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  • Commentary
  • Open Access

Summarized data of genotoxicity tests for designated food additives in Japan

Genes and Environment201840:27

https://doi.org/10.1186/s41021-018-0115-2

  • Received: 21 August 2018
  • Accepted: 5 December 2018
  • Published:

Abstract

The Ministry of Health, Labour and Welfare has carried out genotoxicity tests for food additives used in Japan in cooperation with the Japan Food Additives Association since 1979. Hayashi et al. summarized these data and published a list of 337 designated additives (Shitei-tenkabutsu in Japanese) with genotoxicity test data in 2000. Thereafter, 29 items were eliminated, and 146 items were newly added. Currently, 454 designated additives are allowed to be used as food additives in Japan. This report, based on the Hayashi report, covers the addition of newly derived genotoxicity test data. Routinely, the bacterial reverse mutation test (Ames test), mammalian cell chromosomal aberration test, and in vivo rodent bone marrow micronucleus test have been used for the evaluation of genotoxicity of food additives. In addition to the data from these tests being updated in this report, it newly includes results of transgenic rodent somatic and germ cell gene mutation assays (TGR assays), incorporated in the Organisation for Economic Co-operation and Development (OECD) test guidelines after 2000. We re-evaluated the genotoxicity of 13 designated food additives considering their TGR data.

Keywords

  • Food additives
  • Designated additives
  • Genotoxicity test
  • Ames test
  • Transgenic rodent gene mutation assay

Background

Since 1979, as part of the safety reassessment of food additives, the Ministry of Health, Labour and Welfare (MHLW; prior to January 2001, the Ministry of Health and Welfare) has carried out mutagenicity tests annually in cooperation with the Japan Food Additives Association. In 2000, Dr. M. Hayashi (former Head of Division of Genetics and Mutagenesis at the National Institute of Health Sciences (NIHS)) and colleagues summarized the mutagenicity data for 337 designated additives, 187 existing additives (natural additives), 49 natural fragrances, and seven general food and drink additives from fiscal year (FY) 1979 to FY1998 [1] (hereafter referred to as the “Hayashi report”). Since then, concerning designated additives, 29 items have been eliminated due to abolition of form classification or for other reasons (Table 1), and 146 items have been newly added (Table 2). In this report, which is based on the Hayashi report, data on newly tested items have been added, and mutagenicity data for a total of 454 designated food additives is summarized in Table 3.
Table 1

List of designated food additives eliminated after 2000 (As of October 6, 2016)

Name

Date

Reason

Aluminum Potassium Sulfate (dried) (syn: Burnt Alum)

June 30, 2000

Integrated into “Aluminum Potassium Sulfate”

Ferrous Pyrophosphate

June 30, 2000

Distribution and usage records have not been confirmed

Sodium Sulfite (anhydrous)

June 30, 2000

Integrated into “Sodium Sulfite”

Tetrasodium Pyrophosphate (anhydrous)

June 30, 2000

Integrated into “Tetrasodium Pyrophosphate”

Aluminum Ammonium Sulfate (dried) (syn: Burnt Ammonium Alum)

June 30, 2000

Integrated into “Aluminum Ammonium Sulfate”

Disodium Hydrogen Phosphate (anhydrous) (Disodium Phosphate (anhydrous))

June 30, 2000

Integrated into “Disodium Hydrogen Phosphate”

Sodium Dihydrogen Phosphate (anhydrous) (Monosodium Phosphate (anhydrous))

June 30, 2000

Integrated into “Sodium Dihydrogen Phosphate”

Tertiary Sodium Phosphate (anhydrous)

June 30, 2000

Integrated into “Tertiary Sodium Phosphate”

Choline phosphate

June 30, 2000

Distribution and usage records have not been confirmed

Methyl O-Acetylricinoleate

June 30, 2000

Distribution and usage records have not been confirmed

Citric Acid (anhydrous)

June 30, 2000

Integrated into “Citric Acid”

Ferrous Sulfate (dried)

June 30, 2000

Integrated into “Ferrous Sulfate”

Sodium Acetate (anhydrous)

June 30, 2000

Integrated into “Sodium Acetate”

Sodium Hydroxide (crystal)

June 30, 2000

Integrated into “Sodium Hydroxide”

Sodium Carbonate (crystal)

June 30, 2000

Integrated into “Sodium Carbonate”

Sodium Starch Phosphate

June 4, 2009

Production and usage have not been confirmed

The date and the reasons for disappearance are indicated

Table 2

List of items that have been added to the designated food additives (As of October 6, 2016)

No.a

Name

2

chlorous acid water

4

nitrous oxide

8

calcium l-ascorbate

9

l-ascorbic acid 2-glucoside

13

asparaginase

16

acesulfame potassium

17

acetylated distarch adipate

18

acetylated oxidized starch

19

acetylated distarch phosphate

20

acetaldehyde

24

sodium selenite pentahydrate

25

azoxystrobin

26

advantame

28

β-apo-8′-carotenal

29

(3-amino-3-carboxypropyl) dimethylsulfonium chloride

30

amylalcohol

35

ammonium alginate

36

potassium alginate

37

calcium alginate

44

ammonium isovalerate

46

ion exchange resin

47

isoamylalcohol

51

isoquinoline

54

isovaleraldehyde

55

isobutanol

56

isobutyraldehyde (isobutanal)

57

isopropanol

58

isopentylamine

67

mixture of 2-ethyl-3,5-dimethylpyrazine and 2-ethyl-3,6-dimethylpyrazine

69

2-ethylpyrazine

70

3-ethylpyridine

71

2-ethyl-3-methylpyrazine

72

2-ethyl-5-methylpyrazine

73

2-ethyl-6-methylpyrazine

74

5-ethyl-2-methylpyridine

77

ethers

89

octanoic acid

91

starch sodium octenyl succinate

94

peracetic acid

102

canthaxanthin

106

xylitol (alias xylit)

110

triethyl citrate

113

sodium ferrous citrate (sodium iron citrate)

127

sodium gluconate

128

glutamyl-valyl-glycine

130

monoammonium l-glutamate

132

monocalcium dl- l -glutamate

134

monomagnesium dl-l-glutamate

135

calcium silicate

136

magnesium silicate

140

ketones

150

calcium acetate

156

starch acetate

165

calcium saccharin

168

calcium oxide

169

oxidized starch

172

hypochlorous acid water

174

hypobromous acid water

176

2,3-diethylpyrazine

177

2,3-diethyl-5-methylpyrazine

189

fatty acids

191

aliphatic higher aldehydes (except those generally recognized as highly toxic)

192

aliphatic higher hydrocarbons (except those generally recognized as highly toxic)

193

2,3-dimethylpyrazine

194

2,5-dimethylpyrazine

195

2,6-dimethylpyrazine

196

2,6-dimethylpyridine

201

potassium dl-bitartrate (potassium hydrogen dl-tartrate or potassium hydrogen dl-tartrate)

203

disodium dl-tartrate (disodium dl-tartrate)

226

magnesium hydroxide

227

sucralose (trichlorogalactosucrose)

228

calcium stearate

229

magnesium stearate

231

sodium stearoyl lactylate

236

calcium sorbate

248

thiamine thiocyanate (vitamin B1 thiocyanate)

251

thioethers (except those generally recognized as highly toxic)

252

thiols (thioalcohols) (except those generally recognized as highly toxic)

258

5,6,7,8-tetrahydroquinoxaline

259

2,3,5,6-tetramethylpyrazine

262

terpene hydrocarbons

268

all-racemic -α-tocopheryl acetate

269

R,R,R -α-tocopheryl acetate

272

trimethylamine

273

2,3,5-trimethylpyrazine

276

nisin

277

natamycin

284

carbon dioxide (carbonic acid, gas)

287

potassium lactate

291

neotame

303

valeraldehyde

306

biotin

308

bisbentiamine (benzoylthiamine disulfide)

311

1-hydroxyethylidene-1, 1-diphosphonic acid

313

hydroxycitronellal dimethylacetal

314

hydroxypropyl distarch phosphate

315

hydroxypropyl cellulose

316

hydroxypropyl starch

317

hydroxypropyl methylcellulose

318

piperidine

321

sunflower lecithin

323

pyrazine

325

pyrimethanil

328

pyrrolidine

334

pyrrole

339

2-(3-phenylpropyl) pyridine

340

phenethylamine

341

phenol ethers (except those generally recognized as highly toxic)

342

phenols (except those generally recognized as highly toxic)

343

ferrocyanides (potassium ferrocyanide (potassium hexacyanoferrate (ii)), calcium ferrocyanide (calcium hexacyanoferrate (ii)), sodium ferrocyanide (sodium hexacyanoferrate (ii))

344

butanol

345

butylamine

346

butyraldehyde

350

fludioxonil

352

propanol

353

propionaldehyde

369

2-pentanol (syn: sec-amylalcohol)

370

trans-2-pentenal

371

1-penten-3-ol

372

aromatic alcohols

373

aromatic aldehydes (except those generally recognized as highly toxic)

377

polysorbate 20

378

polysorbate 60

379

polysorbate 65

380

polysorbate 80

381

polyvinylpyrroridone

382

polyvinylpolypyrrolidone

394

5-methylquinoxaline

395

6-methylquinoline

396

6,7-dihydro-5-methyl-5 h-cyclopentapyrazine

398

1-methylnaphthalene

400

2-methypyrazine

401

2-methylbutanol

402

3-methyl-2-butanol

403

2-methylbutyraldehyde

404

trans-2-methyl-2-butenal, (e)-2-methyl-2-butenal

405

3-methyl-2-butenal

406

3-methyl-2-butenol

417

Lactones (except those generally recognized as highly toxic)

422

calcium 5′-ribonucleotide

431

potassium sulfate

439

distarch phosphate

440

monostarch phosphate

443

trimagnesium phosphate (syn: Magnesium phosphate, tribasic)

452

magnesium monohydrogen phosphate

454

phosphated distarch phosphate

anumbers are consistent with those underlined in Table 3

Table 3

List of the results in genotoxicity tests for the 454 designated food additives (As of October 6, 2016)

No.a

Name

Functional classes

CAS#

Molecular weight

Genotoxicity tests

Remarks

Ames

CA

MN

TGR

Others

1

zinc salts (limited to zinc gluconate and zinc sulfate)

Dietary supplement

  

-(13)

-H23 c

    

2 b

chlorous acid water

Sterilizer

13898–47-0

68.45

+FSC1 d

+FSC1

-FSC1

   

3 [20]

sodium chlorite

Bleaching agent etc.

7758-19-2

90.44

+(2)-(12)

+(2)

+(9)

   

4

nitrous oxide

Propellantt

10024–97-2

44.01

-FSC2

     

5

adipic acid

Acidifier

124–04-9

146.14

-(15)

-H22

    

6

sodium nitrite

Color fixative

7632–00-0

69.00

+(1) -(16)

+(1)

-(9)

-H21

 

Target organs for TG in mice: liver, stomach (glandular stomach)

7

l-ascorbic acid (vitamin C)

Antioxidant etc.

50–81-7

176.12

-(1)(10)

-(1)

    

8

calcium l -ascorbate

Dietary supplement etc.

5743-28-2

426.35

-FSC3

     

9

l-ascorbic acid 2-glucoside

Antioxidant etc.

129499–78-1

338.26

-H26

-H26

-FSC4

   

10

l-ascorbic stearate (vitamin C stearate)

Antioxidant etc.

