 gene was replaced by another gene

4846/2203/5726/533
Number of substances for which
17
17
6
40
17
57
9
2
11

correct prediction was made

Predictive value (%)
100.0
73.9
100.0
87.0
65.4
79.2
90.0
40.0
73.3

4846/2203/5726/893
Number of substances for which
17
14
6
37
17
54
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
80.0
60.0
73.3

4846/2203/5726/1119
Number of substances for which
17
14
6
37
17
54
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
80.0
60.0
73.3

4846/2203/5726/1163
Number of substances for which
17
15
6
38
18
56
8
4
12

correct prediction was made

Predictive value (%)
100.0
65.2
100.0
82.6
69.2
77.8
80.0
80.0
80.0

4846/2203/5726/1334
Number of substances for which
17
14
6
37
18
55
8
4
12

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
69.2
76.4
80.0
80.0
80.0

4846/2203/5726/2494
Number of substances for which
17
14
6
37
21
58
8
4
12

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
80.8
80.6
80.0
80.0
80.0

4846/2203/5726/3929
Number of substances for which
17
14
6
37
17
54
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
80.0
60.0
73.3

4846/2203/5726/6620
Number of substances for which
17
14
6
37
18
55
8
4
12

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
69.2
76.4
80.0
80.0
80.0

4846/2203/5726/6742
Number of substances for which
17
14
6
37
16
53
8
2
10

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
80.0
40.0
66.7

TABLE 18

Training substances
Validation substances

Total
Total
Degree of
Total
Total
Degree of

Grp 1
Grp 2
Grp 3
of C+
of C−
consistency
of C+
of C−
consistency

Number of substances

Gene No.
17
23
6
46
26
72
10
5
15

4846/2203/5726/154
Number of substances for which
17
14
6
37
16
53
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
80.0
60.0
73.3

4846/2203/5726/725
Number of substances for which
17
16
5
38
19
57
9
3
12

correct prediction was made

Predictive value (%)
100.0
69.6
83.3
82.6
73.1
79.2
90.0
60.0
80.0

4846/2203/5726/1131
Number of substances for which
17
14
6
37
16
53
10
3
13

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
100.0
60.0
86.7

4846/2203/5726/1562
Number of substances for which
17
14
6
37
19
56
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
73.1
77.8
80.0
60.0
73.3

4846/2203/5726/1741
Number of substances for which
17
14
6
37
16
53
9
2
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
90.0
40.0
73.3

4846/2203/5726/1993
Number of substances for which
17
14
6
37
17
54
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
65.4
75.0
80.0
60.0
73.3

