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WHO FOOD ADDITIVES SERIES 46:Cinnamyl Alcohol and Related Substances

First draft prepared by
Dr A. Mattia
Division of Product Policy, Office of Premarket Approval, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, Washington DC, USA
and
Professor G.I. Sipes
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, USA

Evaluation

Introduction

Estimated daily intake

Metabolic considerations

Application of the Procedure for the Safety Evaluation of Flavouring Agents

Consideration of combined intakes

Conclusions

Relevant background information

Explanation

Additional considerations on intake

Biological data

Absorption, distribution, and excretion

Hydrolysis of esters and acetals

Oxidation and conjunction reactions of cinnamyl alcohol and cinnamaldehyde derivatives

Metabolism of cinnamic acid

Effects of ring and chain substitutuents on metabolism of cinnamyl derivatives

Toxicological studies

Acute toxicity

Short-term and long-term studies of toxicity

Genotoxicity

Reproductive toxicity

References

1. EVALUATION

1.1 Introduction

The Committee evaluated a group of flavouring agents that includes cinnamyl alcohol (No. 647), cinnamaldehyde (No. 656), cinnamic acid (No. 657), and 52 structurally related substances (see Table 1). The evaluations were conducted according to the Procedure for the Safety Assessment of Flavouring Agents. One member of this group, allyl cinnamate (No. 19), had been evaluated previously by the Committee in a separate group of allyl ester flavouring agents examined by the Procedure (Annex 1, reference 122). Cinnamaldehyde (No. 656) was evaluated by the Committee at its eleventh meeting (Annex 1, reference 14), when it established a conditional ADI of 01.25 mg/kg bw. At its twenty-third meeting, the Committee converted the previous conditional ADI to a temporary ADI of 00.7 mg/kg bw (Annex 1, reference 50), which was extended at its twenty-fifth and twenty-eighth meetings (Annex 1, references 56 and 66). At its thirty-fifth meeting, the Committee did not extend the temporary ADI (Annex 1, reference 88) because the data that were required were not available. At its twenty-fifth meeting, the Committee concluded that cinnamyl anthranilate should not be used as a food additive (Annex 1, reference 56). This substance is structurally related to members of the group of cinnamyl alcohol and related flavouring agents but differs in that it is hydrolysed to cinnamyl alcohol and anthranilic acid only slowly, resulting in systemic intake of the intact ester. In contrast, the members of the group of flavouring agents undergo rapid hydrolysis.

Table 1. Summary of the results of safety evaluations of cinnamyl alcohol and related flavouring agentsa

Flavouring agent

No.

CAS no. and structure

Step A3b
Does intake exceed the threshold for human intake?b

Step A4
Is the substance or are its metabolites endogenous?

Step A5
Adequate NOEL for substance or related substance?

Comments

Conclusion based on current intake

Structural class I

3-Phenyl-1-propanol

636

122-97-4

No

N/R

N/R

See note 1.

No safety concern

chemical structure

Europe: 60
USA: 31

 

 

 

 

3-Phenylpropyl formate

637

104-64-3

No

N/R

N/R

See note 2.

No safety concern

chemical structure

Europe: N/D
USA: 0.8

 

 

 

 

3-Phenylpropyl acetate

638

122-72-5

No

N/R

N/R

See note 2.

No safety concern

chemical structure

Europe: 41
USA: 9

 

 

 

 

3-Phenylpropyl propionate

639

122-74-7

No

N/R

N/R

See note 2.

No safety concern

chemical structure

Europe: 0.2
USA: 0.3

 

 

 

 

3-Phenylpropyl isobutyrate

640

103-58-2

No

N/R

N/R

See note 2.

No safety concern

chemical structure

Europe: 4
USA: 16

 

 

 

 

3-Phenylpropyl isovalerate concern

641

5452-07-3

No

N/R

N/R

See note 2.

No safety concern

chemical structure

Europe: 0.01
USA: 0.1

 

 

 

 

3-Phenylpropyl hexanoate concern

642

6281-40-9

No

N/R

N/R

See note 2.

No safety concern

chemical structure

Europe: N/D
USA: 0.4

 

 

 

 

Methyl 3-phenylpropionate concern

643

103-25-3

No

N/R

N/R

See note 2.

No safety concern

chemical structure

Europe: N/D
USA: 3

 

 

 

 

Ethyl 3-phenylpropionate concern

644

2021-28-5

No

N/R

N/R

See note 2.

No safety concern

chemical structure

Europe: 1
USA: 0.07

 

 

 

 

3-Phenylpropionic acid

646

501-52-0

No

N/R

N/R

See note 3.

No safety concern

chemical structure

Europe: 23
USA: 0.5

 

 

 

 

Cinnamyl alcohol

647

104-54-1

Yes

No

Yes.

See note 4.

No safety concern

chemical structure

Europe: 1800
USA: 1900

 

The NOEL of 54 mg/kg bw per day for cinnamyl alcohol (Zaitsev & Rakhmanina, 1974) is > 1000 times the daily intakes of 30 g/kg bw per day in Europe and 32 g/kg bw per day in the USA.

 

 

Cinnamyl formate

649

104-65-4

No

N/R

N/R

See note 5.

No safety concern

chemical structure

Europe: 2
USA: 17

 

 

 

 

Cinnamyl acetate

650

103-54-8

No

N/R

N/R

See note 5.

No safety concern

chemical structure

Europe: 210
USA: 300

 

 

 

 

Cinnamyl propionate

651

103-56-0

No

N/R

N/R

See note 5.

No safety concern

chemical structure

Europe: 4
USA: 25

 

 

 

 

Cinnamyl butyrate

652

103-61-7

No

N/R

N/R

See note 5.

No safety concern

chemical structure

Europe: 3
USA: 2

 

 

 

 

Cinnamyl isobutyrate

653

103-59-3

No

N/R

N/R

See note 5.

No safety concern

chemical structure

Europe: 13
USA: 22

 

 

 

 

Cinnamyl isovalerate

654

140-27-2

No

N/R

N/R

See note 5.

No safety concern

chemical structure

Europe: 5
USA: 8

 

 

 

 

Cinnamyl benzoate

760

5320-75-2

No

N/R

N/R

See note 6.

No safety concern

chemical structure

Europe: N/D
USA: 1

 

 

 

 

Cinnamyl phenylacetate

655

7492-65-1

No

N/R

N/R

See note 7.

No safety concern

chemical structure

Europe: 0.003
USA: 1

 

 

 

 

Cinnamaldehyde

656

104-55-2

Yes

No

Yes.

See note 4.

No safety concern

chemical structure

Europe: 2500
USA: 59 000

 

The NOEL of 620 mg/kg bw per day for cinnamaldehyde (National Toxicology Program, 1995) is > 10 000 and > 600 times the daily intakes of 42 g/kg bw per day in Europe and 990 g/kg bw per day in the USA.

 

 

Cinnamic acid

657

621-82-9

No

N/R

N/R

See note 8.

No safety concern

chemical structure

Europe: 32
USA: 44

 

 

 

 

Methyl cinnamate

658

103-26-4

Yes

No

Yes.

See note 9.

No safety concern

chemical structure

Europe: 2800
USA: 830

 

The NOELs of 54 and 80 mg/kg bw per day for cinnamyl alcohol and ethyl connamate, respectively, are > 1000 times the daily per capita intake of methyl cinnamate in Europe

 

 

Ethyl cinnamate

659

103-36-6

No

N/R

N/R

See note 9.

No safety concern

chemical structure

Europe: 100
USA: 70

 

 

 

 

Propyl cinnamate

660

7778-83-8

No

N/R

N/R

See note 9.

No safety concern

chemical structure

Europe: 0.4
USA: 4

 

 

 

 

Isopropyl cinnamate

661

7780-06-5

No

N/R

N/R

See note 9.

No safety concern

chemical structure

Europe: 19
USA: 3

 

 

 

 

Allyl cinnamate

19

1866-31-5

No

N/R

N/R

See note 9.

No safety concern

chemical structure

Europe: 5
USA: 0.3

 

 

 

 

Butyl cinnamate

663

538-65-8

No

N/R

N/R

See note 9.

No safety concern

chemical structure

Europe: 0.4
USA: 0.2

 

 

 

 

Isobutyl cinnamate

664

122-67-8

No

N/R

N/R

See note 9.

No safety concern

chemical structure

Europe: 1
USA: 3

 

 

 

 

Isoamyl cinnamate

665

7779-65-9

No

N/R

N/R

See note 9.

No safety concern

chemical structure

Europe: 8
USA: 6

 

 

 

 

Heptyl cinnamate

666

10032-08-3

No

N/R

N/R

See note 9.

No safety concern

chemical structure

Europe: 2
USA: 52

 

 

 

 

Cyclohexyl cinnamate

667

7779-17-1

No

N/R

N/R

Hydrolysed to cinnamic acid

No safety concern

chemical structure

Europe: 0.4
USA: 0.04

 

 

(see note 8) and cyclo-hexanol. Cyclo-hexanol is mainly conjugated with glucuronic acid and excreted.

 

Linalyl cinnamate

668

78-37-5

No

N/R

N/R

Hydrolysed to cinnamic acid.

No safety concern

chemical structure

Europe: 7
USA: 3

 

 

(see note 8) and linalool. Linalool undergoes omega and omega-1 oxidation to yield polar, excretable metabolites.

 

Terpinyl cinnamate

669

10024-56-3

No

N/R

N/R

Hydrolysed to cinnamic acid

No safety concern

chemical structure

Europe: 0.01
USA: 0.5

 

 

(see note 8) and terpineol. Terpineol undergoes omea and omea-1 oxidation to yield polar excretable metabolites

 

Benzyl cinnamate

670

103-41-3

No

N/R

N/R

Hydrolysed to cinnamic acid

No safety concern

chemical structure

Europe: 44
USA: 69

 

 

(see note 8) and benzyl alcohol. Benzyl alcohol is oxidized to benzoic acid and excreted as hippuric acid.

 

Phenethyl cinnamate

671

103-53-7

No

N/R

N/R

Hydrolysed to cinnamic acid

No safety concern

chemical structure

Europe: 6
USA: 50

 

 

(see note 8) and phenethyl alcohol. Phenethyl alcohol is oxidized to phenylacetic acid and excreted as the glucuronic acid conjugate.

 

3-Phenylpropyl cinnamate

672

122-68-9

No

N/R

N/R

See notes 1 and 8.

No safety concern

chemical structure

Europe: 0.6
USA: 37

 

 

 

 

Cinnamyl cinnamate

673

122-69-0

No

N/R

N/R

See notes 4

No safety concern

chemical structure

Europe: 2
USA: 36

 

 

 

and 8.

alpha-Amylcinnamyl formate

676

7493-79-0

No

N/R

N/R

See note 10.

No safety concern

chemical structure

Europe: 1.4
USA: 0.5

 

 

 

 

alpha-Amylcinnamyl acetate

677

7493-78-9

No

N/R

N/R

See note 10.

No safety concern

chemical structure

Europe: 3
USA: 260

 

 

 

 

alpha-Amylcinnamyl isovalerate

678

7493-80-3

No

N/R

N/R

See note 10.

No safety concern

chemical structure

Europe: 0.01
USA: 0.5

 

 

 

 

3-Phenyl-4-pentenal

679

939-21-9

No

N/R

N/R

Oxidized to the corresponding acid and excreted

No safety concern

chemical structure

Europe: 1
USA: 2

 

 

 

 

3-(para-Isopropylphenyl)-propionaldehyde

680

7775-00-0

No

N/R

N/R

See note 1.

No safety concern

chemical structure

Europe: N/D
USA: 0.1

 

 

 

 

alpha-Amylcinnamaldehyde dimethyl acetal

681

91-87-2

No

N/R

N/R

See note 10.

No safety concern

chemical structure

Europe: 0.01
USA: 0.007

 

 

 

 

para-Methylcinnam-aldehyde

682

1504-75-2

No

N/R

N/R

See note 4.

No safety concern

chemical structure

Europe: 0.01
USA: 0.9

 

 

 

 

alpha-Methylcinnamaldehyde

683

101-39-3

No

N/R

N/R

See note 4.

No safety concern

chemical structure

Europe: 3
USA: 390

 

 

 

 

para-Methoxycinnamaldehyde

687

1963-36-6

No

N/R

N/R

See note 4.

No safety concern

chemical structure

Europe: 0.04
USA: 0.01

 

 

 

 

ortho-Methoxycinnamal-dehyde

688

1504-74-1

No

N/R

N/R

Oxidized to the corres ponding acid, conju gated and excreted.

No safety concern

chemical structure

Europe: 0.6
USA: 71

 

 

Alternatively, the acid may undergo beta-oxida tion to yield the beta-hyd roxy carboxylic acid derivative, which is also excreted.

 

para-Methoxy-alpha-methyl cinnamaldehyde

689

65405-67-6

No

N/R

N/R

See note 4.

No safety concern

chemical structure

Europe: 0.3
USA: 0.05

 

 

 

 

Structural class II

Cinnamaldehyde ethylene glycol acetal

648

5600-60-6

Yes

No

Yes.

