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WHO FOOD ADDITIVES SERIES: 48

SAFETY EVALUATION OF CERTAIN
FOOD ADDITIVES AND CONTAMINANTS

HYDROXY- AND ALKOXY-SUBSTITUTED
BENZYL DERIVATIVES

First draft prepared by Dr A. Mattia1, Professor A.G. Renwick2, Professor I.G. Sipes3, and M. DiNovi4
1
Division of Product Policy, Office of Premarket Approval, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington DC, USA
2Clinical Pharmacology Group, University of Southampton, Southampton, United Kingdom
3 Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, USA
4Office of Premarket Approval, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington DC, 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

Biochemical data

Absorption, distribution, and elimination

Metabolism

Toxicological studies

Acute toxicity

Short-term studies of toxicity

Long-term studies of toxicity and carcinogenicity

Genotoxicity

Reproductive toxicity

Observations in humans

References

1. EVALUATION

1.1 Introduction

The Committee evaluated a group of flavouring agents1 comprising 46 structurally related substances using the Procedure for the Safety Evaluation of Flavouring Agents (see Figure 1, Introduction). All members of this group are aromatic primary alcohols, aldehydes, carboxylic acids, or their corresponding esters or acetals. The structural feature common to all members of the group is a primary oxygenated functional group bound directly to a benzene ring. The ring also contains hydroxy or alkoxy substituents (see Table 1).

Table 1. Summary of results of safety evaluations of hydroxy- and alkoxy-substituted benzyl derivatives used as flavouring agentsa

Flavouring agent

No.

CAS No. and structure

Step A3b Does intake exceed the threshold for human intake?

Step A4 Is the flavouring agent or are its metabolites endogenous?

Step A5 Adequate margin of safety for the flavouring agent or related substance?

Comments on predicted metabolism

Conclusion based on current intake

Structural class I

4-Hydroxybenzyl alcohol

955

623-05-2

No
Europe: 6
USA: 0.06

NR

NR

See note 1.

No safety concern

4-Hydroxybenzaldehyde

956

123-08-0

No
Europe: 64
USA: 59

NR

NR

See note 1.

No safety concern

4-Hydroxybenzoic acid

957

99-96-7

No
Europe: 19
USA: 17

NR

NR

See note 1.

No safety concern

2-Hydroxybenzoic acid

958

69-72-7

No
Europe: 0.03
USA: 0.03

NR

NR

See note 1.

No safety concern

Butyl para-hydroxy benzoatec

870

94-26-8

No
Europe: ND
USA: 0.03

NR

NR

See note 2.

Evaluation not finalized

Anisyl alcohol

871

105-13-5

No
Europe: 150
USA: 58

NR

NR

See note 1.

No safety concern

Anisyl formate

872

122-91-8

No
Europe: 46
USA: 24

NR

NR

See note 2.

No safety concern

Anisyl acetate

873

104-21-2

No
Europe: 59
USA: 300

NR

NR

See note 2.

No safety concern

Anisyl propionate

874

7549-33-9

No
Europe: ND
USA: 5

NR

NR

See note 2.

No safety concern

Anisyl butyrate

875

6963-56-0

No
Europe: 34
USA: 0.1

NR

NR

See note 2.

No safety concern

Anisyl phenylacetate

876

102-17-0

No
Europe: 0.003
USA: 0.1

NR

NR

See note 4.

No safety concern

Veratraldehyde

877

120-14-9

No
Europe: 140
USA: 55

NR

NR

See note 1.

No safety concern

para-Methoxybenzaldehyde

878

123-11-5

No
Europe: 440
USA: 580

NR

NR

See note 1.

No safety concern

para-Ethoxybenzaldehyde

879

10031-82-0

No
Europe: 0.1
USA: 0.01

NR

NR

See note 1.

No safety concern

Methyl ortho-methoxybenzoate

880

606-45-1

No
Europe: 57
USA: 8

NR

NR

See note 2.

No safety concern

2-Methoxybenzoic acid

881

579-75-9

No
Europe: ND
USA: 0.01

NR

NR

See note 1.

No safety concern

3-Methoxybenzoic acid

882

586-38-9

No
Europe: ND
USA: 0.01

NR

NR

See note 1.

No safety concern

4-Methoxybenzoic acid

883

100-09-4

No
Europe: ND
USA: 0.1

NR

NR

See note 1.

No safety concern

Methyl anisate

884

121-98-2

No
Europe: 1
USA: 0.01

NR

NR

See note 2.