25395–66-8

442.59

-(4)(16)

-(4)

    

11

sodium l-ascorbate (vitamin C sodium)

Antioxidant etc.

134–03-2

198.11

-H20

-H20

-H20

   

12

l-ascorbic palmitate (vitamim C palmitate)

Antioxidant etc.

137–66-6

414.53

-(17)

-H22

    

13

asparaginase

Processing agent

9015–68-3

 

-FSC5

-FSC5

    

14

monosodium l -aspartate

Seasoning etc.

3792-50-5

173.10

-(3)(17)

-(3)

    

15

aspartame (α- l -aspartyl- l -phenylalanine methyl ester)

Sweetener

22839–47-0

294.30

-(16)

-H23

    

16 [21, 22]

acesulfame potassium

Sweetener etc.

55589–62-3

201.24

- (21)

- (22)

    

17

acetylated distarch adipate

Thickening agents etc.

68130–14-3

      

Evaluated as modified starch

18

acetylated oxidized starch

Thickening agents etc.

68187–08-6

      

Evaluated as modified starch

19

acetylated distarch phosphate

Thickening agents etc.

      

Evaluated as modified starch

20

acetaldehyde

Flavoring agent etc.

75–07-0

44.05

-FSC6

+FSC6

    

21

ethyl acetoacetate

Flavoring agent

141–97-9

130.14

-(3)(12)

-(3)

    

22

acetophenone

Flavoring agent

98–86-2

120.15

-(12)

     

23

acetone

Processing agent

67–64-1

58.08

-(1)(17)

+(1)

    

24

sodium selenite pentahydrate

Dietary supplement

10102–18-8

172.94

+FSC7

+FSC7

+FSC7

   

25

azoxystrobin

Preservative etc.

131860–33-8

403.4

-FSC8

 

-FSC8

   

26

advantame

Sweetener

714229–20-6

476.52

-FSC9

 

-FSC9

   

27

anisaldehyde (p-methoxybenzaldehyde)

Flavoring agent

123–11-5

136.15

-(2)(12)

-(2)

    

28

β-apo-8′-carotenal

Food color

1107–26-2

416.64

-FSC10

-FSC10

-FSC10

   

29

(3-amino-3-carboxypropyl) dimethylsulfonium chloride

Flavoring agent

3493-12-7

199.7

-H21

-H21

-H21

   

30

amylalcohol

Flavoring agent

71–41-0

88.15

- H16

- H16

    

31

α-amylcinnamaldehyde (α-amylcinnamic aldehyde)

Flavoring agent

122–40-7

202.29

-(12)

-H24

    

32

dl-alanine

Seasoning etc.

302–72-7

89.09

-(3)(17)

-(3)

    

33

sodium sulfite

Preservative etc.

7757-83-7

126.04

-(1)(12)

-(1)

   

Crystalline form and anhydrous form were used for tests in (1) and (12), respectively.

34

l -arginine l -glutamate

Seasoning etc.

4320–30-3

321.33

-(4)(20)

-(4)

    

35

ammonium alginate

Emulsifier etc.

9005–34-9

 

-FSC11

     

36

potassium alginate

Emulsifier etc.

9005–36-1

 

-FSC12

     

37

calcium alginate

Emulsifier etc.

9005–35-0

      

See compounds with different salt

38 [23]

sodium alginate

Thickening agents

9005–38-3

 

-(2)(8)(18)

-(2)

    

39

propylene glycol alginate

Thickening agents

  

-(2)(13)

-(2)

    

40

benzoic acid

Preservative

65–85-0

122.12

-(2)(11)

-(2)

    

41

sodium benzoate

Preservative

532–32-1

144.10

-(1)(19)

+(1)

-H20

   

42

methyl anthranilate

Flavoring agent

134–20-3

151.16

-(12)

+H22

    

43

ammonia

Processing agent

7664-41-7

17.03

-(18)

     

44

ammonium isovalerate

Flavoring agent

1449430–58-3

323.43

-H17

-H17

    

45

ionone

Flavoring agent

8013-90-9

192.30

-(20)

-H24

   

Mixture of α and β-ionone were used for the assay

46

ion exchange resin

Processing agent

        

47

isoamylalcohol

Flavoring agent

123–51-3

88.15

- H16

- H16

- H16

   

48

isoeugenol

Flavoring agent

97–54-1

164.20

-(12)

-H23

    

49

isoamyl isovalerate

Flavoring agent

659–70-1

172.26

-(3)(12)

-(3)

    

50

ethyl isovalerate

Flavoring agent

108–64-5

130.18

-(3)(12)

-(3)

    

51

isoquinoline

Flavoring agent

119–65-3

129.16

 

+H18

-H21

   

52 [24]

isothiocyanates (except those generally recognized as highly toxic)

Flavoring agent

542–85-8

 

-(19)

    

Ethyl thiocyanate was used for the assay.

53

allyl isothiocyanate (volatile oil of mustard)

Flavoring agent

57–06-7

99.16

-(12)

+H22

    

54

isovaleraldehyde

Flavoring agent

590–86-3

86.13

-FSC13

 

- H17

   

55

isobutanol

Flavoring agent

78–83-1

74.12

-FSC14

- H16

    

56

isobutyraldehyde (isobutanal)

Flavoring agent

78–84-2

72.11

-FSC15

+FSC15

-FSC15

   

57

isopropanol

Processing agent etc.

67–63-0

60.10

-FSC16

 

-FSC16

   

58

isopentylamine

Flavoring agent

107–85-7

87.16

-H18

-H18

-H18

   

59 [25]

l -isoleucine

Dietary supplement

73–32-5

131.17

-(3)(15)

-(3)

    

60

disodium 5′-inosinate (sodium 5′-inosinate)

Seasoning etc.

4691–65-0

392.17

-(1)(17)

+(1)

-H20

   

61

imazalil

Antimolding agent

35554–44-0

297.18

-(20)

     

62

indoles and its derivatives

Flavoring agent

120–72-9 (indole)

 

-(19)-H26

-H23

    

63

disodium 5′-uridylate (sodium 5′-uridylate)

Seasoning etc.

3387–36-8

368.15

-(1)(17)

+(1)

-H22

   

64

γ-undecalactone (undecalactone)

Flavoring agent

104–67-6

184.28

-(3)(12)

-(3)

-(9)

   

65

ester gum

Chewing gum base

  

-(2)

-(2)

    

66

esters

Flavoring agent

  

-(2) or (4)

-(2) or (4)

   

Cinnamyl anthranilate, ethyl caprylate, allyl caproate, ethyl caproate were included.

67

mixture of 2-ethyl-3,5-dimethylpyrazine and 2-ethyl-3,6-dimethylpyrazine

Flavoring agent

55031–15-7

136.20

-FSC17

     

68

ethylvanillin

Flavoring agent

121–32-4

166.17

-(2)(12)

-(2)

    

69

2-ethylpyrazine

Flavoring agent

13925–00-3

108.14

  

+H16

   

70

3-ethylpyridine

Flavoring agent

536–78-7

107.15

 

+H16

-H17

   

71

2-ethyl-3-methylpyrazine

Flavoring agent

15707–23-0

122.17

- H16

- H16

- H16

   

72

2-ethyl-5-methylpyrazine

Flavoring agent

13360–64-0

122.17

- H16

- H16

    

73

2-ethyl-6-methylpyrazine

Flavoring agent

13925–03-6

122.17

- H17

- H17

   

The test substrate was mixture with 2-ethyl-5-methylpyrazine.

74

5-ethyl-2-methylpyridine

Flavoring agent

104–90-5

121.18

-FSC18

 

-FSC18

   

75

calcium disodium ethylenediaminetetraacetate (calcium disodium edta)

Antioxidant

62–33-9

410.30

-(13)

-H22

    

76

disodium ethylenediaminetetraacetate (disodium, EDTA)

Antioxidant

6381-92-6

372.24

-(13)

     

77

ethers

Flavoring agent

        

78

erythorbic acid (isoascorbic acid)

Antioxidant

89–65-6

176.12

+(2) -(17)

-(2)

-(9)

-H21

 

Target organs for TGR in mice:liver, stomach (glandular stomach)

79

sodium erythorbate (sodium isoascorbate)

Antioxidant

6381–77-7

216.12

-(1)(14)

-(1)

    

80

ergocalciferol (calciferol or vitamin D2)

Dietary supplement

50–14-6

396.65

-(1)(20)

-(1)

    

81

ammonium chloride

Raising agent

12125–02-9

53.49

-(2)(16)

+(2)

-(9)

   

82

potassium chloride

Seasoning

7447-40-7

74.55

-(17)

-H23

    

83

calcium chloride

Tofu coagulator etc.

10043–52-4

110.98

-(1)(12)

-(1)

    

84

ferric chloride

Dietary supplement

10025–77-1

270.29

-(4)(15)

-(4)

    

85

magnesium chloride

Tofu coagulator etc.

7791-18-6

203.30

-(4)(5)(18)

-(4)

    

86

hydrochloric acid

Processing agent

7647–01-0

36.46

-(16)

     

87

eugenol

Flavoring agent

97–53-0

164.20

-(2)(14)

+(2)

-(9)

   

88

octanal (capryl aldehyde or octyl aldehyde)

Flavoring agent

124–13-0

128.21

-(14)

-H22

    

89

octanoic acid

Flavoring agent

124–07-2

 

-FSC19

     

90

ethyl octanoate (ethyl caprylate)

Flavoring agent

106–32-1

172.26

-(14)

-H25

    

91

starch sodium octenyl succinate

Thickening agents etc.

 

-FSC20

     

92

o -phenylphenol and sodium o -phenylphenate

Antimolding agent

90–43-7

170.21

-(1)

    

o -phenylphenol was negative in CA

93

sodium oleate

Film-forming agent

143–19-1

304.44

-(14)

     

94

peracetic acid

Preservative etc.

79–21-0

 

+FSC21

+FSC21

-FSC21

   

95

hydrogen peroxide

Sterilizer

7722-84-1

34.01

-(1)(16)

+(1)

-H21

   

96

benzoyl peroxide

Flour treatment agent

94–36-0

242.23

-(1)(12)

-(1)

    

97

sodium caseinate

Processing agent

9005-46-3

 

-(5)(18)

-(5)

    

98

ammonium persulfate

Flour treatment agent

7727-54-0

228.20

-(2)(12)

-(2)

    

99

calcium carboxymethylcellulose (calcium cellulose glycolate)

Thickening agents

9050-04-8

 

-(4)

-(4)

    

100

sodium carboxymethylcellulose (sodium cellulose glycolate)

Thickening agents

9004-32-4

 

-(1)(13)

-(1)

    

101

β-carotene

Food color etc.

7235-40-7

536.88

-(1)(13)

±(1)

    

102

canthaxanthin

Food color

514–78-3

564.82

-FSC22

-FSC22

-FSC22

   

103

isoamyl formate

Flavoring agent

110–45-2

116.16

-(3)(16)

-(3)

    

104

geranyl formate

Flavoring agent

105–86-2

182.26

-(14)

-H24

    

105

citronellyl formate

Flavoring agent

105–85-1

 

-(20)

-H24

    

106 [26]

xylitol

Sweetener

87–99-0

152.15

- (23)

 

- (23)

   

107

disodium 5′-guanylate (sodium 5′-guanylate)

Flavoring agent etc.