 

No safety concern

chemical structure

Europe: 690
USA: 0.007

 

The NOEL of 620 mg/kg bw per day for cinnamaldehyde is > 10 000 times the daily per capita intake of cinnamldehyde ethylene glycol acetal in Europe

Hydrolysed to the corresponding alcohol and aldehyde

 

alpha-Amylcinnamyl alcohol

674

101-85-9

No

N/R

N/R

 

No safety concern

chemical structure

Europe: 4
USA: 1

 

 

Oxidized to alpha-amylcin namaldehyde, which is further oxidized to alpha- amylcinnamic acid and excreted.

 

5-Phenylpentanol

675

10521-91-2

No

N/R

N/R

See note 1.

No safety concern

chemical structure

Europe: N/D
USA: 0.1

 

 

 

 

alpha-Butylcinnamal-dehyde

684

7492-44-6

No

N/R

N/R

See note 11.

No safety concern

chemical structure

Europe: 0.01
USA: 0.07

 

 

 

 

alpha-Amylcinnamal-dehyde

685

122-40-7

No

N/R

N/R

See note 11.

No safety concern

chemical structure

Europe: 25
USA: 23

 

 

 

 

alpha-Hexylcinnamal-dehyde

686

101-86-0

No

N/R

N/R

See note 11

No safety concern

chemical structure

Europe: 87
USA: 11

 

 

 

 

CAS: Chemical Abstracts Service; N/D: no intake data reported; N/R: not required for evaluation because consumption of the substance was determined to be of no safety concern at step A3 of the Procedure.

a Step 2: All of the substances in this group are expected to be metabolized to innocuous products.

b The thresholds for human intake for classes I and II are 1800 g/day and 540 g/day, respectively. All intake values are expressed in g/day.

Notes to Table 1

1. Oxidized to yield the corresponding acid, which undergoes further side-chain beta-oxidation and cleavage to yield the benzoic acid derivative. It then conjugates with glycine and/or glucuronic acid, and is excreted in the urine.

2. Hydrolysed to the corresponding acid and alcohol. The acid is completely oxidized and the alcohol, 3-phenyl-1-propanol, is further metabolized and excreted (see note 1).

3. Undergoes side-chain beta-oxidation and cleavage to yield the corresponding benzoic acid derivative; it then conjugates with glycine and/or glucuronic acid and is excreted in the urine.

4. Oxidized to cinnamic acid or its corresponding derivative and further oxidized to benzoic acid or its corresponding derivative, which is excreted as hippuric acid or its corresponding derivative.

5. Hydrolysed to cinnamyl alcohol and the corresponding carboxylic acid. Cinnamyl alcohol is oxidized and excreted (see note 4); the carboxylic acid is either completely oxidized or conjugated and excreted primarily in the urine.

6. Hydrolysed to cinnamyl alcohol and benzoic acid. Cinnamyl alcohol is oxidized to cinnamic acid, which is further oxidized to benzoic acid (see note 4).

7. Hydrolysed to cinnamyl alcohol and phenylacetic acid. Cinnamyl alcohol is oxidized to cinnamic acid, which is further oxidized to benzoic acid (see note 4). Phenylacetic acid is excreted as the glucuronic acid conjugate.

8. Undergoes beta-oxidation and is excreted as hippuric acid

9. Rapidly hydrolysed to cinnamic acid (see note 8) and the corresponding alcohol. The corresponding alcohol is completely oxidized.

10. Hydrolysed to alpha-amylcinnamyl alcohol (No. 674) and the corresponding acid, which is excreted; alpha-amylcinnamyl alcohol is oxidized to alpha-amylcinnamaldehyde, which is further oxidized to alpha-amylcinnamic acid and excreted.

11. Oxidized to the corresponding acid and excreted.

Twenty-two of the 55 flavouring agents in this group are natural components of foods. Concentrations of cinnamaldehyde of up to 750 g/kg have been detected in oils from natural sources, such as cinnamon, cinnamomum, and cassia leaves; however, these agents are consumed predominantly as flavouring agents (Maarse et al., 1996).

1.2 Estimated daily intake

The total annual volume of production of the 55 cinnamyl compounds considered here is approximately 60 t in Europe (International Organization of the Flavor Industry, 1995) and 480 t in the USA (National Academy of Sciences, 1987; Lucas et al., 1999; see Table 2). Approximately 30% of the total annual volume in Europe and over 93% of that in the USA is accounted for by cinnamaldehyde (No. 656), while 54% of the total annual volume in Europe is accounted for by the use of cinnamyl alcohol (No. 647) and methyl cinnamate (No. 658). The estimated intakes in Europe are 2.5 mg/day of cinnamaldehyde, 1.8 mg/day of cinnamyl alcohol, and 2.8 mg/day of methyl cinnamate. The estimated intakes in the USA are 59 mg/day of cinnamaldehyde, 1.9 mg/day of cinnamyl alcohol, and 0.83 mg/day of methyl cinnamate. The intakes of all the other flavouring agents in the group are in the range 0.003690 g/day, most of the values being at the low end of this range. Production volumes and intake of each substance are reported in Table 2.

Table 2. Annual volumes of production of cinnamyl alcohol and related substances used as flavouring agents in Europe and the USA

Substance (No.)

Most recent annual volume (kg)a

Intakeb

Annual volume in naturally occurring foods (kg)c

Consumption ratiod

g/day

g/kg bw per day

3-Phenyl-1-propanol (636)

Europe

420

60

1

+

NA

USA

236

31

0.5

 

NA

3-Phenylpropyl formate (637)

Europe

N/D

N/D

N/D

NA

USA

6

0.8

0.01

 

NA

3-Phenylpropyl acetate (638)

Europe

289

41

0.7

140

0.5

USA

68

9

0.1

 

2.1

3-Phenylpropyl propionate (639)

Europe

1

0.2

0.003

+

NA

USA

2

0.3

0.005

 

NA

3-Phenylpropyl isobutyrate (640)

Europe

30

4

0.07

NA

USA

123

16

0.3

 

NA

3-Phenylpropyl isovalerate (641)

Europe

0.1

0.01

0.0002

NA

USA

0.5

0.1

0.001

 

NA

3-Phenylpropyl hexanoate (642)

Europe

N/D

N/D

N/D

NA

USA

3

0.4

0.007

 

NA

Methyl 3-phenylpropionate (643)

Europe

N/D

N/D

N/D

NA

USA

23

3

0.05

 

NA

Ethyl 3-phenylpropionate (644)

Europe

10

1

0.02

47

4.7

USA

0.5

0.07

0.001

 

94

3-Phenylpropionaldehyde (645)

Europe

134

19

0.3

+

NA

USA

146

19

0.3

 

NA

3-Phenylpropionic acid (646)

Europe

161

23

0.4

+

NA

USA

4

0.5

0.008

 

NA

Cinnamyl alcohol (647)

Europe

12 543

1 800

30

171

0.00

USA

14 682

1 900

32

 

0.01

Cinnamaldehyde ethylene glycol (648) acetal

Europe

4 821

690

11

NA

USA

0.05

0.007

0.0001

 

NA

Cinnamyl formate (649)

Europe

15

2

0.04

NA

USA

127

17

0.3

 

NA

Cinnamyl acetate (650)

Europe

1 498

210

4

+

NA

USA

2 255

300

5

 

NA

Cinnamyl propionate (651)

Europe

30

4

0.07

NA

USA

191

25

0.4

 

NA

Cinnamyl butyrate (652)

Europe

21

3

0.05

+

NA

USA

17

2

0.04

 

NA

Cinnamyl isobutyrate (653)

Europe

90

13

0.2

NA

USA

164

22

0.4

 

NA

Cinnamyl isovalerate (654)

Europe

32

5

0.08

NA

USA

64

8

0.14

 

NA

Cinnamyl benzoate

Europe

N/D

N/D

N/D

NA

USA

5

1

0.01

 

NA

Cinnamyl phenylacetate (655)

Europe

0.02

0.003

0.05

NA

USA

11

1

0.02

 

NA

Cinnamaldehyde (656)

Europe

17 623

2 500

42

38 642

2.2

USA

451 364

59 000

991

 

0.09

Cinnamic acid (657)

Europe

227

32

0.5

183

0.8

USA

332

44

0.7

 

0.6

Methyl cinnamate (658)

Europe

19 384

2 800

46

57

0.003

USA

6 318

830

14

 

0.009

Ethyl cinnamate (659)

Europe

727

100

2

292

0.4

USA

530

70

1

 

0.6

Propyl cinnamate (660)

Europe

2.6

0.4

0.006

NA

USA

31

4

0.07

 

NA

Isopropyl cinnamate (661)

Europe

135

19

0.3

NA

USA

23

3

0.05

 

NA

Allyl cinnamate (19)

Europe

38

5

0.09

NA

USA

2

0.3

0.004

 

NA

Butyl cinnamate (663)

Europe

3

0.4

0.006

NA

USA

1

0.2

0.003

 

NA

Isobutyl cinnamate (664)

Europe

10

1

0.02

+

NA

USA

21

3

0.05

 

NA

Isoamyl cinnamate (665)

Europe

57

8

0.1

+

NA

USA

46

6

0.1

 

NA

Heptyl cinnamate (666)

Europe

12

2

0.03

NA

USA

391

52

0.9

 

NA

Cyclohexyl cinnamate (667)

Europe

3

0.4

0.006

NA

USA

0.3

0.04

0.001

 

NA

Linalyl cinnamate (668)

Europe

49

7

0.1

NA

USA

19

3

0.04

 

NA

Terpinyl cinnamate (669)

Europe

0.1

0.01

0.0002

NA

USA

4

0.5

0.009

 

NA

Benzyl cinnamate (670)

Europe

310

44

0.7

+

NA

USA

527

69

1

 

NA

Phenethyl cinnamate (671)

Europe

40

6

0.1

NA

USA

382

50

0.8

 

NA

3-Phenylpropyl cinnamate (672)

Europe

4

0.6

0.01

NA

USA

282

37

0.6

 

NA

Cinnamyl cinnamate (673)

Europe

11

2

0.03

+

NA

USA

277

36

0.6

 

NA

alpha-Amylcinnamyl alcohol (674)

Europe

27

4

0.06

NA

USA

9

1

0.02

 

NA

5-Phenylpentanol (675)

Europe

N/D

N/D

N/D

NA

USA

1

0.1

0.002

 

NA

alpha-Amylcinnamyl formate (676)

Europe

10

1.4

0.02

NA

USA

4

0.5

0.009

 

NA

alpha-Amylcinnamyl acetate (677)

Europe

20

3

0.05

NA

USA

1 996

263

4

 

NA

alpha-Amylcinnamyl isovalerate (678)

Europe

0.1

0.01

0.0002

NA

USA

4

0.5

0.009

 

NA

3-Phenyl-4-pentenal (679)

Europe

6

1

0.01

NA

USA

16

2

0.04

 

NA

3-(para-Isopropyl-phenyl)propionaldehyde (680)

Europe

N/D

N/D

N/D

NA

USA

1

0.1

0.002

 

NA

alpha-Amylcinnamaldehyde dimethyl acetal (681)

Europe

0.1

0.01

0.0002

NA

USA

0.05

0.007

0.0001

 

NA

para-Methylcinnamaldehyde (682)

Europe

0.1

0.01

0.0002

NA

USA

7

0.9

0.02

 

NA

alpha-Methylcinnamaldehyde (683)

Europe

20

3

0.05

+

NA

USA

2 932

390

6

 

NA

alpha-Butylcinnamaldehyde (684)

Europe

0.1

0.01

0.0002

NA

USA

0.5

0.07

0.001

 

NA

alpha-Amylcinnamaldehyde (685)

Europe

178

25

0.4

+

NA

USA

173

23

0.4

 

NA

alpha-Hexylcinnamaldehyde (686)

Europe

611

87

1

+

NA

USA

82

11

0.2

 

NA

para-Methoxycinnamaldehyde (687)

Europe

0.3

0.04

0.001

+

NA

USA

0.1

0.01

0.0002

 

NA

ortho-Methoxycinnamaldehyde (688)

Europe

4

0.6

0.01

+

NA

USA

541

71

1

 

NA

para-Methoxy-alpha-methyl-cinnamaldehyde (689)

Europe

2

0.3

0.005

NA

USA

0.4

0.05

0.001

 

NA

Total

Europe

60 000

 

 

 

 

USA

480 000

 

 

 

 

NA, not available; N/D, no intake data reported; +, reported to occur naturally in foods (Maarse et al., 1996), but no quantitative data; , not reported to occur naturally in foods

a From International Organization of the Flavor Industry (1995) and Lucas (1999)

b Intake (g/person per day) calculated as follows: [(annual volume, kg) (1 109 g/kg)/ (population survey correction factor 365 days)], where population (10%, eaters only) = 32 x 106 for Europe and 26 x 106 for the USA. The correction factor = 0.6 for Europe and 0.8 for the USA, representing the assumption that only 60% and 80% of the annual volume of the flavour, respectively, was reported in the poundage surveys (International Organization of the Flavor Industry, 1995; Lucas et al., 1999). Intake (g/kg bw per day) calculated as follows: [(g/person per day)/body weight], where body weight = 60 kg. Slight variations may occur from rounding.

c Quantitative data from Stofberg & Grundschober (1987)

d Calculated as follows: (annual consumption in food, kg)/(most recently reported volume as a flavouring agent, kg)

1.3 Metabolic considerations

Cinnamyl alcohol (No. 647), cinnamaldehyde (No. 656) and its para- and ortho-methoxy derivatives (Nos 687 and 688), cinnamic acid (No. 657) and its corres-ponding methyl ester (No. 658), and the saturated analogue (3-phenylpropionic acid, No. 646) have all been shown to be rapidly absorbed from the gut, metabolized, and excreted primarily in the urine and to a minor extent in the faeces.