No safety concern

Ethyl para-anisate

885

94-30-4

No
Europe: 11
USA: 2

NR

NR

See note 2.

No safety concern

Vanillyl alcohol

886

498-00-0

No
Europe: 6
USA: 6

NR

NR

See note 1.

No safety concern

Vanillind

889

121-33-5

Yes
Europe: 55 000
USA: 150 000

No

Yes; the NOEL of 1000 mg/kg bw per day in a 2-year study in rats is > 100 times the estimated daily intake of vanillin when used as a flavouring agent.

See note 1.

No safety concern

4-Hydroxy-3-methoxy-benzoic acid

959

121-34-6

No
Europe: 29
USA: 26

NR

NR

See note 1.

No safety concern

Vanillin acetate

890

881-68-5

No
Europe: 2
USA: 1

NR

NR

See note 2.

No safety concern

Vanillin isobutyrate

891

20665-85-4

No
Europe: 64
USA: 0.04

NR

NR

See note 2.

No safety concern

Salicylaldehyde

897

90-02-8

No
Europe: 98
USA: 16

NR

NR

See note 1.

No safety concern

2-Hydroxy-4-methyl-benzaldehyde

898

698-27-1

No
Europe: 1
USA: 0.3

NR

NR

See note 1.

No safety concern

Methyl salicylatee

899

119-36-8

Yes
Europe: 490
USA: 44 000

No

Yes; the NOEL of 50 mg/kg bw per day in a 2-year study in dogs is > 100 times the estimated daily intake of methyl salicylate when used as a flavouring agent.

See note 2

No safety concern

Ethyl salicylate

900

118-61-6

No
Europe: 31
USA: 1700

NR

NR

See note 2.

No safety concern

Butyl salicylate

901

2052-14-4

No
Europe: 0.01
USA: 0.0007

NR

NR

See note 2.

No safety concern

Isobutyl salicylate

902

87-19-4

No
Europe: 1
USA: 6

NR

NR

See note 2.

No safety concern

Isoamyl salicylate

903

87-20-7

No
Europe: 49
USA: 7

NR

NR

See note 2.

No safety concern

Benzyl salicylate

904

118-58-1

No
Europe: 30
USA: 29

NR

NR

See note 4.

No safety concern

Phenethyl salicylate

905

87-22-9

No
Europe: 0.2
USA: 4

NR

NR

See note 4.

No safety concern

ortho-Tolyl salicylate

907

617-01-6

No
Europe: ND
USA: 30

NR

NR

See note 4.

No safety concern

2,4-Dihydroxybenzoic acid

908

89-86-1

No
Europe: ND
USA: 6

NR

NR

See note 1.

No safety concern

Structural class II

Vanillyl ethyl ether

887

13184-86-6

No
Europe: 22
USA: 22

NR

NR

See note 1.

No safety concern

Vanillyl butyl ether

888

82654-98-6

No
Europe: ND
USA: 0.1

NR

NR

See note 1.

No safety concern

Ethyl vanillinf

893

121-32-4

Yes
Europe: 6200
USA: 43 000

No

Yes; the NOEL of 500 mg/kg bw per day in a 14-week study in rats is > 100 times the estimated daily intake of ethyl vanillin when used as a flavouring agent.

See note 1.

No safety concern

Vanillin erythro & threo- butan-2,3-diol acetal

960

No
Europe: 4
USA: 3

NR

NR

See note 2.

No safety concern

Ethyl vanillin isobutyrate

953

188417-26-7

No
Europe: 64
USA: ND

NR

NR

See note 2.

No safety concern

Ethyl vanillin propylene glycol acetal

954

68527-76-4

No
Europe: 39
USA: 36

NR

NR

See note 2.

No safety concern

Piperonyl acetate

894

326-61-4

No
Europe: 41
USA: 11

NR

NR

See note 3.

No safety concern

Piperonyl isobutyrate

895

5461-08-5

No
Europe: 0.1
USA: 3

NR

NR

See note 3.

No safety concern

Piperonalg

896

120-57-0

Yes
Europe: 1700
USA: 3200

No

Yes; the NOEL of 250 mg/kg bw per day in a 2-year study in rats is > 100 times the estimated daily intake of piperonal when used as a flavouring agent

See note 3.

No safety concern

Ethyl vanillin beta-d-gluco-pyranoside

892

122397-96-0

No
Europe: ND
USA: 30

NR

NR

See note 2.