5550-12-9

407.18

-(3)(17)

-(3)

    

108

citric acid

Acidifier

77–92-9

192.12

-(1)(15)

-(1)

   

Crystalline form and anhydrous form were used for tests in (1) and (5), respectively.

109

isopropyl citrate

Antioxidant

39413–05-3

 

-(13)

     

110

triethyl citrate

Sweetener

77–93-0

276.28

-H26

-H26

-H26

   

111

monopotassium citrate and tripotassium citrate

Flavoring agent etc.

866–83-1

230.21

-(17)

-(8)-H25

  

Rec assay: +(8)

monopotassium citrate and tripotassium citrate were used in (17), monopotassium citrate was used in (8), tripotassium citrate was used in H25

112

calcium citrate

Dietary supplement etc.

813–94-5

570.49

-(5)(13)

-(5)

    

113

sodium ferrous citrate (sodium iron citrate)

Dietary supplement

50717–86-7

526.01

-H22

+H22

    

114

ferric citrate

Dietary supplement

77–92-9

192.12

-(4)(15)

-(4)

    

115

ferric ammonium citrate

Dietary supplement

1185–57-5

 

-(4)(15)

-(4)

    

116

trisodium citrate (sodium citrate)

Acidifier

68–04-2

258.07

-(17)

-(1)

    

117

glycine

Seasoning etc.

56–40-6

75.07

-(3)(19)

-(3)

   

Crystalline form and powdery form were used for tests in (3), and Crystalline form for (19)

118

glycerol (glycerin)

Processing agent

56–81-5

92.09

-(2)(19)

-(2)

    

119

glycerol esters of fatty acids

Emulsifier

  

-(1)

-(1)

    

120

calcium glycerophosphate

Dietary supplement

27214–00-2

210.14

-(2)(12)

-(2)

    

121

disodium glycyrrhizinate

Sweetener

68797–35-3

899.11

-(1)

+(1)

-(9)

   

122

glucono-delta-lactone (gluconolactone)

Acidifier

90–80-2

178.14

-(1)(15)

-(1)

    

123

gluconic acid

Acidifier

526–95-4

196.16

-(4)(15)

-(4)

    

124

potassium gluconate

Acidifier

299–27-4

234.25

 

-(8)

  

Rec assay: -(8)

 

125

calcium gluconate

Dietary supplement

299–28-5

448.39

-(5)(12)

-(5)

    

126

ferrous gluconate (iron gluconate)

Dietary supplement etc.

299–29-6

446.14

-(19)

+H25

    

127

sodium gluconate

Emulsifier etc.

527–07-1

218.14

     

See substrates with different salt

128

glutamyl-valyl-glycine

Seasoning

38837–70-6

303.31

-FSC23

-FSC23

-FSC23

   

129

l-glutamic acid

Seasoning

56–86-0

147.13

-(5)(19)

-(5)

    

130

monoammonium l-glutamate

Seasoning etc.

7558-63-6

182.18

-FSC24

   

Rec assay: -FSC24

 

131

monopotassium l-glutamate

Seasoning etc.

6382-01-0

203.23

-(17)

-H25

    

132

monocalcium di-l-glutamate

Dietary supplement etc.

69704–19-4

404.38

     

See substrate with different salt

133

monosodium l-glutamate

Seasoning etc.

6106-04-3

187.13

-(1)(19)

-(1)

    

134

monomagnesium di- l-glutamate

Dietary supplement etc.

129160–51-6

388.61

     

See substrate with different salt

135

calcium silicate

Processing agent

38837–70-6

 

-FSC25

-FSC25

    

136

magnesium silicate

Processing agent

1343-88-0

 

-FSC26

     

137

cinnamic acid

Flavoring agent

140–10-3

148.16

-(14)

-H23

    

138

ethyl cinnamate

Flavoring agent

103–36-6

176.21

-(2)(14)

-(2)

    

139

methyl cinnamate

Flavoring agent

103–26-4

162.19

-(14)

+H23

    

140

ketones

Flavoring agent

        

141

geraniol

Flavoring agent

106–24-1

154.25

-(2)(14)

-(2)

    

142

high test hypochlorite

bleaching agent etc.

  

+(3) -(20)

+(3)

-(4)

   

143

succinic acid

Acidifier etc.

110–15-6

118.09

-(1)(15)

-(1)

    

144

monosodium succinate

Seasoning etc.

2922-54-5

140.07

-(19)

-H23-H26

    

145

disodium succinate

Seasoning etc.

150–90-3

162.05

-(1)(19)

±(1)

    

146

cholecalciferol (vitamin D3)

Dietary supplement

67–97-0

384.64

-(3)(20)

-(3)

    

147

sodium chondroitin sulfate

Humectant etc.

12678–07-8

 

-(3)(14)

-(3)

    

148

isoamyl acetate

Flavoring agent

123–92-2

130.18

-(3)(14)

-(3)

    

149

ethyl acetate

Flavoring agent etc.

141–78-6

88.11

-(1)(16)

+(1)

-(9)

   

150

calcium acetate

Dietary supplement etc.

62–54-4

158.17

     

See substrates with different salt

151

geranyl acetate

Flavoring agent

105–87-3

196.29

-(14)

-H24

    

152

cyclohexyl acetate

Flavoring agent

622–45-7

142.20

-(14)

-H25

    

153

citronellyl acetate

Flavoring agent

150–84-5

198.30

-(14)

-H24

    

154

cinnamyl acetate

Flavoring agent

103–54-8

176.21

-(14)

+H24

    

155

terpinyl acetate

Flavoring agent

8007-35-0

196.29

-(14)

-H24

    

156

starch acetate

Thickening agents etc.

9045–28-7

 

-FSC20

-FSC20

-FSC20

   

157

sodium acetate

Acidifier etc.

127–09-3

82.03

-(2)(18)

-(2)

   

Crystalline form was used in (2), and anhydrous form was used in (18).

158

polyvinyl acetate

Chewing gum base etc.

  

-(3)(14)

-(3)

    

159

phenethyl acetate (phenylethyl acetate)

Flavoring agent

103–45-7

164.20

-(15)

-H24

    

160

butyl acetate

Flavoring agent

123–86-4

116.16

-(3)(15)

-(3)

    

161

benzyl acetate

Flavoring agent

140–11-4

150.17

-(4)(15)

-(4)

    

162

l-menthyl acetate

Flavoring agent

2623-23-6

198.30

-(15)

-H22

    

163

linalyl acetate

Flavoring agent

115–95-7

196.29

-(15)

-H23

    

164

saccharin

Sweetener

81–07-2

183.19

-(2)(14)

-(2)

    

165

calcium saccharin

Sweetener

6381-91-5

467.48

 

+FSC27

    

166 [27]

sodium saccharin (soluble saccharin)

Sweetener

128–44-9

205.17

-(1)(11)

+(1)

-H21

   

167

methyl salicylate

Flavoring agent

119–36-8

152.15

-(2)(15)

-(2)

    

168

calcium oxide

Processing agent

1305-78-8

56.08

-FSC28

     

169

oxidized starch

Thickening agents etc.

 

-FSC20

-FSC20

-FSC20

   

170

magnesium oxide

Absorbent etc.

1309–48-4

40.30

-(13)

-H22

    

171 [28]

iron sesquioxide (diiron trioxide or iron oxide red)

Food color

1309–37-1

159.69

-(4)(6)(19)

-(4)

-(8)

 

Rec assay: -(6)

 

172

hypochlorous acid water

Preservative

  

- H6

     

173

sodium hypochlorite (hypochlorite of soda)

Sterilizer etc.

7681–52-9

74.44

+(1) -(12)

+(1)

-(9)

   

174

hypobromous acid water

Sterilizer

13517–11-8

96.91

-FSC29

-FSC29

   

5,5-Dimethylhydantoin was used for the assays.

175

sodium hydrosulfite (hydrosulfite)

bleaching agent etc.

7775–14-6

174.11

-(2)(12)

-(2)

    

176

2,3-diethylpyrazine

Flavoring agent

15707–24-1

136.19

-H15

+H15

+H16

   

177

2,3-diethyl-5-methylpyrazine

Flavoring agent

18138–04-0

150.22

-H17

-H17

-H18

   

178

allyl cyclohexylpropionate

Flavoring agent

2705-87-5

196.29

-(15)

-H22

    

179

l-cystein monohydrochloride

Antioxidant etc.

7048–04-6

175.64

+(4)(14)

+(4)

-(9)

-H21

 

Target organs for TGR in mice:liver, stomach (glandular stomach)

180

disodium 5′-cytidylate (sodium 5′-cytidylate)

Seasoning

6757–06-8

367.16

-(1)(19)

+(1)

-H22

   

181

citral

Flavoring agent

5392–40-5

152.23

-(3)(4)(14)

-(3)(4)

    

182

citronellal

Flavoring agent

106–23-0

154.25

-(15)

-(5)

-(8)

   

183

citronellol

Flavoring agent

106–22-9

156.27

-(15)

-H22

    

184

1,8-cineole (eucalyptol)

Flavoring agent

470–82-6

154.25

-(15)

-H22

    

185 [29]

diphenyl (biphenyl)

Antimolding agent

92–52-4

154.21

-(1)(10)

-(1)

    

186

butylated hydroxytoluene

Antioxidant

128–37-0

220.35

-(10)

-(1)

    

187

dibenzoyl thiamine

Dietary supplement

299–88-7

490.58

-(5)(12)

-(5)

    

188

dibenzoyl thiamine hydrochloride

Dietary supplement

35660–60-7

527.04

-(1)-H26

-(1)+H26

    

189

fatty acids

Flavoring agent

        

190

aliphatic higher alcohols

Flavoring agent

  

-(14)

     

191

aliphatic higher aldehydes (except those generally recognized as highly toxic)

Flavoring agent

        

192

aliphatic higher hydrocarbons (except those generally recognized as highly toxic)

Flavoring agent

        

193

2,3-dimethylpyrazine

Flavoring agent

5910-89-4

108.14

-FSC30

-H16

    

194

2,5-dimethylpyrazine

Flavoring agent

123–32-0

108.14

-FSC31

 

-H16

   

195

2,6-dimethylpyrazine

Flavoring agent

108–50-9

108.14

-FSC32

 

-H16

   

196

2,6-dimethylpyridine

Flavoring agent

108–48-5

107.15

-FSC33

-H18

-H18

   

197

oxalic acid

Processing agent

6153-56-6

126.07

-(3)(13)

-(3)

    

198

potassium bromate

Flour treatment agent

7758-01-2

167.00

+(1) -(12)

+(1)

+(9)

   

199

dl-tartaric acid (dl -tartaric acid)

Acidifier

133–37-9

150.09

-(15)

-H23

    

200

l-tartaric acid (d -tartaric acid)

Acidifier

87–69-4

150.09

-(2)(15)

- (2)

   

Identical to d-tartaric acid

201

potassium dl-bitartrate (potassium hydrogen dl-tartrate or potassium hydrogen dl-tartrate)

Processing agent etc.

  

-H22

-H22

    

202

potassium l-bitartrate (potassium hydrogen l-tartrate or potassium hydrogen d -tartrate)

Raising agent

868–14-4

188.18

-(2)(19)

- (2)

   

Identical to potassium hydrogen d-tartarate

203

disodium dl-tartrate (disodium dl-tartrate)

Processing agent etc.