Esters of cinnamic acid and structurally related aromatic esters have been shown to be hydrolysed rapidly to the component acid and alcohol. The aromatic primary alcohols and aldehydes in this group and those formed by the hydrolysis of esters and acetals are readily oxidized to cinnamic acid or one of its structurally related carboxylic acids. In animals, most carboxylic acids, such as cinnamic acid, are converted to acyl coenzyme A esters. Cinnamoyl coenzyme A undergoes either glycine conjugation catalysed by a glycine N-acyl transferase or beta-oxidation, eventually leading to the formation of benzoyl coenzyme A. This is in turn either conjugated with glycine, yielding hippuric acid, or hydrolysed to yield free benzoic acid, which is then excreted (Nutley et al., 1994).

Cinnamyl derivatives containing alpha-methyl substituents, such as alpha-methylcin-namaldehyde (No. 683), are extensively metabolized by beta-oxidation and cleavage to yield mainly the corresponding hippuric acid derivative. Because ortho-oxygenated ring substituents (e.g. ortho-methoxycinnamaldehyde, No. 688) selectively inhibit oxidation of coenzyme A esters of beta-hydroxyacids via the beta-oxidation pathway, these hydroxyacid derivatives are excreted as glycine conjugates. In contrast, para-oxygenated ring substituents (e.g. para-methoxycinnamaldehyde, No. 687) are oxidized via the beta-oxidation pathway, eventually yielding hippuric acid derivatives.

1.4 Application of the Procedure for the Safety Evaluation of Flavouring Agents

Step 1.

In applying the Procedure for the Safety Evaluation of Flavouring Agents to the above-mentioned substances, the Committee assigned 49 of the 55 substances to structural class I (Cramer et al., 1978). These are simple aromatic compounds with a saturated propyl or unsaturated propenyl side-chain containing a primary oxygenated functional group, which have little toxic potential. The remaining six substances, which are those containing a heterocyclic ring (No. 648) or rings bearing substituents other than 15 carbon aliphatic groups (Nos 674, 675, 684686), were assigned to structural class II.

Step 2.

All the substances in this group are predicted to be metabolized to innocuous products (see section 2.3). The evaluation of these substances therefore proceeded via the left-hand side of the decision-tree.

Step A3.

The estimated daily per capita intakes of 46 of the 49 substances in structural class I and of five of six substances in structural class II are below the threshold for human intake for each structural class (i.e. 1800 g/person per day for class I and 540 g/person per day for class II). According to the Procedure, the safety of these 51 flavouring agents raises no concern when they are used at their currently estimated levels of intake. The intake of cinnamaldehyde (No. 656) is 2.5 mg/person per day in Europe and 59 mg/person per day in the USA. The intake of methyl cinnamate (No. 658) is 2800 g/day in Europe and 830 g/day in the USA. The intake of cinnamaldehyde ethylene glycol acetal (No. 648; structural class II) is 690 g/day in Europe and 0.007 g/day in the USA. The intake of cinnamyl alcohol (No. 647) is 1800 g/person per day in Europe and 1900 g/person per day in the USA. The intakes of these four flavouring agents therefore exceed the threshold for human intake for class I and class II (1800 and 540 g/person per day, respectively). Accordingly, the evaluation of these substances proceeded to step A4.

Step A4.

None of these four flavouring agents is endogenous. Therefore, their evaluation proceeded to step A5.

Step A5.

The NOEL of 54 mg/kg bw per day for cinnamyl alcohol (No. 647) (Zaitsev & Rakhmanina, 1974) is about 1000 times greater than the estimated intake of cinnamyl alcohol from its use as a flavouring agent in Europe (29 g/kg bw per day) and the USA (32 g/kg bw per day). The NOEL of 620 mg/kg bw per day for cinnamaldehyde (No. 656) (National Toxicology Program, 1995) is 10 000 times greater than the estimated daily intake of this substance from use as a flavouring agent in Europe (42 g/kg bw) and about 600 times greater than that in the USA (990 g/kg bw). The NOEL of 54 mg/kg bw per day for cinnamyl alcohol is appropriate for evaluating the safety of methyl cinnamate (No. 658) because cinnamyl alcohol is oxidized to cinnamic acid, which is a product of hydrolysis of methyl cinnamate. In addition, a NOEL of 80 mg/kg bw per day has been identified for a closely related ester, ethyl cinnamate. Both of these NOELs are > 1000 times the daily per capita intake of methyl cinnamate. Cinnamaldehyde ethylene glycol acetate (No. 648) is rapidly hydrolysed to cinnamaldehyde; the NOEL of 620 Mg/kg bw per day for cinnamaldehyde is > 10 000 times the daily per capita intake of cinnamaldehyde ethylene glycol acetal. The Committee therefore concluded that the safety of these substances would not be expected to be of a concern.

Table 1 summarizes the evaluation of cinnamyl alcohol and 54 related substances used as flavouring agents.

1.5 Consideration of combined intakes

In the unlikely event that all foods containing the 50 substances in structural class I were consumed concurrently on a daily basis, the estimated combined intake would exceed the threshold for human intake for class I. In the unlikely event that all foods containing the five substances in structural class II were consumed concurrently on a daily basis, the estimated combined intake would exceed the threshold for human intake for class II. However, all 55 substances in the group are expected to be efficiently metabolized and would not saturate the metabolic pathways. Overall evaluation of the data indicates that combined intake would not raise concern about safety.

1.6 Conclusions

The Committee concluded that the safety of the flavouring agents in this group would not raise concern at the current estimated levels of intake. Other data on the toxicity of cinnamyl derivatives were consistent with the results of the safety evaluation.

2. RELEVANT BACKGROUND INFORMATION

2.1 Explanation

This monograph summarizes data relevant to an evaluation of the safety of cinnamyl alcohol (No. 647), cinnamaldehyde (No. 656), cinnamic acid (i.e., trans-3-phenylpropenoic acid, No. 657), and 52 structurally related substances. All members of this group are primary alcohols, aldehydes, or carboxylic acids or their corresponding esters and acetals. The primary oxygenated functional group is located on a three-carbon chain containing an aromatic ring at position 3 (i.e. a 3-phenylpropyl group). Structural variations among these substances include the presence of unsaturation in the propyl side-chain and/or alkyl, alkoxy, or hydroxy substituents on the aromatic ring. The parent saturated alcohol is 3-phenyl-1-propanol, and the parent unsaturated alcohol is cinnamyl alcohol (3-phenyl-2-propen-1-ol, No. 647).

The group includes 3-phenyl-1-propanol, (No. 636), eight related esters (Nos. 637-644), and the corresponding aldehyde (No. 645) and carboxylic acid (No. 646). It also includes cinnamyl alcohol (No. 647), eight related cinnamyl esters (Nos 649655 and cinnamyl benzoate), cinnamaldehyde (No. 656), an acetal of cinnamaldehyde (No. 648), cinnamic acid (No. 657), and 16 cinnamic acid esters (Nos 658673). Thirteen cinnamyl derivatives contain additional ring side-chain alkyl substituents (Nos 674686), and three contain alkoxy-ring substituents (Nos 687689).

The available data on cinnamyl anthranilate, which is no longer used as a flavouring agent (voluntarily discontinued in 1986), are not presented in this review. A review of the metabolism and the proposed mechanism of toxicity of this agent has been published (Newberne et al., 2000).

2.2 Additional considerations on intake

Cinnamyl compounds are fundamental to plant biochemistry. trans-Cinnamic acid is ubiquitous in the plant kingdom and is required for lignin formation in plants. It is derived from the action of L-phenylalanine ammonia lyase on L-phenylalanine, with the formation of ammonia and cinnamic acid (No. 657). Cinnamic acid is also converted to para-hydroxy cinnamic acid (para-coumaric acid) by plants. para-Coumaric acid is one of the more important precursors of lignins as it can be converted to polyphenolic alcohols, which readily polymerize to form lignin (Goodwin & Mercer, 1972).

Twenty-two of the 55 flavouring substances in this group have been detected as natural components of traditional foods (Maarse et al., 1996; see Table 2). A considerable amount (38 642 kg) of cinnamaldehyde (No. 656) occurs naturally in foods. This agent has been detected in oils derived from natural sources such as cinnamon, cinnamomum, and cassia leaf, at concentrations up to 750 000 mg/kg (Maarse et al., 1996). Quantitative data on natural occurrence have also been reported for 3-phenylpropyl acetate (No. 638), ethyl 3-phenylpropionate (No. 644), cinnamyl alcohol (No. 647), cinnamic acid (No. 657), methyl cinnamate (No. 658), and ethyl cinnamate (No. 659). Generally, the consumption ratios indicate that intake occurs predominately from use of these substances as flavours (i.e. the consumption ratio is < 1) (Stofberg & Kirschman, 1985; Stofberg & Grunschober, 1987).

2.3 Biological data

The cinnamyl derivatives used as flavouring substances are simple aromatic compounds with a propyl side-chain containing a primary oxygenated functional group, and they participate in common routes of absorption, distribution, and metabolism. The members of this group may be hydrolysed to yield the component alcohol, aldehyde, or acid. If the product is an alcohol or aldehyde, it is oxidized to yield the corresponding 3-phenylpropenoic acid or a 3-phenylpropanoic acid derivative which undergoes further side-chain beta-oxidation and cleavage to yield mainly the corresponding benzoic acid derivatives (Figure 2; Williams, 1959). The benzoic acid derivatives are conjugated with glycine and, to a lessor extent, glucuronic acid and excreted primarily in the urine (Snapper et al., 1940). ortho-Alkyl- and ortho-alkoxy-substituted cinnamaldehyde derivatives undergo beta-oxidation to a minor extent, to yield beta-hydroxy-3-phenylpropanoic acid metabolites that are excreted as the glucuronic acid conjugates (Solheim & Scheline, 1973, 1976; Samuelsen et al., 1986).

Figure 2

2.3.1 Absorption, distribution, and excretion

Cinnamyl alcohol (No. 647), cinnamaldehyde (No. 656) and its ortho- and para-methoxy derivatives (Nos 687 and 688), cinnamic acid (No. 657) and its corres-ponding methyl ester (No. 658), and the saturated analogue (3-phenylpropanoic acid, No. 646) have all been shown to be rapidly absorbed from the gut, metabolized, and excreted primarily in the urine and, to a minor extent, in the faeces. Results of studies conducted as long ago as 1909 indicate that cinnamyl derivatives are absorbed, metabolized, and excreted as polar metabolites within 24 h (see Table 3).

Table 3. Metabolism of cinnamyl alcohol and related substances used as flavouring agents

Agent

Species

Route

Dose

% dose in 24-h urine/ faeces

Urinary metabolites (%)


Other

Reference

Benzoic acid (glycine conjugate / free)

Cinnamic acid (glycine conjugate / free)

Cinnamyl alcohol

Rats

Oral

2.5 mmol/kg bw

71/6

52/3

 

1% benzoyl glucuronide

Nutley (1990)

 

Mice

Intraperitoneal

2.5 mmol/kg bw

71/5

32/NR

2/NR

2.4% 3-hydroxy-3-phenyl-propionic acid; 4% benzoyl glucuronide

Nutley (1990)

 

Rabbits

Oral

31.3 g

 

22/NR

NR/43

 

Fischer & Bielig (1940)

Cinnamal-dehyde

Mice

Intraperitoneal

2.5 mmol/kg bw

54/15

11/0.6

1.4/2

 

Nutley (1990)

 

Rats

Oral

2.5 mmol/kg bw

62/16

36/NR

 

10% mercapturic acids; 2.2% 3-hydroxy-3-phenylpropionic acid

Nutley (1990)

 

Rats (M)

Intraperitoneal

2 mg/kg bw

81/7.5

85/0.4

2.2/NR

0.8% benzoyl glucuro-nide

Peters & Caldwell (1994)

 

Rats (M)

Intraperitoneal

250 mg/kg bw

85/0.8

73/1

1/ 0.3

7% benzoyl glucuronide

Peters & Caldwell (1994)

 

Rats (M)

Oral

250 mg/kg bw

91/7

87/0.8

0.3/NR

 

Peters & Caldwell (1994)

 

Rats (F)

Intraperitoneal

2 mg/kg bw

81/8

88/NR

1/NR

 

Peters & Caldwell (1994)

 

Rats (F)

Intraperitoneal

250 mg/kg bw

70/9

84/NR

0.4/NR

2.3% benzoyl glucuro-nide

Peters & Caldwell (1994)

 

Mice (M)

Intraperitoneal

2 mg/kg bw

86/9

72/3

11.3

1% benzoyl glucuronide

Peters & Caldwell (1994)

 