No safety concern

CAS: Chemical Abstracts Service; ND: no data available; NR: 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 flavouring agents 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 mg/day.

c

Further information is required to determine whether this substance is currently used as a flavouring agent.

d

An ADI of 0–10 mg/kg bw was established for vanillin by the Committee at its eleventh meeting (Annex 1, reference 14), which was maintained at the present meeting.

e

An ADI of 0–0.5 mg/kg bw was established for methyl salicylate by the Committee at its eleventh meeting (Annex 1, reference 14), which was maintained at the present meeting. The estimated daily per capita intake of methyl salicylate is 0.7 mg/kg bw when calculated with the usual 10% proportion of eaters. However, an analysis of intake showed that more than 50% of the population would be expected to consume methyl salicylate. Intake calculated with this measured proportion of eaters is 0.1 mg/kg bw.

f

An ADI of 0–3 mg/kg bw was established for ethyl vanillin by the Committee at its forty-fourth meeting (Annex 1, reference 116), which was maintained at the present meeting.

g

An ADI of 0–2.5 mg/kg bw was established for piperonal by the Committee at its eleventh meeting (Annex 1, reference 14), which was maintained at the present meeting.

Notes

1.

Detoxication by excretion in the urine unchanged or as glucuronic acid, glycine, or sulfate conjugates; aldehyde groups undergo oxidation or reduction to the corresponding carboxylic acid or alcohol, respectively, followed by conjugation and excretion; O-dealkylation followed by conjugation and excretion; other minor metabolic routes, probably by intestinal microflora after biliary excretion of conjugates, include decarboxylation and reduction of benzyl groups to the methyl analogue.

2.

Detoxication as in note 1 plus hydrolysis of esters to the corresponding benzyl alcohol or benzoic acid derivatives or acetal hydrolysis to the parent benzaldehyde derivative and simple aliphatic alcohol or glycosidic bond hydrolysis to the corresponding phenolic derivative.

3.

Detoxication as in note 1 plus limited oxidation of the methylenedioxyphenyl group to a catechol, which would undergo conjugation.

4.

Detoxication as in note 1 preceded by hydrolysis to yield mononuclear residues, each of which would be detoxicated as in note 1.

The Committee previously evaluated four members of this group. Ethyl vanillin (No. 893) was evaluated at by the Committee at its eleventh meeting (Annex 1, reference 14), when a conditional ADI of 0–10 mg/kg bw was allocated. At its thirty-fifth meeting, the Committee converted this value to a temporary ADI of 0–5 mg/kg bw (Annex 1, reference 88). At its thirty-ninth meeting, the Committee extended the temporary ADI (Annex 1, reference 101). At its forty-fourth meeting, the Committee allocated an ADI of 0–3 mg/kg bw to ethyl vanillin (Annex 1, reference 116). Vanillin (No. 889) was evaluated by the Committee at its eleventh meeting (Annex 1, reference 14), when an ADI of 0–10 mg/kg bw was established. Methyl salicylate (No. 899) was evaluated at the eleventh meeting (Annex 1, reference 14), when an ADI of 0–0.5 mg/kg bw was allocated. Piperonal (No. 896) was also evaluated at the eleventh meeting, when an ADI of 0–2.5 mg/kg bw was allocated (Annex 1, reference 14).

At its present meeting, the Committee evaluated a group of benzyl derivatives, none of which contains hydroxy- or alkoxy- substituents. Twenty-nine of the 46 members of this group of flavouring agents have been reported to occur naturally in food. Vanillin, a major constituent of natural vanilla, is also present in strawberries and milk. Methyl salicylate, the predominant substituent of oil of wintergreen, is also found in tomatoes and grilled beef. Ethyl vanillin has been detected in raspberries and ginger, while piperonal is found in cooked chicken and pepper (Maarse et al., 1999).

1.2 Estimated daily intake

The total annual production of the 46 flavouring agents in this group is 450 000 kg in Europe (International Organization of the Flavor Industry, 1995) and 1 800 000 kg in the USA (Lucas et al., 1999). Vanillin (No. 889), ethyl vanillin (No. 893), methyl salicylate (No. 899), and piperonal (No. 896), for which ADIs were previously established by the Committee, accounted for approximately 98% of the total annual volume in Europe and 99% in the USA. In Europe, the estimated daily per capita intakes of these compounds were 55 mg of vanillin, 6.2 mg of ethyl vanillin, 0.5 mg of methyl salicylate, and 1.6 mg of piperonal. In the USA, the estimated daily per capita intakes were 150 mg of vanillin, 43 mg of ethyl vanillin, 44 mg of methyl salicylate, and 3.2 mg of piperonal. The estimated daily per capita intakes of two other flavouring agents in this group were in the low milligram range: the estimated intake of ethyl salicylate is 1.7 mg/person per day in the USA and that of para-methoxybenzaldehyde is around 0.5 mg/person per day in both Europe and the USA. The estimated intakes of most of the other 40 flavouring agents were between 1 and 100 ΅g/person per day, and those of 10 were < 1 ΅g/person per day. The annual usage volume of these substances in Europe and the USA is shown in Table 2.