        

204

disodium l-tartrate (disodium l -tartrate)

Seasoning

6106-24-7

194.05

-(1)(19)

+(1)

-(9)

  

Identical to disodium d-tartarate

205

potassium nitrate

Fermentation regulator etc.

7757-79-1

101.10

-(4)(13)

-(4)

    

206

sodium nitrate

Fermentation regulator etc.

7631-99-4

84.99

-(1)(13)

+(1)

-H21

   

207

food red no.2 (amaranth) and its aluminum lake

Food color

915–67-3

604.48

-(1)(18)

+(1)

    

208

food red no.3 (erythrosine) and its aluminum lake

Food color

16423–68-0

897.87

-(1)(18)

+(1)

- (2)

   

209

food red no.40 (allura red ac) and its aluminum lake

Food color

25956–17-6

496.42

-(20)

 

-H20

-H20, -H23

Comet: -H20, -H23

Target organs for Comet in mice:liver, stomach (glandular stomach).Target organs for TGR in mice:liver, stomach (glandular stomach) for (H20), liver, colon for (H23)

210

food red no.102 (new coccine)

Food color

2611–82-7

631.50

-(1)(18)

+(1)

- (2)

-H20

Comet: (+)H20

Target organs for Comet in mice:liver; pseudo positive, stomach (glandular stomach); negative.

Target organs for TGR in mice:liver, stomach (glandular stomach)

211

food red no.104 (phloxine)

Food color

18472–87-2

829.63

-(1)(18)

-(1)

 

-H20

Comet: +H20

Target organs for Comet in mice:liver; pseudo positive, stomach (glandular stomach) in mice; positive.

Target organs for TGR in mice:liver, stomach (glandular stomach)

212

food red no.105 (rose bengale)

Food color

632–69-9

1017.64

-(1)(18)

-(1)

 

-H20

Comet: +H20

Target organs for Comet:liver, stomach (glandular stomach) in mice, positive for both.

Target organs for TGR in mice:liver, stomach (glandular stomach)

213

food red no.106 (acid red)

Food color

3520-42-1

580.65

-(1)(18)

+(1)

- (2)

   

214

food yellow no.4 (tartrazine) and its aluminum lake

Food color

1934-21-0

534.37

-(1)(18)

+(1)

- (9)

   

215

food yellow no.5 (sunset yellow fcf) and its aluminum lake

Food color

2783-94-0

452.37

-(1)(18)

+(1)

-H21

   

216

food green no.3 (fast green fcf) and its aluminum lake

Food color

2353-45-9

808.85

-(1)(18)

-(1)

-(2)

  

High purity sample was used in (1).

217

food blue no.1 (brilliant blue fcf) and its aluminum lake

Food color

3844-45-9

792.85

-(1)(18)

+(1)

-H21

   

218

food blue no.2 (indigo carmine) and its aluminum lake

Food color

860–22-0

466.35

-(1)(18)

+(1)

    

219

sucrose esters of fatty acids

Emulsifier

  

-(1)(20)

±(1)

    

220

silicone resin (polydimethylsiloxane)

Antiforming agent

  

-(2)

- (2)

    

221

cinnamyl alcohol (cinnamic alcohols)

Flavoring agent

104–54-1

134.18

-(16)

+H24

    

222

cinnamaldehyde (cinnamic aldehyde)

Flavoring agent

14371–10-9

132.16

+(3)(14)

+(3)

-(9)

-H22

 

Target organs for TGR:liver, small intestine (Jejunum) in mice

223

potassium hydroxide (caustic potash)

Processing agent

1310-58-3

56.11

-(17)

     

224

calcium hydroxide (slaked lime)

Processing agent etc.

1305-62-0

74.09

-(5)(14)

-(5)

    

225

sodium hydroxide (caustic soda)

Processing agent

1310-73-2

40.00

-(16)

     

226

magnesium hydroxide

Dietary supplement etc.

1309–42-8

58.32

-FSC34

     

227 [30]

sucralose (trichlorogalactosucrose)

Sweetener

56038–13-2

397.64

- (24)

 

- (24)

   

228

calcium stearate

Dietary supplement

1592–23-0

324.56

      

229

magnesium stearate

Emulsifier etc.

557–04-0

591.24

-H13

-H13

-H13

  

See calcium stearate

230

calcium stearoyl lactylate (calcium stearyl lactylate)

Emulsifier

5793-94-2

 

-(3)

-(3)

    

231

sodium stearoyl lactylate

Emulsifier etc.

25383–99-7

378.53

     

See calcium stearoyl lactylate

232

sorbitan esters of fatty acids

Emulsifier

  

-(1)(20)

±(1)

    

233

d-sorbitol (d-sorbit)

Sweetener etc.

50–70-4

182.17

-(1)(11)

-(1)

   

d-Sorbit WP was also used in (1).

234

sorbic acid

Preservative

110–44-1

112.13

-(2)(16)

-(2)

    

235

potassium sorbate

Preservative

24634–61-5

150.22

-(1)(11)

+(1)

-H20

   

236

calcium sorbate

Preservative etc.

7492-55-9

262.32

     

See potassium sorbate

237

ammonium carbonate

Processing agent etc.

506–87-6

96.09

-(19)

-H23

    

238

potassium carbonate (anhydrous)

Processing agent etc.

584–08-7

138.21

-(4)(19)

-(4)

    

239

calcium carbonate

Processing agent etc.

471–34-1

100.09

-(12)

+H23

    

240

ammonium bicarbonate (ammonium hydrogen carbonate)

Raising agent etc.

1066-33-7

79.06

-(4)(19)

-(4)

    

241

sodium bicarbonate (bicarbonate soda or sodium hydrogen carbonate)

Raising agent etc.

144–55-8

84.01

-(2)(19)

-(2)

    

242

sodium carbonate (crystal: carbonate soda, anhydrous: soda ash)

Processing agent

497–19-8

105.99

-(3)(19)

-(3)

   

Crystalline form was used in (3), and anhydrous form was used for in (19).

243

magnesium carbonate

Processing agent

546–93-0

84.31

-(5)(13)

-(5)

    

244

thiabendazole

Antimolding agent

148–79-8

201.25

-(1)(10)

-(1)

    

245

thiamine hydrochloride (vitamin B1 hydrochloride)

Dietary supplement

67–03-8

337.27

-(2)(11)

-(2)

    

246

thiamine mononitrate (vitamin B1 mononitrate)

Dietary supplement

532–43-4

327.36

-(5)(15)

-(5)

    

247

thiamine dicetylsulfate (vitamin B1 dicetylsulfate)

Dietary supplement

  

-(1)

-(1)

    

248

thiamine thiocyanate (vitamin B1 thiocyanate)

Dietary supplement

130131–60-1

341.45

     

See thiamine hydrochloride

249

thiamine naphthalene-1,5-disulfonate (vitamin B1naphthalene-1,5-disulfonate)

Dietary supplement

  

-(2)

-(2)

    

250

thiamine dilaurylsulfate (vitamin B1 dilaurylsulfate)

Dietary supplement etc.

  

-(2)

-(2)

    

251

thioethers (except those generally recognized as highly toxic)

Flavoring agent

        

252

thiols (thioalcohols) (except those generally recognized as highly toxic)

Flavoring agent

        

253

l -theanine

Seasoning

3081-61-6

174.20

-(4)(19)

-(4)

    

254

decanal (decyl aldehyde)

Flavoring agent

112–31-2

156.27

-(3)(16)

-(3)

    

255

decanol (decyl alcohol)

Flavoring agent

112–30-1

158.28

-(16)

-H25

    

256

ethyl decanoate (ethyl caprate)

Flavoring agent

110–38-3

200.32

-(16)

-H25

    

257

sodium iron chlorophyllin

Food color

  

-(1)

-(1)

    

258

5,6,7,8-tetrahydroquinoxaline

Flavoring agent

34413–35-9

134.18

-H16

-H16

    

259

2,3,5,6-tetramethylpyrazine

Flavoring agent

1124–11-4

136.20

-FSC35

     

260

sodium dehydroacetate

Preservative

4418-26-2

208.14

-(1)(11)

+(1)

+(9)

   

261

terpineol

Flavoring agent

8000-41-7

154.25

-(16)

-H22

    

262

terpene hydrocarbons

Flavoring agent

        

263

sodium carboxymethylstarch

Thickening agents

  

-(3)

-(3)

    

264

copper salts (limited to copper gluconate and cupric

Dietary supplement

  

-(13)

     

265

sodium copper chlorophyllin

Food color

28302–36-5

722.13

-(1)(13)

-(1)

    

266

copper chlorophyll

Food color

15739–09-0

 

-(1)

-(1)

    

267

dl -α-tocopherol

Antioxidant

50–02-9

430.71

-(1)(10)

-(1)

    

268

all-racemic-α-tocopheryl acetate

Dietary supplement etc.

  

-FSC36

    

Identical to dl-α-tocopheryl acetate

269

R,R,R -α-tocopheryl acetate

Dietary supplement etc.

       

See all-racemic -α-tocopheryl acetate

270

dl-tryptophan

Dietary supplement etc.

54–12-6

204.23

-(2)(15)

-(2)

    

271

l-tryptophan

Dietary supplement etc.

73–22-3

204.23

-(2)(15)

-(2)

    

272

trimethylamine

Flavoring agent

75–50-3

59.11

-H11

+H11

-H17

   

273

2,3,5-trimethylpyrazine

Flavoring agent

14667–55-1

122.17

-H15

-H15

    

274

dl-threonine

Dietary supplement etc.

80–68-2

119.12

-(2)(15)

-(2)

    

275

l -threonine

Dietary supplement etc.

72–19-5

119.12

-(2)(15)

-(2)

    

276

nisin

Preservative etc.

1414–45-5

3354.07

-FSC37

 

-FSC37

   

277

natamycin

Preservative

7681-93-8

665.73

-FSC38

+FSC38

    

278

sodium methoxide (sodium methylate)

Processing agent

124–41-4

54.02

-(14)

     

279

nicotinic acid (niacin)

Dietary supplement etc.

59–67-6

123.11

-(3)(11)

-(3)

-(9)

   

280

nicotinamide (niacinamide)

Dietary supplement etc.

98–92-0

122.12

-(2)(11)

-(2)

    

281

sulfur dioxide (sulfurous acid, anhydride)

Preservative etc.

7446-09-5

64.06

      

282

chlorine dioxide

Flour treatment agent

10049–04-4

67.45

+(4)(4)

+(4)

+(9)

  

Ames showed positive for liquid form and negative for powdery form, CA showed numerical abnormality for powdery form, liquid form was used in MN

283

silicon dioxide (silica gel)

Filtration aid

14464–46-1

60.08

-(20)

     

284

carbon dioxide (carbonic acid, gas)

Preservative

124–38-9

44.01

      

285

titanium dioxide

Food color

13463–67-7

79.87

-(19)

-H22

    

286

lactic acid

Acidifier

50–21-5

90.08

-(3)(18)

-(3)

   

Samples were distinguished whether in a glass container or a plastic container

287

potassium lactate

Seasoning etc.

996–31-6

128.17

 

-FSC39

  

Rec assay: -FSC39

 

288

calcium lactate

Sweetener etc.

814–80-2

218.22

-(13)

-H23

    

289

iron lactate

Dietary supplement

5905-52-2

233.99

-(18) + (5)

+(5)

-(7)

-H23

 

Target organs for TGR in mice:liver, kidneys

290

sodium lactate

Acidifier etc.