Mice (M)

Intraperitoneal

250 mg/kg bw

81/6

72/NR

8/NR

2% benzoyl glucuronide

Peters & Caldwell (1994)

 

Mice (F)

Intraperitoneal

2 mg/kg bw

71/16

72

13/NR

 

Peters & Caldwell (1994)

 

Mice (F)

Intraperitoneal

250 mg/kg bw

79/10

71/NR

4 /NR

5% benzoyl glucuronide

Peters & Caldwell (1994)

Cinnamic acid

Rats (M)

Oral

0.5 mol to 2.5 mmol/kg bw

85/5

50/2

 

5% benzoyl glucuronide

Caldwell & Nutley (1986)

 

Rats

Intraperitoneal

1822 mg/kg bw

48/25

38/9

9/NR

 

Teuchy & Van Sumere (1971)

 

Rats

Oral

50 mg/kg bw

 

67/NR

 

 

Fahelbum & James (1977)

 

Mice

Intraperitoneal

2.5 mmol/kg bw

90/4

67/6

3/1

5% 3-hydroxy-3-phenyl-propionic acid

Nutley (1990)

 

Rats

Oral

2.5 mmol/kg bw

82/.9

73/2

NR/1

 

Nutley (1990)

 

Mice

Intraperitoneal

0.5 mol to 2.5 mmol/kg bw

85/5a

30/NR

20/NR

 

Caldwell & Nutley (1986)

 

Rats (M)

Oral

0.5 mol to 2.5 mmol/kg bw

85/5a

> 50/2

2/NR

 

Caldwell & Nutley (1986)

 

Rats

Oral

0.0005 mmol/kg bw

74/0.5

71/0.4

NR/0.1

0.2% 3-hydroxy-3-phenyl-propionic acid; 0.4% benzoyl glucuronide

Nutley et al. (1994)

 

Rats

Oral

0.005 mmol/kg bw

73/1

69/0.6

NR/0.1

0.2% 3-hydroxy-3-phen yl- propionic acid; 0.2% benzoyl glucuronide

Nutley et al. (1994)

 

Rats

Oral

0.05 mmol/kg bw

80/0.9

77/0.4

NR/0.1

0.2% 3-hydroxy-3-phen yl- propionic acid; 0.3% benzoyl glucuronide

Nutely et al. (1994)

 

Rats

Oral

0.5 mmol/kg bw

73/0.5

69/0.4

NR/0.1

0.2% 3-hydroxy-3-phenyl- propionic acid; 0.5% benzoyl glucuronide

Nutley et al. (1994)

 

Rats

Oral

2.5 mmol/kg bw

88/1

76/2.3

NR/.3

5% benzoyl glucuronide

Nutley et al. (1994)

 

Mice

Intraperitoneal

0.0005 mmol/kg bw

78/2.3

44/0.8

29

0.6% 3-hydroxy-3-phen yl- propionic acid; 0.1% benzoyl glucuronide

Nutley et al. (1994)

 

Mice

Intraperitoneal

0.005 mmol/kg bw

88/1.5

54/0.7

27

0.3% 3-hydroxy-3-phenyl- propionic acid; 0.2% benzoyl glucuronide

Nutley et al. (1994)

 

Mice

Intraperitoneal

0.05 mmol/kg bw

84/2.4

64/1

0.2/14

0.5% 3-hydroxy-3-phenyl- propionic acid; 0.3% benzoyl glucuronide

Nutley et al. (1994)

 

Mice

Intraperitoneal

0.5 mmol/kg bw

85/3.1

66/.5

0.3/8.2

2.0% 3-hydroxy-3-phenyl- propionic acid; 0.2% benzoyl glucuronide

Nutley et al. (1994)

 

Mice

Intraperitoneal

2.5 mmol/kg bw

93/3.2

57/8.6

2.3/2.4

9.8% 3-hydroxy-3-phenyl- propionic acid; 1.7% benzoyl glucuronide

Nutley et al. (1994)

Methyl cinnamate

Rats

Oral

50 mg/kg bw

 

66

 

5% benzoyl glucuronic acid

Fahelbum & James (1977)

 

Rabbits

Oral

50 mg/kg bw

 

66

 

5% benzoyl glucuronic acid

Fahelbum & James (1977)

 

Rabbits (F)

Oral

500 mg/kg bw

 

56

 

8% benzoyl glucuronic acid

Fahelbum & James (1977)

Ethyl cinnamate

Cats

Intraperitoneal

1.19 g/kg bw

 

55

 

 

Dakin (1909)

3-Phenyl propionic acid

Rabbits

Oral

500 mg/kg bw

 

56

8

para-hydroxyhippuric acid (trace)

Fahelbum & James (1977)

3-Phenyl propionic acid, sodium salt

Cats

Intraperitoneal

1190 mg/kg bw

 

55

 

1% acetophenone

Dakin (1909)

 

Dogs

Subcutaneous

120 mg/kg bw

 

77/NR

 

 

Raper & Wayne (1928)

 

Dogs

Subcutaneous

500 and 700 mg/kg bw

 

 

5067/NR

 

Dakin (1909)

 

Dogs

Oral

5, 6, or 7.4 g

 

1421/NR

2835

 

Quick (1928)

 

Foxes

Subcutaneous

1280 mg

 

77

 

 

Raper & Wayne (1928)

ortho-Methoxy-cinnamaldehyde

Rats

Oral

239 mg/kg bw

91

trace/trace

24/3.2

22% 3-hydroxy-3-(ortho- methoxy-phenyl) propio nic acid; 37% 2-(ortho- methoxy-phenyl) propionyl glycine

Samuelson et al. (1986)

NR, not reported

a Collected over 3 days

In groups of male Fischer 344 rats, 83% of an oral dose of 2.5 mmol/kg bw of [3-14C-d5]-cinnamyl alcohol (335 mg/kg bw), 77% of a dose of [3-14C-d5]-cinnamaldehyde (330 mg/kg bw), and 79% of a dose of [3-14C-d5]-cinnamic acid (370 mg/kg bw) were excreted mainly in the urine within 24 h. Excretion in the faeces accounted for only minor amounts of the administered alcohol (6.1%), aldehyde (16%), and acid (0.9%). More than 90% of the administered dose of any of the three substances was recovered in the urine and faeces within 72 h. Administration of the same doses of the parent alcohol, aldehyde, or acid to groups of CD-1 mice by intraperitoneal injection resulted in a similar pattern of excretion in the urine and faeces at 24 h (75%, 80%, and 93%, respectively) and 72 h (> 93%) (Nutley, 1990).

The tissue distribution and excretion of cinnamaldehyde (No. 656) were studied in groups of eight male Fischer 344 rats pretreated with single daily oral doses of 5, 50, or 500 mg/kg bw of cinnamaldehyde by gavage for 7 days and the same single oral dose of [3-14C]cinnamaldehyde 24 h later. Further groups received no pretreatment but the same single doses. The radiolabel was distributed primarily to the gastrointestinal tract, kidneys, and liver in all groups. After 24 h, > 80% of the radiolabel was recovered in the urine and < 7% in the faeces from all rats, regardless of dose. In all groups, a small amount of the dose was distributed to fat. Radiolabel was still present in animals killed 3 days after receiving 50 or 500 mg/kg bw. In animals pretreated with the two lower doses, the main urinary metabolite was hippuric acid, with small amounts of cinnamic and benzoic acid. In those pretreated with the high dose, benzoic acid was the major metabolite, suggesting that saturation of the glycine conjugation pathway occurs with repeated high doses of cinnamaldehyde (Sapienza et al., 1993).

The effect of dose and sex on the disposition of [3-14C]cinnamaldehyde was studied in Fischer 344 rats and CD-1 mice. More than 85% of doses of 2.0 and 250 mg/kg bw administered to groups of four male and four female rats and six male and six female mice by intraperitoneal injection was recovered in the urine and faeces within 24 h, and > 90% was recovered by 72 h. Of a dose of 250 mg/kg bw of [3-14C]cinnamaldehyde administered orally to Fischer 344 rats, 98% was recovered from the urine (91%) and faeces (7%) within 24 h (Peters & Caldwell, 1994). The effect of dose on the disposition of [3-14C-d5]-cinnamic acid was also studied in Fischer 344 rats and CD-1 mice. Five doses of cinnamic acid in the range 0.00052.5 mmol/kg bw were given orally to groups of four rats or by intraperitoneal injection to groups of four mice. After 24 h, 7388% of the radiolabel was recovered in the urine of rats and 7893% in the urine of mice; after 72 h, 85100% of the radiolabel was recovered from rats and 89100% from mice, mainly in the urine (Caldwell & Nutley, 1986). Only trace amounts of radiolabel were present in the carcass, indicating that cinnamic acid was readily and quantitatively excreted at all doses. The parent alcohol, aldehyde, and acid therefore appear to undergo rapid absorption, metabolism, and excretion, independently of dose up to 250 mg/kg bw, species, sex, and mode of administration (Nutley et al., 1994).

Eleven persons each received a single intravenous dose of cinnamic acid (No. 657) equivalent to 5 mg/kg bw. Analysis of blood showed that 100% of the dose was present within 2.5 min and none after 20 min (Quarto di Palo & Bertolini, 1961).

An oral dose of 1.5 mmol/kg bw (240 mg/kg bw) methyl cinnamate (No. 658) was rapidly and almost completely (95%) absorbed from the gut in rats. The agent was partially hydrolysed to cinnamic acid in the stomach (9%) and gut (40%), and the rate of absorption of cinnamic acid and methyl cinnamate from the gut was similar. No ester was detected in the peripheral blood of dosed rabbits or rats. Only traces were detected in portal and heart blood taken from the rats, indicating that almost complete hydrolysis of methyl cinnamate had occurred during intestinal absorption (Fahelbum & James, 1977).

After administration of a single oral dose of 57 mg of ring-deuterated 3-phenyl-propionic acid to one person, deuterobenzoic acid corresponding to 110% of the dose was isolated from alkaline hydrolysed urine within 100 min (Pollitt, 1974).

These studies indicate that cinnamyl derivatives can be anticipated to be rapidly absorbed, metabolized, and excreted, mainly in the urine, within 24 h.

2.3.1.1 Hydrolysis of esters and acetals

In general, esters containing an aromatic ring system are expected to be hydrolysed in vivo. The hydrolysis is catalysed by classes of enzymes recognized as carboxylesterases or esterases (Heymann, 1980), the most important of which are the A-esterases. In mammals, A-esterases occur in most tissues of the body (Anders, 1989; Heymann, 1980) but predominate in hepatocytes (Heymann, 1980). Acetals are rapidly hydrolysed in acidic media (Morgareidge, 1962).

Esters of cinnamic acid (No. 657) and structurally related aromatic esters have been shown to hydrolyse rapidly to the component acid and alcohol. Oral administration of a single dose of 50 mg/kg bw of methyl cinnamate (No. 658) resulted in the urinary excretion, after 24 h, of hippuric acid (66%) and benzoylglucuronide (5%). This distribution of metabolites, which was nearly identical to that for cinnamic acid, indicates that rapid hydrolysis of the ester in vivo precedes metabolism of the acid (Fahelbum & James, 1977). Ethyl cinnamate (No. 659) administered subcutaneously to a cat also produced only cinnamic acid metabolites in the urine (Dakin, 1909). Incubation of benzyl cinnamate (No. 670) or benzyl acetate with simulated intestinal fluid (pH 7.5; pancreatin) at 37 C for 2 h resulted in 80% and 50% hydrolysis, respectively (Grundschober, 1977). Incubation of the structurally related aromatic acetal, 2-phenylpropanal dimethyl acetal (1 mmol/L) with simulated gastric juice at 37 C resulted in 97% hydrolysis within 1 h. Under the same experimental conditions, benzaldehyde propylene glycol acetal (1 mmol/L) was 97% hydrolysed within 5 h (Morgareidge, 1962).

2.3.1.2 Oxidation and conjugation reactions of cinnamyl alcohol (No. 647) and cinnamaldehyde derivatives

The aromatic primary alcohols and aldehydes used as flavouring substances or formed by the hydrolysis of esters and acetals are readily oxidized to a cinnamic acid derivative (see Figure 2). Human NAD+-dependent alcohol dehydrogenase catalyses oxidation of primary alcohols to aldehydes (Pietruszko et al., 1973), and isoenzyme mixtures of NAD+-dependent aldehyde dehydrogenase (Weiner, 1980) catalyse oxidation of aldehydes to carboxylic acids. Aromatic alcohols and aldehydes have been reported to be excellent substrates for alcohol dehydrogenase (Sund & Theorell, 1963) and aldehyde dehydrogenase (Feldman & Wiener, 1972), respec-tively. The urinary metabolites of cinnamyl alcohol (No. 647) and cinnamaldehyde (No. 656) are mainly those derived from metabolism of cinnamic acid (see Figure 2).