Table 2. Annual volumes of use of hydroxy- and alkoxy-substituted benzyl derivatives used as flavouring agents in Europe and the USA

Substance (No.)

Most recent annual volume (kg)

Intakea

Annual volume in naturally occurring foods (kg)b

Consumption ratioc

 

 

΅g/day

΅g/kg bw per day

 

 

4-Hydroxybenzyl alcohol (955)

Europe

43

6

0.1

+

NA

USA

0.4

0.06

0.001

 

NA

4-Hydroxybenzaldehyde (956)

Europe

450

64

1

3 600

8

USA

450

59

1

 

8

4-Hydroxybenzoic acid (957)

Europe

130

19

0.3

23 000

180

USA

130

17

0.3

 

180

2-Hydroxybenzoic acid (958)

Europe

0.2

0.03

0.0005

20 000

100 000

USA

0.2

0.03

0.0005

 

100 000

Butyl para-hydroxybenzoate (870)

Europe

NR

NA

NA

–

NA

USA

0.2

0.03

0.0005

 

NA

Anisyl alcohol (871)

Europe

1 100

150

2.5

+

NA

USA

440

58

1

 

NA

Anisyl formate (872)

Europe

320

46

0.8

–

NA

USA

180

24

0.4

 

NA

Anisyl acetate (873)

Europe

410

59

1

+

NA

USA

2 300

300

5

 

NA

Anisyl propionate (874)

Europe

NR

NA

NA

–

NA

USA

37

5

0.08

 

NA

Anisyl butyrate (875)

Europe

240

34

0.6

–

NA

USA

1

0.1

0.002

 

NA

Anisyl phenylacetate (876)

Europe

0.02

0.003

0.00005

–

NA

USA

0.5

0.1

0.002

 

NA

Veratraldehyde (877)

Europe

1 000

140

2.3

+

NA

USA

410

55

1

 

NA

para-Methoxybenzaldehyde (878)

Europe

3 000

440

7.3

77

0.03

USA

4 400

580

10

 

0.02

para-Ethoxybenzaldehyde (879)

Europe

0.6

0.1

0.002

+

NA

USA

0.1

0.01

0.0002

 

NA

Methyl ortho-methoxybenzoate (880)

Europe

400

57

1

25

0.06

USA

64

8

0.1

 

0.4

2-Methoxybenzoic acid (881)

Europe

NR

NA

NA

+

NA

USA

0.1

0.01

0.0002

 

NA

3-Methoxybenzoic acid (882)

Europe

NR

NA

NA

+

NA

USA

0.1

0.01

0.0002

 

NA

4-Methoxybenzoic acid (883)

Europe

NR

NA

NA

120

NA

USA

1

0.1

0.002

 

120

Methyl anisate (884)

Europe

8

1

0.02

33

4

USA

0.05

0.01

0.0002

 

660

Ethyl para-anisate (885)

Europe

75

11

0.2

+

NA

USA

16

2

0.03

 

NA

Vanillyl alcohol (886)

Europe

44

6

0.1

+

NA

USA

46

6

0.1

 

NA

Vanillin (889)

Europe

385 000

55 000

920

20 000

0.05

USA

1 140 000

150 000

2 500

 

0.02

4-Hydroxy-3-methoxybenzoic acid (959)

Europe

200

29

0.5

100 000

500

USA

200

26

0.4

 

500

Vanillin acetate (890)

Europe

15

2

0.03

+

NA

USA

6

1

0.02

 

NA

Vanillin isobutyrate (891)

Europe

450

64

1

–

NA

USA

0.3

0.04

0.0007

 

NA

Salicylaldehyde (897)

Europe

690

98

1.6

66 000

96

USA

120

16

0.3

 

550

2-Hydroxy-4-methyl benzaldehyde (898)

Europe

5

1

0.02

+

NA

USA

2

0.3

0.005

 

NA

Methyl salicylate (899)

Europe

3 400

490

8

2 500

0.7

USA

340 000

44 000

730

 

0.07

Ethyl salicylate (900)

Europe

220

31

0.5

9

0.04

USA

13 000

1 700

28

 