72–17-3

112.06

-(4)(19)

-(4)

    

291

neotame

Sweetener etc.

165450–17-9

378.46

-FSC40

-FSC40

-FSC40

   

292

γ-nonalactone (nonalactone)

Flavoring agent

104–61-0

 

-(16)

-H22

    

293

potassium norbixin

Food color

33261–80-2

456.66

-(1)

-(1)

    

294

sodium norbixin

Food color

33261–81-3

424.45

-(1)

-(1)

    

295

vanillin

Flavoring agent

121–33-5

152.15

-(2)(16)

-(2)

    

296

isobutyl p -hydroxybenzoate

Preservative

4247–02-3

194.23

-(1)(16)

-(1)

    

297

isopropyl p -hydroxybenzoate

Preservative

4191-73-5

180.20

-(1)(16)

-(1)

    

298

ethyl p -hydroxybenzoate

Preservative

120–47-8

166.17

-(1)(16)

+(1)

    

299

butyl p -hydroxybenzoate

Preservative

94–26-8

194.23

-(1)(10)

-(1)

    

300

propyl p -hydroxybenzoate

Preservative

94–13-3

180.20

-(16)

+H25

    

301

p -methylacetophenone

Flavoring agent

122–00-9

134.18

-(16)

+H24

    

302

l -valine

Dietary supplement etc.

72–18-4

117.15

-(3)(18)

-(3)

    

303

valeraldehyde

Flavoring agent

110–62-3

86.13

-FSC41

 

-H17

   

304

calcium pantothenate

Dietary supplement

137–08-6

476.53

-(3)(12)

-(3)

    

305

sodium pantothenate

Dietary supplement

75033–16-8

 

-(5)(11)

-(5)

    

306

biotin

Dietary supplement

58–85-5

244.31

-FSC42

    

identical to d-biotin

307

l -histidine monohydrochloride

Dietary supplement

7048–02-4

209.63

-(5)

-(5)

    

308

bisbentiamine (benzoylthiamine disulfide)

Dietary supplement

2667-89-2

770.92

-(4) -H23

-(4) ± H23

    

309

vitamin A (retinol)

Dietary supplement

68–26-8

286.45

-(20)

-H23

    

310

vitamin a fatty acids esters (retinol esters of fatty acids esters)

Dietary supplement

  

-(1)

-(1)

-(9)

   

311

1-hydroxyethylidene-1, 1-diphosphonic acid

Processing agent

2809-21-4

 

-FSC19

-FSC19

    

312

hydroxycitronellal

Flavoring agent

107–75-5

172.26

-(20)

+H24

    

313 [31]

hydroxycitronellal dimethylacetal

Flavoring agent

141–92-4

218.33

- (25)

 

- (25)

   

314

hydroxypropyl distarch phosphate

Thickening agents etc.

5324-00-8

      

Evaluated as modified starch

315

hydroxypropyl cellulose

Emulsifier etc.

9004–64-2

 

-FSC43

     

316

hydroxypropyl starch

Thickening agents etc.

68130–14-3

      

Evaluated as modified starch

317

hydroxypropyl methylcellulose

Emulsifier etc.

9004–65-3

 

-H12

-H12

-H12

   

318

piperidine

Flavoring agent

110–89-4

85.15

-FSC44

-H17

-H17

   

319

piperonal (heliotropine)

Flavoring agent

120–57-0

150.13

+(16)

 

-H22

-H23

 

Target organs for TGR in mice:liver, kidneys

320

piperonyl butoxide

Insecticide

51–03-6

338.44

-(1)(19)

-(1)

    

321

sunflower lecithin

Emulsifier

8002–43-5

 

-FSC45

-FSC45

    

322

acetic acid, glacial

Acidifier

64–19-7

60.05

-(4)(17)

-(4)

    

323

pyrazine

Flavoring agent

290–37-9

80.09

-FSC46

+FSC46

-H18

   

324

pyridoxine hydrochloride (vitamin B6)

Dietary supplement

58–56-0

205.64

-(2)(11)

-(2)

    

325

pyrimethanil

Preservative etc.

131341–86-1

199.26

-FSC47

 

-FSC47

 

Rec assay: -FSC47

 

326

potassium pyrosulfite (potassium hydrogen sulfite or potassium metabisulfite)

Preservative etc.

16731–55-8

222.33

-(1)(12)

-(1)

    

327

sodium pyrosulfite (sodium metabisulfite, acid sulfite of soda)

Preservative etc.

7681–57-4

190.11

-(3)(19)

-(3)

   

Described as sodium bisulfite, anhydrous

328

pyrrolidine

Flavoring agent

123–75-1

71.12

-FSC48

+H17

-H18

   

329

potassium pyrophosphate (tetrapotassium pyrophosphate)

Processing agent

7320–34-5

330.34

-(15)

-H22

    

330

calcium dihydrogen pyrophosphate (acid calcium pyrophosphate)

Dietary supplement etc.

14866–19-4

216.04

-(20)

-H23

    

331

disodium dihydrogen pyrophosphate (acid disodium pyrophosphate)

Processing agent

7758-16-9

221.94

-(4)(20)

-(4)

    

332

ferric pyrophosphate

Dietary supplement etc.

1332-96-3

745.21

-(4)(11)

-(4)

    

333

sodium pyrophosphate (tetrasodium pyrophosphate)

品質改良剤

7722-88-5

265.90

-(5)(15)

-(5)

    

334

pyrrole

Flavoring agent

109–97-7

67.09

-FSC49

+H18

-H21

   

335

l -phenylalanine

Dietary supplement etc.

63–91-2

165.19

-(2)(18)

-(2)

    

336

isoamyl phenylacetate

Flavoring agent

102–19-2

206.28

-(16)

-H25

    

337

isobutyl phenylacetate

Flavoring agent

102–13-6

192.25

-(19)

-H24

    

338

ethyl phenylacetate

Flavoring agent

101–97-3

164.20

-(2)(16)

-(2)

    

339

2-(3-phenylpropyl) pyridine

Flavoring agent

2110-18-1

197.28

-H17

+H17

-H18

   

340

phenethylamine

Flavoring agent

64–04-0

121.18

-H17

+H17

-H18

   

341

phenol ethers (except those generally recognized as highly toxic)

Flavoring agent

        

342

phenols (except those generally recognized as highly toxic)

Flavoring agent

        

343

ferrocyanides (potassium ferrocyanide (potassium hexacyanoferrate (ii)), calcium ferrocyanide (calcium hexacyanoferrate (ii)), sodium ferrocyanide (sodium hexacyanoferrate (ii)))

Processing agent etc.

13943–58-3, 13821–08-4, 13601–19-9

422.39, 508.29, 484.06

   

Evaluated as potassium ferrocyanide and sodium ferrocyanide

344

butanol

Processing agent etc.

71一36–3

74.12

-H15

-H15

-H15

   

345

butylamine

Flavoring agent

109–73-9

73.14

-FSC50

+H17

-H18

   

346

butyraldehyde

Flavoring agent

123–78-8

72.11

-FSC51

+FSC51

-FSC51

-FSC51

   

347

butylated hydroxyanisole

Antioxidant

25013–16-5

180.24

-(1)(10)

-(1)

-(3)

   

348

fumaric acid

Acidifier

110–17-8

116.07

-(4)(17)

-(4)

    

349

monosodium fumarate (sodium fumarate)

Acidifier

5873-57-4

138.05

-(5)(19)

-(5)

    

350

fludioxonil

Preservative etc.

131341–86-1

248.19

-FSC52

+FSC52

-FSC52

   

351

furfurals and its derivatives (except those generally recognized as highly toxic)

Flavoring agent

  

-(17)

-H23

   

Furfural was used for the assay.

352

propanol

Processing agent etc.

71–23-8

60.09

-FSC53

     

353

propionaldehyde

Flavoring agent

123–38-6

58.08

-FSC54

 

-H17

   

354

propionic acid

Flavoring agent etc.

79–09-4

74.08

-(16)

-H22

    

355

isoamyl propionate

Flavoring agent

105–68-0

144.21

-(3)(16)

-(3)

    

356

ethyl propionate

Flavoring agent

105–37-3

102.13

-(3)(16)

-(3)

    

357

calcium propionate

Preservative

4075-81-4

186.22

-(2)(16)

-(2)

    

358

sodium propionate

Preservative

137–40-6

96.06

-(1)(11)

-(1)

    

359

benzyl propionate

Flavoring agent

122–63-4

164.20

-(16)

-H24

    

360

propylene glycol

Quality sustainer etc.

57–55-6

76.09

-(1)(14)

+(1)

-(9)

   

361

propylene glycol esters of fatty acids

Emulsifier

  

-(2)(20)

-(2)

    

362

hexanoic acid (caproic acid)

Flavoring agent

142–62-1

116.16

-(12)

-H24

    

363

allyl hexanoate (allyl caproate)

Flavoring agent

123–68-2

156.22

-(17)

-(4) -H22

    

364

ethyl hexanoate (ethyl caproate)

Flavoring agent

123–66-0

144.21

-(17)

-(4) -H24

    

365

ethyl heptanoate (ethyl enanthate)

Flavoring agent

106–30-9

158.24

-(12)

-H24

    

366

l -perillaldehyde

Flavoring agent

18031–40-8

150.22

-(3)(19)

+(3)

-(9)

   

367

benzyl alcohol

Flavoring agent

100–51-6

108.14

-(3)(17)

-(3)

-(9)

   

368

benzaldehyde

Flavoring agent

100–52-7

106.12

-(17)

-H22

    

369

2-pentanol (sec-amylalcohol)

Flavoring agent

6032-29-7

88.15

-H16

-H16

    

370

trans-2-pentenal

Flavoring agent

1576-87-0

84.12

  

-H17

   

371

1-penten-3-ol

Flavoring agent

616–25-1

86.13

+H16

-H16

-H17

  

Ames showed positive only in T1537, whose maximum number of the revertants was s within the range of negative control.

372

aromatic alcohols

Flavoring agent

        

373

aromatic aldehydes (except those generally recognized as highly toxic)

Flavoring agent

        

374

propyl gallate

Antioxidant

121–79-9

212.20

-(1) + (13)

+(1)

-H21

-H21

 

Target organs for TGR in mice:liver, stomach (glandular stomach)

375

sodium polyacrylate

Thickening agents etc.

9003–04-7

 

-(2)(13)

-(2)

    

376

polyisobutylene (butyl rubber)

Chewing gum base

9003–27-4

 

-(3)

-(3)

    

377

polysorbate 20

Emulsifier etc.

9005-64-5

1227.72

     

See the data on substances with different molecular weight

378

polysorbate 60

Emulsifier etc.

9005-67-8

1311.90

-FSC55

-FSC55

    

379

polysorbate 65

Emulsifier etc.

9005-71-4

1842

-FSC55

+FSC55

-FSC55

   

380

polysorbate 80

Emulsifier etc.

9005-65-6

1309.68

-FSC55

-FSC55

-FSC55

 

Rec assay: -FSC55

 

381

polyvinylpyrroridone

Thickening agents etc.

9003-39-8

 

-FSC56

     

382

polyvinylpolypyrrolidone

Processing agent

25249–54-1

 

-FSC57

     

383

polybutene (polybutylene)

Chewing gum base

9003–28-5

 

-(4)(20)

-(4)

    

384

potassium polyphosphate

Processing agent

68956–75-2

 

-(20)

-H22

    

385

sodium polyphosphate

Processing agent

68915–31-1

 

-(3)(5)(15)

-(3)(5)

   

Sodium tripolyphosphate was used in (3).