In four rats given cinnamyl alcohol (No. 647) orally at a dose of 335 mg/kg bw, 52% was recovered in the urine within 24 h as the glycine conjugate of benzoic acid (hippuric acid). Ten minor metabolites cumulatively accounted for about 10% of the dose. When cinnamyl alcohol (No. 647) was administered to mice by intraperitoneal injection, hippuric acid was the major urinary metabolite (Nutley, 1990).

trans-[3-14C]Cinnamaldehyde was given at doses of 2 and 250 mg/kg bw by intraperitoneal injection to male and female Fischer 344 rats and CD-1 mice, and doses of 250 mg/kg bw were administered by oral gavage to male rats and mice only. In both species, the major urinary metabolites were formed from oxidation of cinnamaldehyde (No. 656) to cinnamic acid (No. 657), which was subsequently oxidized in the -oxidation pathway. The major urinary metabolite was hippuric acid (7175% in mice and 7387% in rats), and this was accompanied by small amounts of metabolites, including 3-hydroxy-3-phenylpropionic acid (0.44%), benzoic acid (0.43%), and benzoyl glucuronide (0.87.0%). The glycine conjugate of cinnamic acid was formed to a considerable extent only in the mice (413%). Glutathione conjugation of cinnamaldehyde competes to a small extent with the oxidation pathway. Approximately 69% of either dose was excreted within 24 h as glutathione conjugates of cinnamaldehyde. The authors concluded that the excretion pattern and metabolic profile of cinnamaldehyde in rats and mice are not systematically affected by sex, dose size, or route of administration (Peters & Caldwell, 1994).

The toxicokinetics of cinnamaldehyde (No. 656) has been investigated in male Fischer 344 rats. Cinnamaldehyde (limit of detection, < 0.1 g/ml) and cinnamic acid (No. 657; < 1 g/ml) were not detectable in the plasma of groups of three to six rats given a single oral dose of 50 mg/kg bw by gavage in corn oil. At doses of 250 and 500 mg/kg bw, the plasma concentrations of cinnamaldehyde and cinnamic acid were approximately 1 and > 10 g/ml, respectively. The bioavailability of cinnamaldehyde was calculated to be < 20% at both doses. A dose-dependent increase in the amount of hippuric acid, the major urinary metabolite, occurred 6 h after gavage and continued over the next 18 h. Only small amounts of cinnamic acid were excreted in the urine either free or as the glucuronic acid conjugate. The hippuric acid recovered in the urine over 50 h accounted for 7281% over doses ranging from 50 to 500 mg/kg bw (Yuan et al., 1992).

Approximately 15% of an oral dose of 250 mg/kg bw of cinnamaldehyde (No. 656) administered to rats by gavage was excreted in the urine as two mercapturic acid derivatives, N-acetyl-S-(1-phenyl-3-hydroxypropyl)cysteine and N-acetyl-S-(1-phenyl-2-carboxyethyl)cysteine, in a ratio of 4:1. Approximately 9% of an oral dose of 125 mg/kg bw of cinnamyl alcohol (No. 647) was excreted in the urine as N-acetyl-S-(1-phenyl-3-hydroxypropyl)cysteine (Delbressine et al., 1981).

2.3.1.3 Metabolism of cinnamic acid (No. 657)

In animals, aromatic carboxylic acids such as cinnamic acid (No. 657) that enter the cell are converted to acyl coenzyme A (CoA) esters. Cinnamoyl CoA either conjugates with glycine, a reaction catalysed by N-acyl transferase, or undergoes -oxidation eventually leading to the formation of benzoyl CoA. The reactions which form benzoic acid from cinnamic acid are reversible but the equilibrium favours formation of the benzoic acid CoA ester. Benzoyl CoA is in turn conjugated with glycine, yielding hippuric acid, or the CoA thioester is hydrolysed to yield free benzoic acid, which is then excreted (Nutley et al., 1994). CoA thioesters of carboxylic acids are obligatory intermediates in amino acid conjugation reactions (Hutt & Caldwell, 1990). The reactions in this sequence are of historical significance in biochemistry, since it was studies on cinnamic acid and fatty acids that revealed the beta-oxidation pathway of fatty acid catabolism (Nutley et al., 1994). Regardless of dose or species, the beta-oxidation pathway is the predominant pathway of metabolic detoxication of cinnamic acid in animals (see Table 3).

Six doses in the range 0.00052.5 mmol/kg (0.08400 mg/kg bw) of [14C]- or [14C/5H2]-cinnamic acid (No. 657) were administered orally to male Fischer 344 rats or by intraperitoneal injection to male CD-1 mice. In both species, 84101% was recovered within 72 h, most (7393%) being recovered from the urine within 24 h. The metabolites identified at all doses included hippuric acid, benzoyl glucuronide, 3-hydroxy-3-phenyl-propionic acid, benzoic acid, and unchanged cinnamic acid. The major metabolite at all doses was hippuric acid (4477%). At the highest dose tested (2.5 mmol/kg bw), the percentage of hippuric acid decreased while the percentages of benzoyl glucuronide and benzoic acid increased. The formation of larger amounts of benzoyl glucuronide (0.55%) and free benzoic acid (0.42%) at doses > 0.5 mmol/kg bw provides evidence that saturation of the glycine conjugation pathway occurs at these doses. The fact that the excretion of 3-hydroxy-3-phenyl-propionic acid differed little over the dose range (0.20.9%) supports the conclusion that the capacity of the beta-oxidation pathway is not limited at doses of cinnamic acid up to 2.5 mmol/kg bw in male rats (Nutley et al., 1994). The increasing role of glucuronic acid conjugation relative to glycine conjugation as the dose increases is a general trend observed in the metabolism of carboxylic acids (Caldwell et al., 1980).

In mice, glycine conjugation of cinnamic acid (No. 657) competes with the beta-oxidation pathway, but only at low doses. As the dose was increased from 0.0005 to 2.5 mmol/kg bw, the urinary concentration of hippuric acid increased from 44 to 67%, while that of cinnamoylglycine decreased from 29 to 2.4%. These results suggest that gylcine N-acetyl transferase has a stronger affinity but a lower capacity for cinnamic acid than for benzoic acid. At the highest dose, 2.5 mmol/kg bw, the amount of free benzoic acid excreted was increased from 0.8 to 8.6%, suggesting that the capacity of glycine conjugation of benzoyl CoA is also limited in mice. At all doses, mice excreted only a small proportion of benzoyl glucuronide, indicating that this conjugation reaction is of minimal importance in this species (Nutley et al., 1994).

Eleven volunteers received a single intravenous dose of cinnamic acid (No. 657) equivalent to 5 mg/kg bw. In blood plasma, 100% of the dose was found within 2.5 min and 0% after 20 min. Ninety minutes after dosing, hippuric acid, cinnamoyl-glucuronide, and benzoylglucuronide were found in a ratio of 74:24:1.5 (Quarto di Palo & Bertolini, 1961).

2.3.1.4 Effects of ring and chain substituents on metabolism of cinnamyl derivatives

The position and size of the substituent play a role in the metabolism of cinnamyl derivatives. Those containing alpha-methyl substituents (e.g. alpha-methylcinnamaldehyde, No. 683) are extensively metabolized via beta-oxidation and cleavage to yield mainly the corresponding hippuric acid derivative. A benzoic acid metabolite was isolated from the urine of dogs given either alpha-methylcinnamic acid or alpha-methylphenylpropionic acid (Kay & Raper, 1924). Larger substituents located at the alpha- or beta-position inhibited beta-oxidation to some extent (Deuel, 1957; Kassahun et al., 1991), in which case there may be direct conjugation of the carboxylic acid with glucuronic acid, followed by excretion. While alpha-methylcinnamic acid undergoes oxidation to benzoic acid, alpha-ethyl- and alpha-propylcinnamic acids are excreted unchanged (Carter, 1941). alpha-Ethylcinnamic alcohol and alpha-ethylcinnamaldehyde administered orally to rabbits resulted in urinary excretion of alpha-ethylcinnamic acid and of small amounts of benzoic acid (Fischer & Bielig, 1940). These observations suggest that alpha-methylcinnam-aldehyde undergoes oxidation to benzoic acid, while higher homologues are excreted primarily unchanged or as the conjugated form of the cinnamic acid derivative.

ortho-ring substituents (e.g. ortho-methoxycinnamaldehyde, No. 688) selectively inhibit oxidation of CoA esters of beta-hydroxyacids within the beta-oxidation pathway, and the hydroxyacid derivative is excreted as a glycine conjugate. The metabolism of ortho-methoxycinnamaldehyde ceases after formation of the beta-hydroxy derivative (Samuelsen et al., 1986).

The glycine conjugates of ortho-methoxycinnamic and ortho-methoxyphenyl-propionic acids are the principal urinary metabolites of ortho-methoxycinnamaldehyde in rats. Relatively large amounts of the beta-hydroxylated phenylpropionic acid derivatives were also detected, but only traces of benzoic and hippuric acid derivatives (products of further beta-oxidation) were excreted. The detection of relatively large amounts of a beta-hydroxylated derivative suggests that this metabolite is not readily oxidized, perhaps because of steric hindrance of the ortho substituent (Solheim & Scheline, 1973).

In contrast, para-ring substituents (e.g. 3-(para-isopropyl-phenyl)propional-dehyde, No. 680, and para-methylcinnamaldehyde, No. 682) may not affect metabolism via beta-oxidation significantly. In male albino rats, para-methoxycinnamic acid was metabolized primarily to para-methoxybenzoic acid and its corresponding glycine conjugate (Solheim & Scheline, 1973). Similar results were reported with 3,4-dimethoxycinnamic acid (which is meta and para substituted) (Solheim & Scheline, 1976). The structurally related substance para-tolualdehyde has been reported to be metabolized to para-methylbenzoic acid with no apparent oxidation of the methyl group (Williams, 1959). These observations indicate that the presence of side-chain alkyl substituents with more than one carbon atom and of ortho-ring substituents inhibits the beta-oxidation pathway. In these cases, the parent acid (cinnamic acid derivative) or an intermediary beta-oxidation metabolite (e.g. beta-hydroxy-3-phenylpropanoic acid derivative) is excreted as the glycine or glucuronic acid conjugate.

2.3.2 Toxicological studies

2.3.2.1 Acute toxicity

LD50 values after oral administration have been reported for 39 of the 55 substances in this group. In rats, the values were in the range 1500 to > 5000 mg/kg bw (Jenner et al., 1964; Moreno, 1971; Weir & Wong, 1971; Keating, 1972; Levenstein & Wolven, 1972; Moreno, 1972; Denine & Palanker, 1973; Moreno, 1973; Russell, 1973; Moreno, 1974; Opdyke, 1974; Wohl, 1974; Zaitsev & Rakhmanina, 1974; Levenstein, 1975; Moreno, 1975; Levenstein, 1976; Moreno, 1976, 1977, 1981, 1982; Schafer et al., 1983), demonstrating that the acute toxicity of these substances after oral administration is low. In rats, the values were 900 to > 5000 mg/kg bw (Draize et al., 1948; Harada & Ozaki, 1972; Zaitsev & Rakhmanina, 1974; Levenstein, 1975; Schafer & Bowles, 1985), and in guinea-pigs they were 3100 to > 5000 mg/kg bw (Draize et al., 1948; Zaitsev & Rakhmanina, 1974).

2.3.2.2 Short-term and long-term studies of toxicity

Toxicological studies have been reported for 11 substances in the group. The results of those with the parent alcohol cinnamyl alcohol (No. 647), the corresponding aldehyde, two cinnamate esters, two alpha-alkyl substituted cinnamaldehyde derivatives, two alkoxy-substituted cinnamaldehyde derivatives, and a mixture of five cinnamyl derivatives are described below and summarized in Table 4.

Table 4. Results of short-term studies of toxicity and long-term studies of toxicity and carcinogenicity on cinnamyl alcohol and related substances used as flavouring agents administered orally

No.

Substance

Species; sex

No. test groupsa/ no. per groupb

Duration

NOEL (mg/kg bw per day)

Reference

647

Cinnamyl alcohol

Rat; M

1/12

4 months

54c

Zaitsev & Rakhmanina (1974)

656

Cinnamaldehyde

Rat; M

1/12

4 months

68c

Zaitsev & Rakhmanina (1974)

656

Cinnamaldehyde

Rat; M, F

3/10

12 weeks

230c

Trubeck Laboratories (1958a)

656

Cinnamaldehyde

Rat; M, F

2/24

12 weeks

100c,d

Trubek Laboratories (1958b)

656

Cinnamaldehyde

Rat; M, F

4/20

13 weeks

620

National Toxicology Program (1995)

656

Cinnamaldehyde

Rat; M, F

3/20

16 weeks

120c

Hagan et al. (1967)

658

Methyl cinnamate

Rat; M, F

2/24

12 weeks

3c,d

Trubeck Laboratories (1958b)

659

Ethyl cinnamate

Rat; M

1/12

4 months

80c

Zaitsev & Rakhmanina (1974)

659

Ethyl cinnamate

Rat; M, F

2/24

12 weeks

3c,d

Trubeck Laboratories (1958b)

668

Linalyl cinnamate

Rat; M, F

3/20

17 weeks

500c

Hagan et al. (1967)

670

Benzyl cinnamate

Rat; M, F

2/20

19 weeks

500c

Hagan et al. (1967)

670

Cinnamyl cinnamate

Rat; M, F

2/24

12 weeks

3c,d

Trubeck Laboratories (1958b)

683

alpha-Methylcinnamaldehyde

Rat; M

3/10

90 days

220c

Trubeck Laboratories (1958c)

683

alpha-Methylcinnamaldehyde

Rat; M, F

2/24

12 weeks

3c,d

Trubeck Laboratories (1958b)

685

alpha-Amylcinnamaldehyde

Rat; M, F

3/30

14 weeks

290c (M)
320c (F)

Carpanini et al. (1973)

685

alpha-Amylcinnamaldehyde

Rat; M, F

1/30

90 days

6.1c (M)
6.6c (F)

Oser et al. (1965)

688

ortho-Methoxycinnamaldehyde

Rat; M, F

1/2032

90 days

47c (M)
52c (F)

Posternak et al. (1969)

689

para-Methoxy-alpha-methyl-cinnamaldehyde

Rat; M, F

1/2032

90 days

2.4c (M)
2.7c (F)

Posternak et al. (1969)

M, male; F, female

a Total number of test groups does not include control animals.

b Total number per test group includes both male and female animals.

c Study performed with either a single dose or multiple doses that had no adverse effect; the value is therefore the highest dose tested.

d The substance was administered as a component of a mixture.