4846/2203/5726/2546
Number of substances for which
17
14
6
37
16
53
8
2
10

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
61.5
73.6
80.0
40.0
66.7

4846/2203/5726/3776
Number of substances for which
17
14
6
37
18
55
8
3
11

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
69.2
76.4
80.0
60.0
73.3

4846/2203/5726/4095
Number of substances for which
17
16
6
39
17
56
8
3
11

correct prediction was made

Predictive value (%)
100.0
69.6
100.0
84.8
65.4
77.8
80.0
60.0
73.3

4846/2203/5726/4973
Number of substances for which
17
15
6
38
16
54
8
2
10

correct prediction was made

Predictive value (%)
100.0
65.2
100.0
82.6
61.5
75.0
80.0
40.0
66.7

4846/2203/5726/5634
Number of substances for which
17
15
6
38
14
52
8
4
12

correct prediction was made

Predictive value (%)
100.0
65.2
100.0
82.6
53.8
72.2
80.0
80.0
80.0

4846/2203/5726/5878
Number of substances for which
17
14
6
37
22
59
9
3
12

correct prediction was made

Predictive value (%)
100.0
60.9
100.0
80.4
84.6
81.9
90.0
60.0
80.0

4846/2203/5726/6026
Number of substances for which
17
16
6
39
19
58
8
4
12

correct prediction was made

Predictive value (%)
100.0
69.6
100.0
84.8
73.1
80.6
80.0
80.0
80.0

4846/2203/5726/6303
Number of substances for which
17
17
6
40
16
56
9
4
13

correct prediction was made

Predictive value (%)
100.0
73.9
100.0
87.0
61.5
77.8
90.0
80.0
86.7

Comparative Example 1

Among the genes in the combination of the genes which gave the highest predictive value in Example 1, one gene was replaced by a gene which was not selected in Examination Example 1, and the carcinogenicity of each of the training substances and the validation substances was predicted. The results are shown in Table 19. Incidentally, the gene used in the replacement for the gene in Example 1 in this Comparative Example is a gene randomly selected from the genes which were not selected in Examination Example 1.

TABLE 19

Training substances
Validation substances

Total
Total
Degree of
Total
Total
Degree of

Grp 1
Grp 2
Grp 3
of C+
of C−
consistency
of C+
of C−
consistency

Number of substances

17
23
6
46
26
72
10
5
15

No. 4846 gene was replaced by another gene

1161/2203/813
Number of substances for which
5
17
3
25
24
49
3
5
8

correct prediction was made

Predictive value (%)
29.4
73.9
50.0
54.3
92.3
68.1
30.0
100.0
53.3

1507/2203/813
Number of substances for which
5
17
3
25
24
49
3
5
8

correct prediction was made

Predictive value (%)
29.4
73.9
50.0
54.3
92.3
68.1
30.0
100.0
53.3

1856/2203/813
Number of substances for which
5
17
3
25
24
49
3
5
8

correct prediction was made

Predictive value (%)
29.4
73.9
50.0
54.3
92.3
68.1
30.0
100.0
53.3

No. 2203 gene was replaced by another gene

4846/2864/813
Number of substances for which
12
4
4
20
20
40
5
4
9

correct prediction was made

Predictive value (%)
70.6
17.4
66.7
43.5
76.9
55.6
50.0
80.0
60.0

4846/3525/813
Number of substances for which
12
4
4
20
19
39
5
4
9

correct prediction was made

Predictive value (%)
70.6
17.4
66.7
43.5
73.1
54.2
50.0
80.0
60.0

4846/4186/813
Number of substances for which
12
5
4
21
19
40
5
4
9

correct prediction was made

Predictive value (%)
70.6
21.7
66.7
45.7
73.1
55.6
50.0
80.0
60.0

No. 813 gene was replaced by another gene

4846/2203/5066
Number of substances for which
13
16
4
33
20
53
5
4
9

correct prediction was made

Predictive value (%)
76.5
69.6
66.7
71.7
76.9
73.6
50.0
80.0
60.0

4846/2203/5877
Number of substances for which
13
16
4
33
20
53
5
4
9

correct prediction was made

Predictive value (%)
76.5
69.6
66.7
71.7
76.9
73.6
50.0
80.0
60.0

4846/2203/6027
Number of substances for which
13
16
4
33
20
53
5
4
9

correct prediction was made

Predictive value (%)
76.5
69.6
66.7
71.7
76.9
73.6
50.0
80.0
60.0

From Tables 15 and 16, it is found that, in Example 1, the predictive value for the carcinogens in Group 1 is 80% or more. On the other hand, in Comparative Example 1 in which the No. 4846 gene was replaced by another gene, as is apparent from Table 19, the predictive value for the carcinogens in Group 1 was 29.4%. Similarly, in the case where the No. 2203 gene was replaced by another gene, the predictive value for the carcinogens in Group 2 significantly decreased; and in the case where the No. 813 gene was replaced by another gene, the predictive value for the carcinogens in Group 3 significantly decreased.

Further, the predictive value for the validation substances with carcinogenicity was from 60 to 100% in Example 1, however, it was from 30 to 50% in Comparative Example 1.

METHOD FOR PREDICTION ABOUT CARCINOGENICITY OF SUBSTANCE IN RODENT

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