386

d -borneol

Flavoring agent

464–43-7

154.25

-(17)

-H22

    

387

maltol

Flavoring agent

118–71-8

126.11

-(4) + (17)

+(4)

+(9)

-H21

 

Target organs for TGR in mice:liver, stomach (glandular stomach)

388

d-mannitol (d-mannite)

Antisticking agent etc.

69–65-8

182.17

-(3)(13)

-(3)

    

389

potassium metaphosphate

Processing agent

7790–53-6

118.07

-(2)(15)

-(2)

    

390

sodium metaphosphate

Processing agent

10361–03-2

101.96

-(3)(15)

-(3)

    

391

dl-methionine

Dietary supplement etc.

59–51-8

149.21

-(18)

-H22

    

392

l-methionine

Dietary supplement

63–68-3

149.21

-(18)

-H23

    

393

methyl n-methylanthranilate

Flavoring agent

85–91-6

165.19

-(17)

-H22

    

394

5-methylquinoxaline

Flavoring agent

13708–12-8

144.17

-H15

+H15

-H15

   

395

6-methylquinoline

Flavoring agent

91–62-3

143.19

  

-H16

   

396

6,7-dihydro-5-methyl-5 h-cyclopentapyrazine

Flavoring agent

23747–48-0

134.18

-H16

-H16

-H17

   

397

methyl cellulose

Thickening agents etc.

9004–67-5

 

-(3)(5)(14)

-(3)

    

398

1-methylnaphthalene

Flavoring agent

90–12-0

142.20

-FSC58

+H17

-H18

-FSC58

 

Target organs for TGR in mice:lung

399

methyl β-naphthyl ketone

Flavoring agent

93–08-3

170.21

-(17)

-H24

    

400

2-methypyrazine

Flavoring agent

109–08-0

94.11

-FSC59

 

-H16

   

401

2-methylbutanol

Flavoring agent

137–32-6

88.15

-H15

-H15

-H15

   

402

3-methyl-2-butanol

Flavoring agent

598–75-4

88.15

-H17

-H17

    

403

2-methylbutyraldehyde

Flavoring agent

96–17-3

86.13

-FSC60

 

-H17

   

404

trans −2-methyl-2-butenal, (e)-2-methyl-2-butenal

Flavoring agent

497–03-0

84.12

+H17

+H17

-H18

   

405

3-methyl-2-butenal

Flavoring agent

107–86-8

84.12

+H16

+H16

-H17

   

406

3-methyl-2-butenol

Flavoring agent

556–82-1

86.13

+H16

+H16

+H17

  

The result of MN was pseudo positive.

407

methyl hesperidin (soluble vitamin P)

Dietary supplement

11013–97-1

624.59

-(3)(12)

-(3)

    

408

dl-menthol (dl-peppermint camphor)

Flavoring agent

89–78-1

156.27

-(1)(20)

-(1)

    

409

l-menthol (peppermint camphor)

Flavoring agent

2216–51-5

156.269

-(4)(17)

-(4)

    

410

morpholine salts of fatty acids

Coating agent

  

-(2)

-(2)

    

411

folic acid

Dietary supplement

59–30-3

441.404

-(5)(11)

-(5)

    

412

butyric acid

Flavoring agent

107–92-6

88.106

-(3)(17)

-(3)

    

413

isoamyl butyrate

Flavoring agent

106–27-4

158.241

-(3)(4)(17)

-(3)(4)

    

414

ethyl butyrate

Flavoring agent

105–54-4

116.16

-(3)(4)(17)

-(3)(4)

    

415

cyclohexyl butyrate

Flavoring agent

1551-44-6

170.252

-(17)

-H25

    

416

butyl butyrate

Flavoring agent

109–21-7

144.214

-(17)

-H24

    

417

lactones (except those generally recognized as highly toxic)

Flavoring agent

        

418

l-lysine l-aspartate

Dietary supplement etc.

  

-(5)

-(5)

    

419

l-lysine monohydrochloride

Dietary supplement etc.

657–27-2

182.65

-(5)(18)

-(5)

    

420

l-lysine l-glutamate

Dietary supplement etc.

  

-H23

-H23

    

421

linalool

Flavoring agent

78–70-6

154.25

-(3)(17)

-(3)

    

422

calcium 5′-ribonucleotide

Seasoning

  

-H22

-H22

    

423

disodium 5′-ribonucleotide (sodium 5′-ribonucleotide)

Seasoning

  

-(3)

-(3)

    

424

riboflavin (vitamin B2)

Dietary supplement etc.

83–88-5

376.369

-(1)(6)(11)

+(1)

-(9)

 

Rec assay: -(6)

 

425

riboflavin tetrabutyrate (vitamin B2 tetrabutyrate)

Dietary supplement etc.

752–56-7

656.733

-(4)(18)

-(4)

    

426

riboflavin 5′-phosphate sodium (riboflavin phosphate sodium, vitamin B2 phosphate sodium)

Dietary supplement etc.

130–40-5

478.33

-(3)(18)+(5)

-(3)

-(7)

   

427

sulfuric acid

Processing agent

7664-93-9

98.072

-(16)

     

428

aluminum ammonium sulfate (crystal: ammonium alum, desiccated: burnt ammonium alum)

Raising agent etc.

7784–25-0

237.15

-(14)

-H20

-H20

   

429

aluminum potassium sulfate (crystal: alum or potassium alum, desiccated: burnt alum)

Raising agent etc.

10043–67-1

258.21

-(3)(14)

-(3)

-H20

   

430

ammonium sulfate

Processing agent

7783-20-2

132.14

-(18)

-H23

    

431

potassium sulfate

Seasoning etc.

7778–80-5

174.25

     

See substances with different salt

432

calcium sulfate

Tofu coagulator etc.

7778–18-9

172.17

-(13)

-H23

    

433

ferrous sulfate

Dietary supplement etc.

13463–43-9

151.91

-(3)(19)

+(3)

-(9)

   

434

sodium sulfate

Processing agent

7757-82-6

142.036

-(18)

-H23

    

435

magnesium sulfate

Tofu coagulator etc.

7487-88-9

120.361

-(3)(18)

-(3)

   

Dried sample was also used in (3)

436

dl-malic acid (dl -malic acid)

Acidifier

6915-15-7

134.087

-(2)(17)

-(2)

    

437

sodium dl-malate (sodium dl -malate)

Acidifier etc.

138–09-0

178.051

-(4)(19)

-(4)

    

438

phosphoric acid

Acidifier

7664-38-2

97.994

-(18)

-H23

    

439

distarch phosphate

Thickening agents etc.

 

-FSC20

-FSC20

-FSC20

   

440

monostarch phosphate

Thickening agents etc.

 

-FSC20

-FSC20

-FSC20

   

441

tripotassium phosphate (potassium phosphate, tribasic)

Processing agent

7778-53-2

212.264

-(19)

     

442

tricalcium phosphate (calcium phosphate, tribasic)

Processing agent etc.

7758-87-4

310.18

-(13)

     

443

trimagnesium phosphate

Processing agent

10233–87-1

262.86

     

See substances with different salt

444

diammonium hydrogen phosphate (diammnonium phosphate or ammnonium phosphate, dibasic)

Processing agent

7783-28-0

132.056

-(18)

-H23

    

445

ammonium dihydrogen phosphate (ammonium phosphate,monobasic or monoammonium phosphate)

Processing agent

7722-76-1

115.025

-(5)(18)

+(5)

-(7)

   

446

dipotassium hydrogen phosphate (dipotassium phosphate or potassium phosphate, dibasic)

Processing agent

7758-11-4

174.174

-(19)

     

447

potassium dihydrogen phosphate (monopotassium

Processing agent

7778–77-0

136.084

-(18)

-H22

    

448

calcium monohydrogen phosphate (calcium phosphate,

Processing agent

136.06

136.06

-(5)(12)

-(5)

    

449

calcium dihydrogen phosphate (calcium phosphate,

Processing agent

7758-23-8

234.05

-(3)(14)

-(3)

    

450

disodium hydrogen phosphate (disodium phosphate)

Processing agent

7558-79-4

141.958

-(3)(19)

-(3)

   

Crystal form and anhydrous form were used in (3) and in (19), respectively.

451

sodium dihydrogen phosphate (monosodium phosphate)

Processing agent

7558-80-7

119.976

-(18)

     

452

magnesium monohydrogen phosphate

Processing agent etc.

7782–75-4

174.33

     

See compounds with different salt

453

trisodium phosphate (sodium phosphate, tribasic)

Processing agent

7601-54-9

163.94

-(19)

    

Anhydrous substrate was also used in the assay

454

phosphated distarch phosphate

Thickening agents etc.

      

See substrate with different salt

aNumbers are consistent with those shown in Tables 1 and 2

bUnderlined bold style means that the items are added as indicated in Table 2

c(H12) means Heisei era 12, and indicates that it referred to the report on food inspection expenses implemented in 2000 (=Heisei 12). The same applies to H13, H16, H17, H18, H20, H21, H22, H23, H24, H25, H26

d(FSC) indicates Risk assessment reports in Food Safety Commission of Japan. See http://www.fsc.go.jp/fsciis/evaluationDocument/list?itemCategory=000 (Additional file 1)

How the data were summarized

The following set of three tests has traditionally been used to evaluate mutagenicity of food additives: reverse mutation assay (Ames test) using bacteria; chromosomal aberration test using cell culture (CA); and micronucleus test using mice (MN). The Hayashi report summarized the data from the results of these three tests. Two new tests suitable for the evaluation of food additives were subsequently added in the OECD Genotoxicity Test Guidelines. The two adopted test guidelines are: “Genetic mutation test using transgenic rodent somatic and germ cells (TG 488)” (the TGR test); and “In vivo mammalian alkaline comet assay (TG 489)” (the comet test). Results using these assays are included in this paper (Table 3). While both tests have advantages for the evaluation of genotoxicity in specific tissues, the TGR test is intended as a mutagenicity test (similar to the Ames test) therefore the weighting of TGR results is generally higher due to its high correlation with carcinogenicity.

When searching for items that had not been subjected to mutagenicity testing, it was felt desirable that all test outcomes were discovered without exception. Thus, in Table 3, data from journals other than Hayashi report are included; for example, data published in the Annual Report of the Tokyo Metropolitan Research Laboratory of Public Health (originally published in Japanese). Reports that were surveyed, including journals other than Hayashi report, are listed at the end of this paper. Results of the three tests used initially are mainly copied from the Hayashi report (also published in Japanese). Data reevaluated or added after the Hayashi report include results from outsourced testing laboratories accredited by the MHLW as part of the “Projects for safety of food additives.”

In the main Table, the superscript symbols “H22” and “H23” indicate results commissioned in tests conducted in Fiscal Year 2010 (FY2010) and FY2011, respectively. Test results from the Risk Assessment Reports prepared by the Food Safety Commission (FSC) are also included; these are indicated by superscript “FSC”, and the URL for the Risk Assessment Reports of the item is given in the reference list. Note that reference numbers in the text are not given in numeric order but in the order of appearance in Table 3. The eliminated and newly added substances are listed in Tables 1 and 2, respectively. In Table 3, the item numbers from Table 2 are shown underlined.