Cinnamyl alcohol (No. 647), cinnamaldehyde (No. 656), and ethyl cinnamate (No. 659)

Sunflower oil solutions containing cinnamyl alcohol (No. 647; 0.2 ml/100 g bw) providing a dose of 54 mg/kg bw per day, cinnamaldehyde (No. 656) providing a dose of 68 mg/kg bw per day, or ethyl cinnamate (No. 659) providing a dose of 80 mg/kg bw per day, equivalent to 0.02 of the LD50 for the respective substance, was administered to groups of 12 male white rats (strain not identified) by oral intubation once daily for 4 months. Liver function was tested at days 40 and 140. Increased (26%) blood serum fructose diphosphate aldolase activity was observed in the groups given cinnamyl alcohol (No. 647) and ethyl cinnamate (No. 659) on day 140, but the activities of serum cholinesterase and alanine aminotransferase and the concentration of SH groups in serum showed no change from control values. The authors concluded that none of the three cinnamyl derivatives caused pronounced pathological changes in rat liver; however, the study was described by the authors as preliminary (Zaitsev & Rakhmanina, 1974).

Cinnamaldehyde (No. 656)

Groups of 10 male and 10 female Osborne-Mendel rats were maintained on a diet containing cinnamaldehyde (No. 656) at a concentration of 0 (control), 1000, 2500, or 10 000 mg/kg, equivalent to 50, 120, and 500 mg/kg bw per day, for 16 weeks. Body weights and food intake, recorded weekly, did not differ significantly between treated and control animals. Haematological parameters at termination were normal. At necropsy, no differences in the weights of the major organ were found. Gross examination of the tissues showed no remarkable changes. Histological examination of three to four male and female animals at the high dose revealed slight hepatic cellular swelling and slight hyperkeratosis of the squamous epithelium of the stomach. The NOEL was 120 mg/kg bw per day (Hagan et al., 1967).

Groups of five male and five female rats were maintained on a diet containing cinnamaldehyde (No. 656) at concentrations calculated to result in a daily intake of 0 (control), 58, 110, or 230 mg/kg bw for 12 weeks. General condition, behaviour, body weight, food intake, and efficiency of food use were recorded regularly, and no statistically significant differences were seen between treated and control animals. Haematological examination after 12 weeks revealed normal blood haemoglobin concentration, and urine analysis revealed the absence of glucose and only traces of albumin in males (attributed to the possible presence of semen). At necropsy, no significant difference in liver or kidney weights was seen between treated and control groups. Gross examination revealed occasional respiratory infection in animals in all groups (Trubeck Laboratories, 1958a).

In a 13-week study, groups of 10 male and 10 female Fischer 344/N rats were given diets containing 0, 1.25, 2.5, 5.0, or 10% microencapsulated trans-cinnamaldehyde (No. 656), equal to 0, 620, 1250, 2500, or 5000 mg/kg bw per day. Necropsies were performed on all survivors, and tissues from animals at the two highest doses and the control group were examined histologically. There were no early deaths and no treatment-related clinical toxicity. The mean terminal body weights of untreated controls and vehicle controls were similar, but the mean body weights of animals at the three higher doses were decreased. The food consumption of treated animals was depressed during the first week, possible because of unpalata-bility. No overt haematological effects were seen. The clinical chemical parameters that were increased by treatment included bile salt concentration and alanine transaminase activity (in males and females at the highest dose), suggesting mild cholestasis. Microscopic examination showed no morphological alterations to the liver. Gross and microscopic examination of the stomach and forestomach indicated irritation at all doses of trans-cinnamaldehyde. The NOEL was 620 mg/kg bw per day (National Toxicology Program, 1995).

Mixture of cinnamaldehyde (No. 656), methyl cinnamate (No. 658), ethyl cinnamate (No. 659), cinnamyl cinnamate (No. 673), and alpha-methyl-cinnamaldehyde (No. 683)

A mixture of flavourings containing 900 mg/kg cinnamaldehyde (No. 22) and 25 mg/kg each of methyl cinnamate (No. 656), ethyl cinnamate (No. 659), cinnamyl cinnamate (No. 673), and alpha-methyl-cinnamaldehyde (No. 683) was added to the diet of groups of 12 rats of each sex for 12 weeks, resulting in daily intakes of 110 mg/kg bw for males and 120 mg/kg bw for females, which were equivalent to 100 mg/kg bw of cinnamaldehyde and 3 mg/kg bw of each of the other components. Weekly measurements of body weight and food intake revealed a statistically nonsignificant decrease in weight gain in treated males when compared with controls. The efficiency of food use was statistically significantly decreased in treated males (p < 0.01) and females (p < 0.05) when compared with their respective controls. At week 12, blood haemoglobin, urinary sugar, and urinary albumin concentrations, measured in three animals of each sex, were normal. At necropsy, the weights of the liver, kidney, and brain were within normal limits. Gross examination revealed no obvious differences between treated and control groups. The results of histological examinations were not reported (Trubeck Laboratories, 1958b).

Linalyl cinnamate (No. 668) and benzyl cinnamate (No. 670)

Groups of 10 male and 10 female Osborne-Mendel rats were fed a diet containing linalyl cinnamate (No. 668) at a concentration of 0 (control), 1000, 2500, or 10 000 mg/kg (equivalent to 0, 50, 120, or 500 mg/kg bw per day) for 17 weeks, and groups of five animals of each sex were given benzyl cinnamate (No. 670) at a concentration of 0 (control), 1000, or 10 000 mg/kg of diet (equivalent to 0, 50, or 500 mg/kg bw per day) for 19 weeks. The diets were prepared weekly; analysis of old diet preparations revealed a 4% weekly loss of linalyl cinnamate, but the dietary loss of benzyl cinnamate was not determined. No significant differences in body weight or food intake, recorded weekly, were seen between treated and control animals. Haematological examination at termination revealed no significant difference between treated and control animals, and no difference in the weights of the major organs was found at necropsy. Gross examination of the tissue of animals given either agent showed no remarkable change. Histological examination of three to four animals of each sex at the high dose of linalyl cinnamate and in the control group revealed no treatment-related lesions. The tissues of animals given benzyl cinnamate were not examined histologically (Hagan et al., 1967).

ortho-Methoxycinnamaldehyde (No. 688) and para-methoxy-alpha-methyl-cinnamaldehyde (No. 689)

Groups of 1016 Charles River CD rats of each sex were maintained on diets containing ortho-methoxycinnamaldehyde (No. 688) at concentrations calculated to result in daily intakes of 0 (control) or 47 mg/kg bw for males and 52 mg/kg bw for females or para-methoxy-alpha-methycinnamaldehyde at concentrations calculated to result in daily intakes of 2.4 mg/kg bw for males and 2.7 mg/kg bw for females, for 90 days. The control groups received basal diets only. The animals were housed in pairs of the same sex and given access to water and food ad libitum. The concentration of the test material in the diet was adjusted during the study to maintain constant dietary intakes. Clinical observations were recorded daily, and food consumption and body weights were determined weekly. Haematological parameters and blood urea were determined in 50% of the animals at week 7 and on all animals at week 13. After 90 days, all animals were killed and necropsied, and the livers and kidneys were weighed. A wide range of tissues and organs from each animal were preserved, and major organs and tissues were examined histologically. No differences in growth, food intake, haematological or clinical chemical parameters, organ weights, or organ appearance were observed between treated and control animals (Posternak et al., 1969).

alpha-Methylcinnamaldehyde (No. 683)

Groups of five rats of each sex were maintained on a diet containing alpha-methylcinnamaldehyde (No. 683) at concentrations calculated to result in average daily intakes of 0, 58, 120, or 220 mg/kg bw for 90 days. Growth and food intake were recorded weekly, as were the results of regular examinations for physical appearance, behaviour, and efficiency of food use. At week 12, urine samples were collected and analysed for the presence of sugar and albumin, and blood samples were taken for determination of haemoglobin. No statistically significant differences were found between treated and control animals, and no differences in liver or kidney weights were seen (Trubeck Laboratories, 1958c).

alpha-Amylcinnamaldehyde (No. 685)

Groups of 15 male and 15 female CFE rats were maintained on a diet containing alpha-amylcinnamaldehyde (No. 685) at a concentration of 0 (control), 80, 400, or 4000 mg/kg for 14 weeks. Additional groups of five male and five female rats were maintained on diets containing 400 or 4000 mg/kg of the agent for 2 and 6 weeks. The mean dietary intakes over 14 weeks were reported to be 0, 6.1, 30, and 290 mg/kg bw per day for males and 0, 6.7, 35, and 320 mg/kg bw per day for females. Measurement of body weight and food and water consumption revealed no significant differences between treated and control groups. Evaluations of haemoglobin content, haematocrit, erythrocyte and leukocyte counts, and individual leukocyte counts and of blood chemistry at 2, 6, and 14 weeks revealed normal values. Reticulocyte counts, performed only on controls and animals at the high dose, showed no significant difference. Urine analysis performed during the final week of treatment revealed no difference in cell content or renal concentration. At autopsy, a statistically significant increase in the relative weight of the liver was seen in males (p < 0.01) and females (p < 0.05) at 4000 mg/kg of diet after 14 weeks, the stomach weights of males at 400 mg/kg of diet were decreased after 6 weeks, and the relative weight of the kidneys was increased in males (p < 0.01) at 4000 mg/kg after 14 weeks. The increases in relative organ weights were not associated with histological lesions. Microscopic examination of tissues from all major organs revealed no histopathological changes that could be associated with administration of the agent (Carpanini et al., 1973).

Groups of 15 male and 15 female FDRL rats were maintained on a diet containing alpha-amylcinnamaldehyde (No. 685) at concentrations calculated to result daily intakes of 6.1 mg/kg bw for males and 6.6 mg/kg bw for females, for 90 days. Body weight, food consumption, and general condition were recorded regularly. Haematological and clinical chemical parameters were measured in eight rats of each sex at week 6 and in all animals at week 12. The measurements of growth, haematology, and clinical chemistry and histopathology at necropsy gave no evidence of toxic effects (Oser et al., 1965).

2.3.2.3 Genotoxicity

Numerous studies of the genotoxicity of the substances in this group of flavouring substances have been reported and are summarized in Table 5 and described below.

Table 5. Studies of genotoxicity with cinnamyl alcohol and related substances used as flavouring agents

No.

Agent

End-point

Test object

Maximum concentration

Result

Reference

In vitro

645

3-Phenylpropion-aldehyde

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537

3 mol/plate
(402 g/plate)

Negativea

Florin et al. (1980)

645

3-phenylpropion-aldehyde

Sister chromatid exchange

Chinese hamster ovary cells

33.3 mol/L
(4468 g)

Negativeb

Sasaki et al. (1989)

647

cinnamyl alcohol

Reverse mutationc

S. typhimurium TA1537, TA1535

3000 g/plate

Negativea

Sekizawa & Shibimoto (1982)

647

cinnamyl alcohol

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

21 g/disc

Negativeb

Oda et al. (1979)

647

cinnamyl alcohol

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

1.0 mg/disc
(1000 g/disc)

Positivea

Sekizawa & Shibimoto (1982)

647

cinnamyl alcohol

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

10 l/disc
(10 400 g/disc)

Positiveb

Yoo (1986

647

cinnamyl alcohol

Mutation

E. coli WP2 uvrA

3000 g/plate

Negativeb

Sekizawa & Shibimoto (1982)

647

cinnamyl alcohol

Mutation

E. coli WP2 uvrA

4.0 mg/plate
(4000 g/plate)

Negativeb

Yoo (1986)

647

cinnamyl alcohol

Sister chromatid exchange

Chinese hamster ovary cells

33.3 mol/L
(4468 g)

Negativeb

Sasaki et al. (1989)

650

cinnamyl acetate

Sister chromatid exchange

Chinese hamster ovary cells

33.3 mol/L
(5868 g)

Negativeb

Sasaki et al. (1989)

656

Cinnamaldehyde

Reverse mutationc

S. typhimurium TA1537, TA1538, TA98, TA100, TA1535

600 g/plate

Negativea

Sekizawa & Shibamoto (1982)

656

trans-cinnamaldehyde

Reverse mutation

S. typhimurium TA1537, TA98, TA100, TA1535

10 mg/plate
(10,000 g/plate)

Negativea

Prival et al. (1982)

656

cinnamaldehyde

Reverse mutation

S. typhimurium TA104 (with preincubation)

0.8 mol
(105 g)

Negativea

Marnett et al. (1985)

656

Cinnamaldehyde

Reverse mutation

S. typhimurium TA1537, TA92, TA94, TA98, TA100, TA1535 (with preincubation)

0.5 mg/plate
(500 g/plate)

Positivea,d

Ishidate et al. (1984)

656

trans-Cinnamaldehyde

Reverse mutation

S. typhimurium TA1537, TA92, TA94, TA98, TA100, TA1535 (with plate incorporation and preincubation)

500 g/plate

Negativea

Lijinsky & Andrews (1980)

656

trans-Cinnamaldehyde

Reverse mutation

S. typhimurium TA1537, TA1538, TA98, TA100, TA1535 (with plate incorporation and preincubation)

500 g/plate

Negativea

Kasamaki et al. (1982)

656

cinnamaldehyde

Reverse mutation

S. typhimurium TA97, TA98, TA100 (with preincubation)

1 mg/ml
(1000 g/ml)

Negativea

Azizan & Blevins (1995)

656

trans-cinnamaldehyde

Reverse mutation

S. typhimurium TA98, TA100, TA104 (with preincubation)

Not reported

Negativea

Kato et al. (1989)

656

trans-cinnamaldehyde

Reverse mutation

S. typhimurium TA1537, TA98, TA100, TA1535 (with preincubation)

100 g/plate

Negativea

Mortelmans et al. (1986)

656

trans-cinnamaldehyde

Reverse mutation

S. typhimurium TA100 (with preincubation)

5 mol/plate
(661 g/plate)

Negativea

Neudecker et al. (1983)

656

cinnamaldehyde

Mutation

E. coli WP2 uvrA

600 g/plate

Negativeb

Sekizawa & Shibimoto (1982)

656

cinnamaldehyde

Mutation

E. coli WP2 uvrA

0.8 mg/plate
(800 g/plate)

Negativeb

Yoo (1986)

656

cinnamaldehyde

DNA repair

B. subtilis M45 (rec-)

0.2 mg/disk
(200 g/disc)

Positiveb

Sekizawa & Shibimoto (1982)

656

cinnamaldehyde

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

10 l/disc
(10,500 g/disc)

Positiveb

Yoo (1986)

656

cinnamaldehyde

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

10 l/disc
(10,500 g/disc)

Positivea

Kuroda et al. (1984)

656

cinnamaldehyde

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

21 g/disc

Negativeb

Oda et al. (1979)

656

cinnamaldehyde

Sister chromatid exchange

Chinese hamster ovary cells

33.3 mol/L
(4401 g)

Negativeb

Sasaki et al. (1987)

656

cinnamaldehyde

Chromosomal aberration

Chinese hamster fibroblasts

0.015 mg/ml
(15 g/ml)

Positiveb

Ishidate et al. (1984)

656

cinnamaldehyde

Chromosomal aberration

Chinese hamster B241 cells

20 nmol/L
(2.6 g)

Positiveb

Kasamaki & Urasawa (1985)

656

cinnamaldehyde

Chromosomal aberration

Chinese hamster B241 cells

10 nmol/L
(1.3 g)

Positivea

Kasamaki et al. (1982)

656

trans-cinnamaldehyde

Chromosomal aberration

Chinese hamster ovary cells

18.3 g/ml
100 g/ml

Negativeb
Negativee

Galloway et al. (1987)

656

trans-cinnamaldehyde

Sister chromatid exchange

Chinese hamster ovary cells

6.8 g/ml

Weakly positiveb

Galloway et al. (1987)

 

 

 

 

91.8 g/ml

Weakly positivee

 

656

Cinnamaldehyde

DNA strand breaks

Mouse L1210 lymphoma cells

500 mol
(66 080 g)

Positiveb

Eder et al. (1993)

656

Cinnamaldehyde

Cytotoxicity

Mouse L1210 lymphoma cells

10 g/ml

Positiveb

Moon & Pack (1983)

656

Cinnamaldehyde

Mutation

Chinese hamster V79 cells

100 mol/L
(13 216 g)

Negativeb

Fiorio & Bronzetti (1994)

656

Cinnamaldehyde

Micronucleus formation

Hep-G2 cells

500 g/ml

Weakly positiveb

Sanyal et al. (1997)

657

Cinnamic acid

Reverse mutation

S. typhimurium TA1537, TA1538, TA98, TA100, TA1535 (with plate incorporation and preincubation)

1000 g

Negativea

Lijinsky & Andrews (1980)

657

Cinnamic acid

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

25 g/disc

Negativeb

Oda et al. (1979)

657

Cinnamic acid

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

2.0 mg/disc
(2000 g/disc)

Negativeb

Yoo (1986)

657

Cinnamic acid

Sister chromatid exchange

Chinese hamster ovary cells

33.3 mol/L
(4934 g)

Positiveb

Sasaki et al. (1989)

658

Methyl cinnamate

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

20 g/disc

Negativeb

Oda et al. (1979)

658

Methyl cinnamate

Sister chromatid exchange

Chinese hamster ovary cells

33.3 mol/L
(5401 g)

Positiveb

Sasaki et al. (1989)

659

Ethyl cinnamate

Reverse mutation

S. typhimurium TA1537, TA92, TA94, TA98, TA100,TA1535 (with preincubation)

5.0 mg/plate
(5000 g/plate)

Negativea

Ishidate et al. (1984)

659

Ethyl cinnamate

Chromosomal aberration

Chinese hamster fibroblasts

0.063 mg/ml
(63 g/ml)

Equivocalb

Ishidate et al. (1984)

659

Ethyl cinnamate

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

20 g/disc

Negativeb

Oda et al. (1979)

659

Ethyl cinnamate

Sister chromatid exchange

Chinese hamster ovary cells

33.3 mol/L
(5868 g)

Positiveb

Sasaki et al. (1989)

19

Allyl cinnamate

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

3.6 mg/plate
(3600 g/plate)

Negativea

Wild et al. (1983)

667

Cyclohexyl cinnamate

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

3.6 mg/plate
(3600 g/plate)

Negativea

Wild et al. (1983)

670

Benzyl cinnamate

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537

3 mol/plate
(715 g/plate)

Negativea

Florin et al. (1980)

670

Benzyl cinnamate

DNA repair

B. subtilis M45 (rec-) and H17 (rec+)

1.0 mg/disc
(1000 g/disc)

Negativeb

Yoo (1986)

674

alpha-Amylcinnamyl alcohol

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

3.6 mg/plate
(3600 g/plate)

Negativea

Wild et al. (1983)

683

alpha-Methylcinnamal-dehyde

Reverse mutation

S. typhimurium TA100(with preincubation)

4 mol/plate
(585 g/plate)

Negativea

Neudecker et al. (1983)

683

alpha-Methylcinnamal-dehyde

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537 (with preincubation)

500 g/plate

Negativea

Mortelmans et al. (1986)

683

alpha-Methylcinnamal-dehyde

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

3.6 mg/plate
(3600 g/plate)

Negativea

Wild et al. (1983)

683

alpha-Methylcinnamal-dehyde

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

3.6 mg/plate
(3600 g/plate)

Negativea

Wild et al. (1983)

685

alpha-Amylcinnamal-dehyde

Reverse mutation

S. typhimurium TA97, TA102 (with preincubation)

1.0 mg/plate
(1000 g/plate)

Negativea

Fujita & Sasaki (1987)

686

alpha-Hexylcinnamal-dehyde

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

3.6 mg/plate
(3600 g/plate)

Negativea

Wild et al. (1983)

688

ortho-Methoxycinnamal-dehyde

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537 (with preincubation)

666 g/plate

Positivea

Mortelmans et al. (1986)

689

para-Methoxy-alpha-methyl- cinnamaldehyde

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537, TA1538

3.6 mg/plate
(3600 g/plate)

Negativea

Wild et al. (1983)

In vivo

656

trans-Cinnamaldehyde

Sex-linked recessive lethal mutation

D. melanogaster

800 mg/kg od diet
(800 g/g)

Negative

Woodruff et al. (1985)

19

Allyl cinnamate

Sex-linked recessive lethal mutation

D. melanogaster

1 mmol/L
(188 000 g)

Negative

Wild et al. (1983)

674

alpha-Amylcinnamyl alcohol

Sex-linked recessive lethal mutation

D. melanogaster

45 mmol/L
(9 194 000 g)

Negative

Wild et al. (1983)

683

alpha-Methylcinnamal-dehyde

Sex-linked recessive lethal mutation

D. melanogaster

5 mmol/L
(731 000 g)

Negative

Wild et al. (1983)

685

alpha-Amylcinnamal-dehyde

Sex-linked recessive lethal mutation

D. melanogaster

10 mmol/L
(2 023 000 g)

Negative

Wild et al. (1983)

686

alpha-Hexylcinnamal-dehyde

Sex-linked recessive lethal mutation

D. melanogaster

10 mmol/L
(2 163 000 g)

Negative

Wild et al. (1983)

656

Cinnamaldehyde

Unscheduled DNA synthesis

Rat and mouse hepatocytes

1 000 000 g/kg bw

Negative

Mirsalis et al. (1989)

656

Cinnamaldehyde

Micronucleus formation

Mouse bone-marrow cells

500 000 g/kg bw

Negative

Hayashi et al. (1984, 1988)

656

trans-Cinnamaldehyde

Micronucleus formation

Rat and mouse hepatocytes

1 700 000 g/kg bw (mice)
1 100 000 g/kg bw (rats)

Positive

Mereto et al. (1994)

656

trans-Cinnamaldehyde

Micronucleus formation

Rat and mouse bone marrow

1 700 000 g/kg bw (mice)
1 100 000 g/kg bw (rats)

Negative

Mereto et al. (1994)

656

Cinnamaldehyde

Nuclear anomaliesf

Rat and mouse fore- stomach mucosal cells

1 700 000 g/kg bw (mice)

Negative

Mereto et al. (1994)

 

 

 

 

1 100 000 g/kg bw (rats)

Positive

 

656

trans-Cinnamaldehyde

DNA fragmentation

Rat hepatocytes and gastric mucosal cells

1 100 000 g/kg bw

Negative

Mereto et al. (1994)

656

Cinnamaldehyde

Hyperplastic foci

Rat hepatocytes

500 000 g/kg bw per dayg

Positive

Mereto et al. (1994)

19

Allyl cinnamate

Micronucleus formation

Mouse bone-marrow cells

282 000 g/kg bw

Negative

Wild et al. (1983)

674

alpha-Amylcinnamyl alcohol

Micronucleus formation

Mouse bone-marrow cells

510 000 g/kg bw

Negative

Wild et al. (1983)

683

alpha-Methylcinna-maldehyde

Micronucleus formation

Mouse bone-marrow cells

438 000 g/kg bw

Negative

Wild et al. (1983)

685

alpha-Amylcinnamal-dehyde

Micronucleus formation

Mouse bone-marrow cells

1 213 000 g/kg bw

Negative

Wild et al. (1983)

686

alpha-Hexylcinna-maldehyde

Micronucleus formation

Mouse bone-marrow cells

657 000 g/kg bw

Negative

Wild et al. (1983)

a With and without metabolic activation

b Without metabolic activation

c Method included both plate incorporation (without metabolic activation) and preincubation method (with metabolic activation)

d Positive results in strain TA100 only

e With metabolic activation

f Includes % micronuclei, pyknosis, and karyorrhexis

g Rats were initiated with N-nitrosodiethylamine then given cinnamaldehyde by oral gavage for 14 consecutive days.

In vitro

Cinnamaldehyde (trans- and unspecified stereochemistry), cinnamyl alcohol (No. 647) (trans- and unspecified stereochemistry), cinnamic acid (No. 657), alpha-methylcinnamaldehyde (No. 683), cinnamyl acetate (No. 650), benzyl cinnamate (No. 670), cyclohexyl cinnamate (No. 667), alpha-amylcinnamaldehyde (No. 685), alpha-hexylcinnamaldehyde (No. 686), para-methoxy-alpha-methylcinnamaldehyde (No. 689), and 3-phenylpropionaldehyde (No. 645) generally did not cause reverse mutation in Salmonella typhimurium strains TA92, TA94, TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538, and TA2637. The assays were performed at concentrations up to the level of cytotoxicity, both in the absence and presence of metabolic activation obtained from the livers of Aroclor 1254- or methylcholanthrene-induced Sprague-Dawley rats or Syrian hamsters (Dunkel & Simon, 1980; Eder et al., 1980; Florin et al., 1980; Lijinsky & Andrews, 1980; Lutz et al., 1980; Eder et al., 1982a,b; Kasamaki et al., 1982; Lutz et al., 1982; Prival et al., 1982; Sekizawa & Shibamoto, 1982; Neudecker et al., 1983; Wild et al., 1983; Ishidate et al., 1984; Huang et al., 1985; Marnett et al., 1985; Mortelmans et al., 1986; Fujita & Sasaki, 1987; Tennant et al., 1987; Kato et al., 1989; Eder et al., 1991; Dillon et al., 1992; Azizan & Blevins, 1995).