Commentary for 13 items newly subjected to the TGR test

1) Five items positive for Ames and chromosome aberration tests while negative for in vivo micronucleus test

Sodium nitrite (No. 6 in Table 3)

Ames testing was performed at the highest dose of 10 mg/plate using TA1535, TA98, TA1537, TA94, TA92 in FY1979, and positive results were obtained with TA100 and TA1535 regardless of S9mix [2]. Subsequently, using TA97 and TA102, a statistically significant increase in the number of revertants (maximum dose 10 mg/plate) was reported in both strains regardless of S9mix [3]. However, the result is given as negative in Table 3 since the number of reverted colonies did not reach twice of the number for the negative control. Many positive results of Ames tests were reported in this item, thus, mutagenicity was suspected for this substance [4].

For chromosomal abnormalities, the chromosome aberration test using Chinese Hamster lung (CHL) was performed at a maximum dose of 1.0 mg/mL without S9mix, and strong induction of structural abnormalities was reported [2]. Subsequently, in vivo bone marrow micronucleus tests using ddY mice were carried out under three conditions: a single intraperitoneal (i.p.) dose of 200 mg/kg body weight; four i.p. doses at 50 mg/kg body weight at 24-h intervals; and a single oral dose of 400 mg/kg body weight [5], all results being reported as negative.

In FY2009, TGR testing using gpt delta mice was performed in the liver and glandular stomach for confirmation of in vivo mutagenicity. These organs were selected because the liver metabolizes many substances and is highly sensitive in this assay, and the glandular stomach is the organ first exposed to the substance under test with oral administration. The mutagenicity that was previously observed in the Ames test did not occur in vivo, since the TGR results were negative in both organs after 28 days of administration via drinking water at a maximum dose of 5000 mg/kg body weight [H21(FY2009)].

l-Cysteine monohydrochloride (No. 179 in Table 3)

Ames testing was carried out using TA100, TA98, TA2637, TA94, at a maximum dose of 10 mg/plate with or without S9mix in FY1982. Positive results were reported for TA100 with S9mix, and for TA2637 with and without S9mix [6]. Chromosomal aberration tests were performed using CHL cells without S9mix and structural abnormalities were induced (maximum dose was 2 mg/mL) [6]. Subsequently, the Ames test was conducted at Tokyo Metropolitan Research Laboratories of Public Health (TMRL) using TA97 and TA102 with and without S9mix at a maximum dose of 10 mg/plate, and positive results were reported under all conditions [7]. Since genotoxicity was detected in vitro, bone marrow micronucleus test using ddY mice was conducted in 1986. The test results were negative for single i.p. doses of 125, 250 and 500 mg/kg body weight [5].

In FY2009, TGR testing using gpt delta mice was performed in the liver and glandular stomach for confirmation of in vivo mutagenicity; the results were negative in both organs following oral gavage for 28 days at a maximum dose of 1000 mg/kg body weight [H22(FY2010)].

Despite positive in vitro results, it was concluded that l-cysteine hydrochloride is not genotoxic in living organisms because results were negative in in vivo MN and TGR tests.

Cinnamaldehyde (No. 222 in Table 3)

Ames tests were performed at a maximum dose of 0.5 mg/plate with and without S9mix using TA100, TA1535, TA98, TA1537, TA92, TA97 in FY1981. Only TA100 showed positive results regardless of the metabolic activation in this report [8]. In the chromosome aberration test simultaneously carried out using CHL cells, structural abnormalities were induced without S9mix (maximum dose 0.015 mg/mL) [8]. Subsequently, Ames tests were carried out at TMRL using TA97 and TA102 at a maximum dose of 0.1 mg/plate; results were negative regardless of metabolic activation [7]. Since genotoxicity was detected in vitro, in vivo bone marrow MN testing was conducted in FY1986. Mice (ddY) were given a single i.p. injection at 125, 250 and 500 mg/kg body weight and the results were negative [5].

We conducted TGR tests in the liver and small intestine (jejunum) using gpt delta mice to confirm in vivo mutagenicity in FY2010 and FY2011. The reason for choosing the small intestine as the target organ is that it is the first in the gastrointestinal tract to be exposed to substances administered orally. Mice were dosed by oral gavage at 125, 250, 500 and 1000 mg/kg body weight for 28 days, and mutagenicity was investigated for the animals dosed at 500 and 1000 mg/kg body weight. Negative results were obtained in both organs [H22(FY2010)].

Despite showing genotoxicity in vitro, it was concluded that cinnamaldehyde does not show genotoxicity in living organisms because results were negative in in vivo MN and TGR tests.

Iron lactate (No. 289 in Table 3)

Ames testing was carried out in FY1983 and the results were positive without S9mix in TA97, TA102 and TA2637 at the highest dose of 5.0 mg/plate, and negative in TA100 and TA98 with and without S9mix [9]. Chromosomal aberration testing using CHL cells conducted in the same year induced structural abnormalities without S9mix (maximum dose 2.5 mg/mL) [9]. Subsequently, Ames tests were conducted at TMRL using TA97 and TA102, yielding negative results with and without S9mix [10]. The maximum dose in this study, 1.0 mg/plate, is considered to be insufficient. In vivo MN testing using ddY mice was conducted in FY1986 (single i.p. administration of 30 mg/kg body weight, and four separate i.p. doses of 7.5 mg/kg body weight/day), with negative results [11].

In FY2011, TGR tests in the liver and kidney were carried out using gpt delta mice to confirm in vivo mutagenicity. The reason for using the kidney as the target organ is that nephrotoxicity was observed in macroscopic examinations. After doses of 250, 500 and 1000 mg/kg body weight for 28 days by oral gavage, mutation was investigated at doses of 500 and 1000 mg/kg body weight. Since the results were negative in both organs [H23(FY2011)], it was concluded that iron lactate does not induce mutation in vivo.

Propyl gallate (No. 374 in Table 3)

Ames testing was carried out in TA100, TA98, TA1537 at 500 μg/plate in FY1979, and negative results were obtained regardless of metabolic activation [2]. Subsequently, Ames tests were carried out at TMRL using TA97 and TA102, and at the maximum dose of 0.1 mg/plate it was found that TA102 showed a statistically significant increase in the number of revertants regardless of metabolic activation [12]. TA100 and TA1535 mainly detect base substitution occurring in GC base pairs, while TA102 mainly reveals substitution in AT base pairs. Thus, these results (negative in TA100 and TA1535, positive in TA102) suggest that propyl gallate is reactive with AT base pairs. The negative results for TA98, TA1537 and TA97 indicate that the probability of inducing a frameshift mutation is low. Five out of six tests showed positive results at doses higher than 50 μg/mL, but an unusual pattern was shown regarding dose correlation for which the mechanism is unknown.

Since the above results suggested that propyl gallate induces base substitution with AT base pairs in vitro, TGR testing was performed in liver and glandular stomach using gpt delta mice in FY2009 [H21(FY2009)]. Repeated administration over 28 days produced negative results for both organs at the highest dose of 1000 mg/kg body weight. Thus, the mutagenicity of propyl gallate was detected in vitro, but not considered to be detected in vivo.

After chromosomal aberration testing in FY1979 it was reported that structural abnormalities were induced in CHL after 24 h treatment at a dose of 0.04 mg/mL without S9mix [2]. To investigate the risk of chromosomal aberration, in vivo bone marrow MN testing was conducted in FY2009, with negative results at the maximum dose of 1000 mg/kg body weight (administered twice) [H21(FY2009)]. Therefore, although chromosomal abnormalities were detected in vitro, they were not in vivo.

From the results detailed above, propyl gallate was considered to be non-genotoxic to living bodies.

2) Two items negative for chromosome aberration and in vivo micronucleus tests while positive for Ames test

Erythorbic acid (isoascorbic acid)(No. 78 in Table 3)

This substance was positive only in TA100 regardless of S9mix (highest dose 50 mg/plate) in the Ames test using the strains TA100, TA98, TA1535, TA98, TA1537, TA92 and TA94 in FY1980 [13]. In chromosomal aberration tests using CHL cells, a negative result was reported at the highest dose of 0.25 mg/mL without S9mix [13]. In the Ames test conducted at TMRL using TA97 and TA102, a statistically significant increase in the number of revertants was reported in both strains regardless of S9mix (maximum dose 10 mg/plate) [14]. However, the result is given as negative in Table 3 since the number of revertants did not reached twice of the number of the negative control. Subsequently, an in vivo bone marrow MN test using ddY mice was performed, and this substance showed negative when administered in a single dose of 1500 mg/kg body weight (at the maximum) or as four treatments (at 24-h intervals) at 750 mg/kg body weight (at the maximum).

Thereafter, TGR testing using gpt delta mice was conducted for liver and glandular stomach (maximum dose 1000 mg/kg body weight for 28 days by gavage) in FY2009 in order to investigate in vivo mutagenicity. Neither point mutation nor deletion mutation was induced in either organ [H21(FY2009)]. It was concluded that there are no concerns for genotoxicity of erythorbic acid to living bodies.

Piperonal (No. 319 in Table 3)

In Ames testing at TMRL using TA97 and TA102 this substance showed positive results in TA97 without S9mix at the highest dose of 1 mg/plate [3]. It is reported that a statistically significant increase was observed with S9mix, but the level did not reach twice that of the negative control. There are no reports on chromosomal aberration tests. MN testing using ICR mice was carried out in FY2010, and the results were negative in bone marrow after oral administration of 250, 500 and 1000 mg/kg body weight (two doses at 24-h intervals) [H22(FY2010)]. In FY2010–11, TGR testing using gpt delta mice was performed in the liver and kidney in order to confirm in vivo mutagenicity at doses of 250, 500 and 1000 mg/kg body weight for 28 days by gavage. The results were negative for both organs at doses of 500 and 1000 mg/kg body weight [H23(FY2011)].

From the above results it was concluded that piperonal does not show genotoxicity in living organisms.

3) One item positive in all three tests (Ames, chromosomal aberration and in vivo micronucleus tests)

Maltol

In 1982, Ames testing using TA100, TA98, TA2637, and TA94 was carried out for maltol at a maximum dose of 10.0 mg/plate, and the results were negative both with and without S9mix [6]. Chromosomal aberration testing was conducted in the same year, and it was reported that structural abnormalities were induced in CHL cells at the highest dose (0.075 mg/mL) without S9mix [6]. Subsequently, Ames testing was performed at TMRL with TA97 and TA102, at the highest dose of 10.0 mg/plate with and without S9mix. Induction of colony formation at a reversion level almost double that of the negative control was observed in TA97 at a dose of 1 mg/plate without S9mix. Positive judgment has been reported in a micronucleus test using bone marrow of ddY mice, 24 h after single i.p. administration of 125, 250 and 500 mg/kg body weight [5]. Since the usage of this item is limited to fragrances, there is no possibility of exposure in vivo at a concentration equivalent to the dose at which chromosomal abnormality was detected in vitro.

In FY2009, TGR testing using gpt delta mice was performed in the liver and glandular stomach for confirmation of in vivo gene mutagenicity. The results were negative in both organs at doses of 400, 200, 100 and 50 mg/kg body weight for 28 days by gavage [H21(FY2009)].

From the above, it seems that there is no concern of genotoxicity in maltol for living bodies.

4) Five items for which the Ames test was negative

1-Methylnaphthalene

In FY2005 this substance was reported to have induced structural abnormalities in a chromosome aberration test using CHL cells [H17(FY2005)] while in vivo bone marrow micronucleus testing conducted in FY2006 reported negative results in a two-dose study of 1000 mg/kg body weight at the maximum [H18(FY2006)].

Regarding mutagenicity, in Ames tests using several strains of Salmonella typhimurium conducted from 1980 to 2002, all results were negative, whereas a weak positive result was reported in the forward mutation test using S. typhimurium (maximum dose 0.992 mg/mL, 2-h exposure) [FSC58]. In theory, the Ames test, which is a reverse mutation test, can detect only specific point mutations while a forward mutation test can detect mutations of any type. Thus, it would be problematic for the negative results of the Ames tests to be taken as completely eliminating the concerns about mutagenicity arising from the result of the forward mutation tests. Subsequently, a TGR test in gpt delta mice (males and females) was performed on the lungs. The reason that the lungs were selected as the target organ was that weak carcinogenicity was observed in the lungs of mice in the 81-week chronic toxicity–carcinogenicity combination test reported in 1993. TGR tests were conducted at doses of 170 and 280 mg/kg body weight for females and 120 and 220 mg/kg body weight for males by dietary administration for 13 weeks, the results being negative in all conditions ([15], FSC58).

From the above, 1-methylnaphthalene is considered to have no concerns of genotoxicity for living bodies.

Food Red No. 40

In FY1995 at TMRL negative results (maximum dose 10 mg/plate) in Ames tests with TA97 and TA102 with and without S9mix were reported [16]. Chromosomal aberration tests have not been carried out. Subsequently, in vivo micronucleus tests using CD1 mice was performed in FY2008, and results of single oral gavage of 500, 1000 and 2000 mg/kg body weight were reported to be negative in bone marrow [H20(FY2008)].

In FY2008 and FY2011, comet and TGR tests using mice were conducted to examine in vivo DNA damage inducibility and mutagenicity, respectively. In the comet test, CDF1 mice were administered two doses of 500, 1000 and 2000 mg/kg body weight by oral gavage with a 24-h interval. The results were negative for both liver and glandular stomach (H20(FY2008), [17]). In addition, another comet assay using ICR mice was carried out with two oral gavage administrations (24-h interval) at doses of 500, 1000 and 2000 mg/kg body weight. The results were negative in both stomach and colon, while an increase without dose correlation was observed in liver [H23(FY2011)]. TGR testing was conducted using the Muta™Mouse, orally gavaged at doses of 250, 500 and 1000 mg/kg body weight for 28 days; mutagenicity in the liver and glandular stomach was not observed [H20(FY2008)]. Furthermore, TGR tests using gpt delta mice were carried out and the results were negative for mutagenicity in the large intestine following oral gavage for 28 days at doses of 250, 500 and 1000 mg/kg body weight [H23(FY2011)].

From the above, it seems that there is no concern of genotoxicity of Food Red No. 40 for living bodies.

Food Red No. 102

In 1979 negative results were reported following Ames tests carried out with and without S9mix conditions using TA100, TA1535, TA98, TA1537, TA92 and TA94 (maximum dose 5.0 mg/plate) [2]. In chromosomal aberration tests using CHL cells carried out in the same year, induction of structural abnormalities was observed with S9mix (maximum dose of 4.0 mg/mL) [2]. Subsequently, Ames tests were conducted at TMRL using TA97 and TA102, and the results were negative with and without S9mix (maximum dose 10 mg/plate) [10]. Since chromosomal abnormalities were induced in vitro, micronucleus tests using ddY mice were carried out in FY1980. Results were negative for two sets of conditions in bone marrow: single i.p. administration of 300, 600 and 1200 mg/kg body weight; and four i.p. doses of 300 mg/kg body weight [13].

In FY2008, comet and TGR tests using mice were carried out to examine in vivo DNA damage inducibility and mutagenicity, respectively [H20(FY2008)]. Comet tests were carried out by oral gavage (twice, at 24-h intervals) at doses of 500, 1000 and 2000 mg/kg body weight using CDF1 mice, and the results were judged as negative in both liver and glandular stomach. The TGR tests were carried out in Muta™Mouse using oral gavage at 250, 500 and 1000 mg/kg body weight for 28 days, and the results were negative in both liver and glandular stomach.

From the above, Food Red No.102 is considered not to have concerns of genotoxicity to living bodies.

Food Red No. 104

In 1979, negative results in Ames tests carried out using TA100, TA1535, TA98, TA1537, TA92 and TA94 with and without S9mix (maximum dose 5 mg/plate) were reported [2]. In the same year, chromosomal aberration tests using CHL cells were conducted without S9mix, and the results were negative (maximum dose 0.25 mg/mL) [2]. Ames tests were also conducted at TMRL with TA97 and TA102, and the results were negative with and without S9mix (maximum dose 1 mg/plate) [10]. Micronucleus testing using mice was not performed because both in vitro tests were negative.

In FY2008, comet and TGR tests using mice were carried out to confirm in vivo DNA damage inducibility and mutagenicity, respectively. Comet tests for liver and glandular stomach were performed by oral gavage (twice, 24-h interval) at doses of 250, 500 and 1000 mg/kg body weight on CDF1 mice. Results showed false positive in the liver and positive in glandular stomach [H20(FY2008)]. TGR tests were conducted using Muta™Mouse with oral gavage at 250, 500 and 1000 mg/kg body weight for 28 days and liver and glandular stomach were examined for mutation induction; the results were negative in both [H20(FY2008)].

Based on the above results, it is likely that the DNA damage detected in the comet tests would not reach the level necessary to produce mutation. The negative results in liver and glandular stomach in TGR tests support this view, and it seems likely that the DNA damage is repaired in mouse body. Therefore, Food Red No. 104 is considered not to induce genotoxicity (mutagenicity) in vivo.

Food Red No. 105

In FY1978 results were negative in Ames tests (maximum dose 5.0 mg/plate) with and without S9mix using TA100, TA1535, TA98, TA1537, TA92 and TA94 [2]. In the same year, chromosomal aberration tests using CHL cells were also carried out (S9mix only) and the results were negative (maximum dose 0.25 mg/mL) [2]. Subsequently, Ames tests were carried out at TMRL using TA97 and TA102 (maximum dose 1 mg/plate) with and without S9mix, with negative results [10]. Micronucleus tests using mice were not carried out because both in vitro tests were negative.

In FY2008, in order to examine in vivo DNA damage inducibility and mutagenicity, comet and TGR tests were conducted, respectively, in mice. The comet test was positive in both liver and glandular stomach for oral administration (twice, 24-h interval) at doses of 250, 500 and 1000 mg/kg body weight for CDF1 mice, and were examined [H20(FY2008)]. The TGR test was conducted using Muta™Mouse with oral gavage at 250, 500 and 1000 mg/kg body weight for 28 days. Mutation induction in the liver and glandular stomach was tested for, both results being negative [H20(FY2008)].

Since the TGR tests performed in mouse liver and glandular stomach were negative, the DNA damage detected in the comet test is considered to have been repaired in vivo. Thus, there is a high possibility that such DNA damage would not lead to mutation. In conclusion, there are no concerns that Food Red No. 105 induces genotoxicity (mutagenicity) in vivo.

Discussion

The standard genotoxicity tests are carried out to detect gene mutation by Ames test using bacteria, and to detect chromosomal abnormalities by an in vitro chromosomal aberration test using cell culture and an in vivo micronucleus test using mice. Chromosomal abnormalities in chromosomal aberration tests are observed as morphological abnormalities in chromosomes during the interphase of cell division because damaged DNA is not normally replicated and the abnormalities persist. Such structural abnormalities are lethal for cells in many cases, and the majority of chromosomal abnormalities are not inherited by the next generation. Similarly, micronuclei in the micronucleus test also transiently appear in daughter cells after cell division, and disappear after the next cell division. Therefore, chromosomal abnormalities and micronuclei are indicators that DNA has been exposed to genotoxic substances, not a cause of cancer in cells. The fragmentation of DNA observed in the comet test is also transient, thus the comet test is also an indicator test. On the other hand, gene mutation is irreversible and permanent. Gene mutations arising in oncogenes or tumor suppressor genes have the possibility to cause cell transformation and initiate cancer-forming cells. Therefore, genetic mutation is a direct trigger of cancer, and it is highly correlated with carcinogenicity in rodents compared to other genotoxic end points [18], while the chromosome aberration test and micronucleus test have high false positive rates and low correlation with carcinogenicity tests [19].

The TGR test, which is an in vivo gene mutation test, is thus recommended when chemical substances have shown positive results in chromosome aberration tests, micronucleus tests, and comet tests. In particular, when comet and TGR tests are carried out on the same target tissue, if the results differ between the two, the results of the TGR tests should be given priority. The TGR test is also useful for follow-up of the same gene mutation test, the Ames test. A false positive reaction sometimes occurs in the Ames test because of bacteria-specific conditions such as drug metabolism, in vitro test-specific reactions using rat S9, as well as nonspecific reactions due to non-physiological conditions differing from the in vivo situation. Confirmation of an indication of mutagenicity with the Ames test by the TGR test in the living body is important on both scientific and safety grounds.

Among the 13 designated food additives covered in the Commentary section, eight items were positive for the Ames test, but the TGR test showed negative results for all of them. As a result, the possibility that these eight food additives exhibit genetic toxicity (especially mutagenicity, which is problematic for living bodies) is eliminated. This knowledge is important to ensure human safety. The TGR test took effect with publication of the OECD Guideline TG488 in 2011 and therefore was not available for implementation at the time of the Hayashi report (2000). We expect the safety of other food additives to be confirmed as TGR test results are accumulated.

Postscript

In this report, we summarized the data for the most widely used substances in the classification of designated food additives in Japan. Currently we are summarizing the results of genotoxicity tests conducted at MHLW for existing food additives, a group of the next most widely used food additives, in the same way. The reports will be updated from time to time since additions and deletions of items are considered likely in the future.

Abbreviations

CA: 

Chromosomal aberration test

CHL: 

Chinese hamster lung

FSC: 

Food Safety Commission

FY: 

Fiscal year

MHLW: 

The Ministry of Health, Labour and Welfare

MN: 

Micronucleus test

NIHS: 

National Institute of Health Sciences

OECD: 

The Organisation for Economic Co-operation and Development

TGR: 

Transgenic rodent gene mutation assay

TMRL: 

Tokyo Metropolitan Research Laboratories of Public Health

Declarations

Acknowledgements

We would like to thank all the authors in the references, and the researchers who conducted the tests and provided the data. The authors wish to express their gratitude to the Food Safety Standards and Evaluation Division, Pharmaceutical Safety and Environmental Health Bureau, Ministry of Health, Labour and Welfare of Japan and Japan Food Additives Association for providing genotoxicity test data of food additives.

Funding

This work was supported by Health and Labour Sciences Research Grants (H28-Food-General-001 and H30-Food-General-003) from Ministry of Health, Labour and Welfare of Japan.

Availability of data and materials

Not applicable

Authors’ contributions

MH conceived of the study and participated in its design and coordination. MY collected the data, created a detailed table and wrote the manuscript. Both authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Department of Applied Chemistry, National Defense Academy, 1-10-20, Hashirimizu, Yokosuka-shi Kanagawa, 239-8686, Japan
(2)
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki-ku, Kawasaki-shi Kanagawa, 210-9501, Japan

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