Weakly positive or positive results were reported for cinnamaldehyde (No. 656) in S. typhimurium strain TA100 with the pre-incubation method (Dillon et al., 1992; Ishidate et al., 1984), but most other studies in this strain, including a recent study with a prolonged pre-incubation time (120 min) and others in which the standard plate incorporation method was used gave no evidence of mutagenicity (Sasaki & Endo, 1978; Lijinsky & Andrews, 1980; Eder et al., 1982a,b; Kasamaki et al., 1982; Lutz et al., 1982; Prival et al., 1982; Sekizawa & Shibamoto, 1982; Neudecker et al., 1983; Marnett et al., 1985; Mortelmans et al., 1986; Kato et al., 1989; Eder et al., 1991; Azizan & Blevins, 1995).

Negative or weakly positive results were obtained in S. typhimurium with pre-incubation with ortho-methoxycinnamaldehyde (No. 688) (Eder et al., 1991; Mortelmans et al., 1986). The weakly positive results in strain TA100 with metabolic activation were obtained with two different activation systems (Mortelmans et al., 1986). Negative results were obtained in strains TA1535, TA1537, and TA98 both with and without metabolic activation. In the study with strain TA100, negative results were reported in the absence of metabolic activation (Eder et al., 1991). No standard plate incorporation assay was available for ortho-methoxycinnamaldehyde, which might be expected to behave similarly to the other cinnamyl compounds on the basis of structural and metabolic similarities.

The results of assays for mutation in Escherichia coli strains WP2uvrA, PQ37, and Sd-4-73, including several in which the pre-incubation method was used, were negative with cinnamaldehyde (No. 656), cinnamyl alcohol (No. 647), cinnamic acid (No. 657), alpha-methylcinnamaldehdye (No. 683), and alpha-amylcinnamaldehyde (No. 685) (Szybalski, 1958; Sekizawa & Shibamoto, 1982; Ohta et al., 1986; Yoo, 1986; Kato et al., 1989; Eder et al., 1991, 1993). In the rec assay in Bacillus subtilis, positive results were reported with cinnamaldehyde (No. 656) and cinnamyl alcohol (No. 647), whereas cinnamic acid (No. 657), ethyl cinnamate (No. 659), methyl cinnamate (No. 648), and benzyl cinnamate (No. 670) gave negative results in all such tests (Oda et al., 1979; Sekizawa & Shibamoto, 1982; Kuroda et al., 1984; Yoo, 1986).

Assays with isolated mammalian cells gave mixed but generally positive results for cinnamyl esters overall. Equivocal to positive results were obtained for cinnamaldehyde (No. 656) in the assay for forward mutation in L5178Y mouse lymphoma cells with and without metabolic activation, but the reports of these tests did not provide sufficient detail of the method, concentrations tested, or cytotoxic effects to allow adequate evaluation of the results (Rudd et al., 1983; Palmer, 1984). In L1210 mouse lymphoma cells, DNA strand breaks were observed only at cytotoxic concentrations of cinnamaldehyde (Eder et al., 1993).

The results of tests for the induction of sister chromatid exchange in Chinese hamster ovary cells exposed to cinnamaldehyde (No. 656) were negative at low concentrations and weakly positive at concentrations approaching cytotoxic levels, suggesting only weak activity (Galloway et al., 1987; Sasaki et al., 1987). A dose-dependent increase in the frequency of sister chromatid exchange was reported only when cultures were pre-treated with mitomycin C (Sasaki et al., 1987); however, the activity in conjunction with mitomycin contributes little to an evaluation of potential sister chromatid exchange activity. Cinnamaldehyde at concentrations < 15 g/ml was reported to induce chromosomal aberrations in Chinese hamster fibroblasts and B241 cells tested with and without metabolic activation (Kasamaki et al., 1982; Ishidate et al., 1984; Kasamaki & Urasawa, 1985). However, higher concentrations did not induce chromosomal aberrations in Chinese hamster ovary cells in the presence or absence of metabolic activation in a well-conducted, repeated assay (Galloway et al., 1987).

The results of assays for cell transformation with cinnamaldehyde (No. 656) were positive at near-cytotoxic concentrations or after multiple generations of growth and negative in human HAIN-55 cells (Kasamaki et al., 1987; Matthews et al., 1993). Subcutaneous injection of the transformed cells into nude mice led to the formation of nodules at the site of injection and neoplastic growth in the spleen (Kasamaki et al., 1987). Negative results were obtained with cinnamaldehyde (No. 656) in Chinese hamster V79 cells (Fiorio & Bronzetti, 1994), while a weak increase in the incidence of micronucleated Hep-G2 cells was reported by Sanyal et al. (1997).

The results obtained with the other cinnamyl compounds in isolated mammalian cells were, in general, comparable to those obtained with cinnamaldehyde (No. 656). Sister chromatid exchange was not observed in Chinese hamster ovary cells exposed to cinnamyl alcohol (No. 647), cinnamic acid (No. 657), ethyl cinnamate (No. 659), methyl cinnamate (No. 658), cinnamyl acetate (No. 650), or 3-phenylpropionaldehyde (No. 645). Pretreatment with mitomycin C increased the incidence of sister chromatid exchange in assays with cinnamic acid (No. 657), methyl cinnamate (No. 658), and ethyl cinnamate (No. 659) but not cinnamyl alcohol (No. 647), cinnamyl acetate (No. 650), or 3-phenylpropionaldehyde (No. 645) (Sasaki et al., 1989). Palmer (1984) reported reproducible, dose-related increases in the incidence of reversions in L5178Y mouse lymphoma cells, with and without metabolic activation, after treatment with cinnamyl alcohol (No. 647), cinnamic acid (No. 657), cinnamyl cinnamate (No. 673), and ortho-methoxycinnamaldehyde (No. 688).

The results of assays in L5178Y mouse lymphoma cells at the Tk+/ locus have yielded equivocal results. The positive results were seen at near-lethal concentrations in studies in which this was reported. The results of assays with simple aliphatic and aromatic substances were not consistent with the results of other, standard assays for genotoxicity (Tennant et al., 1987; Heck et al., 1989). Culture conditions of low pH and high osmolality, which may pertain with substances that have a potentially acidifying effect on the culture medium (aldehydes, carboxylic acids, lactones, and hydrolysed esters), have been shown to produce false-positive results in this and other assays (Heck et al., 1989). Other reports of positive responses in the mouse lymphoma cell assay lacked information on the concentration tested and on cytolethality (Rudd et al., 1983; Palmer, 1984).

In vivo

Most of the results of tests of the administration of cinnamyl compounds in vivo pertains to cinnamaldehyde (No. 656). An increase in the frequency of sex-linked recessive lethal mutations was reported when Drosophila melanogaster were injected with cinnamaldehyde at 20 000 mg/kg of diet, but no increase in the frequency of mutations was seen when D. melanogaster were fed 800 mg/kg of diet for 3 days. Reciprocal translocations were not observed in either assay (Woodruff et al., 1985). No increase in the frequency of unscheduled DNA synthesis was found in the hepatocytes of rats or mice given cinnamaldehyde at 1000 mg/kg bw by oral gavage (Mirsalis et al., 1989). The frequency of micronuclei was not increased when rats or mice were given 1700 mg/kg bw or 1100 mg/kg bw, respectively, of cinnamaldehyde by oral gavage (Mereto et al., 1994) or when mice were given 500 mg/kg bw by intraperitoneal injection (Hayashi et al., 1984, 1988). The frequency of micronucleated bone-marrow cells in mice that had been exposed to X-rays decreased after injection of 500 mg of cinnamaldehyde (Sasaki et al., 1990).

An increase in the frequency of micronucleated cells was reported in rat and mouse hepatocytes and in rat (but not mouse) forestomach cells after the animals had received up to 1100 (rats) or 1700 (mice) mg/kg bw of cinnamaldehyde by oral gavage. No increase in the frequency of micronuclei in liver or forestomach was observed at doses 850 mg/kg bw, and no DNA fragmentation was observed in rat hepatocytes or gastric mucosal cells. The incidence and size of gamma-glutamyl transferase-positive foci were increased in hepatocytes of rats pretreated with N-nitrosodiethylamine and then given cinnamaldehyde at 500 mg/kg bw per day by oral gavage for 14 days (Mereto et al., 1994).

The positive findings with cinnamaldehyde (No. 656) in rat forestomach and in the livers of both rats and mice treated in vivo are not consistent with the results of the standard assays in bone marrow and were observed at doses that far exceeded those resulting from intake of cinnamaldehyde in foods. Cinnamaldehyde given at oral doses 500 mg/kg bw depleted hepatocellular glutathione concentrations (Swales & Caldwell, 1991, 1992, 1993), and the increases in micronucleus frequency were found at doses that appeared to affect cellular defence mechanisms, such as glutathione depletion. As the micronucleus formation was dose-dependent, induction of micronuclei may be a threshold phenomenon which occurs at intakes orders of magnitude greater than that of cinnamaldehyde as a flavouring agent. Furthermore, the bolus doses resulting from gavage probably resulted in much greater exposure of both the forestomach and the liver than administration in a dietary admixture. The author of the study in which these results were obtained (Mereto et al., 1994) acknowledged these facts and concluded that their data did not justify the conclusion that cinnamaldehyde is clastogenic. In view of the apparent threshold for micronucleus induction and the lack of activity in other studies in vivo, the effects induced by the bolus dose in the liver and forestomach are considered irrelevant to the evaluation of the safety of cinnamaldehyde when used as a flavouring agent.

Wild et al. (1983) reported negative results in tests for sex-linked recessive lethal mutation in D. melanogaster and in an assay for micronucleus formation in mouse bone-marrow cells after administration of alpha-methylcinnamaldehyde (No. 683), allyl cinnamate (No. 19), alpha-amylcinnamyl alcohol (No. 674), alpha-amylcinnamaldehyde (No. 685), or alpha-hexylcinnamaldehyde (No. 686).

2.3.2.4 Reproductive toxicity

Cinnamyl alcohol (No. 647)

Groups of 14 or 15 female rats were given cinnamyl alcohol (No. 647) orally at a dose of 53.5 mg/kg bw on day 4 (implantation) or days 1012 (organogenesis) of gestation. On day 20 of gestation, all animals were killed, and their fetuses were removed for examination. Fetal body weight, length, and the number surviving did not differ significantly between treated and control groups. Histological examination revealed a slight reduction in skeletal ossification of the extremities. Examination of the saggital sections revealed no anomalies in relation to palatal structure, eyes, brain, or other internal organs (Maganova & Zaitsev, 1973).

In a second study, groups of 14 or 15 female rats were given cinnamyl alcohol (No. 647) orally at a dose of 53.5 mg/kg bw per day throughout gestation. On day 20 of gestation, 50% of the treated and control animals were killed, and their fetuses were removed for examination. Fetal body weight, liver nucleic acids, number of survivors, and bone development did not differ significantly between test and control groups. The remaining females from both groups were allowed to deliver normally. Again, the body weights, number surviving, and size and general development of offspring at birth or at 1 month did not differ significantly between treated and control groups (Zaitsev & Maganova, 1975).

Cinnamaldehyde (No. 656)

Groups of 1416 rats were given cinnamaldehyde (No. 656) at a dose of 5, 25, or 250 mg/kg bw per day by gavage in olive oil on days 717 of gestation. A control group was included, but it was not stated whether they received the olive oil vehicle. The fetal abnormalities observed included poor cranial ossification at all doses; increased incidences of dilated pelvis, reduced renal papillae, and dilated ureters at the low and intermediate doses; and an increased number of fetuses with two or more abnormal sternebrae at the intermediate dose. These effects were not dose-related and might be attributable to the decrease in maternal weight gain at the two higher doses (Mantovani et al., 1989).

Cinnamic acid (No. 657)

Groups of 14 or 15 female rats were given cinnamic acid (No. 657) orally at a dose of 0, 5, or 50 mg/kg bw per day throughout gestation. On day 20 of gestation, 50% of the females in all groups were killed, and their fetuses were removed for examination. No significant differences in fetal body weight, number of survivors, bone development, or hepatic nucleic acids were found between treated and control groups. The remaining females in both treated and control groups were allowed to deliver normally on days 2223 of gestation. No significant differences in the body weights, size, number surviving, or general development of offspring at birth or at 1 month were found between treated and control groups (Zaitsev & Maganova, 1975).

Conclusion

Cinnamyl alcohol (No. 647) and related compounds lack direct mutagenic or genotoxic activity, as indicated by the negative results obtained in bacterial test systems. The mixed results in the assay for DNA repair and in various studies of antimutagenicity were associated with cytotoxicity, as noted by Sekizawa & Shibamoto (1982). Evidence of genotoxic activity was found in isolated mammalian cells, the cinnamyl compounds inducing chromosomal aberrations and/or mutations in the presence or absence of metabolic activation; however, the reported activity in vitro was not seen as mutagenic, clastogenic, or genotoxic activity in vivo.

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    See Also:
       Toxicological Abbreviations
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