0.0007

Butyl salicylate (901)

Europe

0.1

0.01

0.0002

+

NA

USA

0.005

0.0007

0.00001

 

NA

Isobutyl salicylate (902)

Europe

8

1

0.02

–

NA

USA

43

6

0.1

 

NA

Isoamyl salicylate (903)

Europe

340

49

0.8

14 000

41

USA

55

7

0.1

 

250

Benzyl salicylate (904)

Europe

210

30

0.5

+

NA

USA

220

29

0.5

 

NA

Phenethyl salicylate (905)

Europe

1

0.2

0.003

–

NA

USA

32

4

0.07

 

NA

ortho-Tolyl salicylate (907)

Europe

NR

NA

NA

–

NA

USA

230

30

0.5

 

NA

2,4-Dihydroxybenzoic acid (908)

Europe

NR

NA

NA

+

NA

USA

45

6

0.1

 

NA

Vanillyl ethyl ether (887)

Europe

165

22

0.4

–

NA

USA

165

22

0.4

 

NA

Vanillyl butyl ether (888)

Europe

NR

NA

NA

–

NA

USA

1

0.1

0.002

 

NA

Ethyl vanillin (893)

Europe

44 000

6 200

100

–

NA

USA

330 000

43 000

720

 

NA

Vanillin erythro & threo-butan-2,3-diol acetal (960)

Europe

25

4

0.07

–

NA

USA

25

3

0.05

 

NA

Ethyl vanillin isobutyrate (953)

Europe

450

64

1

–

NA

USA

NR

NA

NA

 

NA

Ethyl vanillin propylene glycol acetal (954)

Europe

280

39

0.7

–

NA

USA

280

36

0.6

 

NA

Piperonyl acetate (894)

Europe

280

41

0.7

+

NA

USA

82

11

0.2

 

NA

Piperonyl isobutyrate (895)

Europe

0.7

0.1

0.002

–

NA

USA

26

3

0.05

 

NA

Piperonal (896)

Europe

12 000

1 700

28

+

NA

USA

24 000

3 200

53

 

NA

Ethyl vanillin beta-d-glucopyranoside (892)

Europe

NR

NA

NA

–

NA

USA

230

30

0.5

 

NA

Total

Europe

450 000

 

 

 

 

USA

1 900 000

 

 

 

 

NA, not applicable; NR, not reported; +, reported to occur naturally in foods (Maarse et al., 1999), but quantitative data were not available; -, not reported to occur naturally in foods

a

Intake expressed as ΅g/person per day calculated as follows: [(annual volume, kg) x (1 x 109 ΅g/kg)/ (population x survey correction factor x 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 expressed as ΅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.

b

Quantitative data from Stofberg & Grundschober (1987)

c

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

1.3 Metabolic considerations

The aromatic esters in this group are expected to be appreciably hydrolysed through the catalytic activity of the intestinal carboxylesterases, especially beta-esterases, to benzyl alcohol or benzoic acid derivatives before absorption (Heymann, 1980; Anders, 1989). Likewise, acetals of substituted benzaldehyde derivatives are hydrolysed in gastric and intestinal fluids to yield benzaldehyde and component aliphatic alcohols. The resulting hydroxy- and alkoxy-substituted benzyl derivatives are rapidly absorbed in the gut, metabolized in the liver, and excreted in urine (Davison, 1971; Abdo et al., 1985; Temellini et al., 1993).

Once absorbed, benzyl derivatives are oxidized to the corresponding benzoic acid derivative, which is subsequently excreted unchanged or as sulfate or glucuronide conjugates (Sammons & Williams, 1946; Davison, 1971; Scheline, 1972). Piperonal is oxidized to piperonylic acid and excreted mainly as the glycine conjugate (Kamienski & Casida, 1970; Klungsoyr & Scheline, 1984). Minor metabolic detoxication pathways include O-demethylation, reduction, and decarboxylation. These pathways arise during enterohepatic cycling of conjugated benzyl metabolites and subsequent intestinal bacterial action (Strand & Scheline, 1975).

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

Step 1 Thirty-six of the 46 flavouring agents in this group were assigned to structural class I (Cramer et al., 1978). They are simple substituted aromatic compounds that are expected to be hydrolysed to aromatic aldehydes and simple aliphatic alcohols (Nos 870–875, 877–886, 889–891, 897–903 and 955–959), or they contain two aromatic rings that are expected to be hydrolysed to mononuclear residues with simple functional groups (Nos 876, 904, 905, and 907). The remaining 10 flavouring agents (Nos 887, 888, 892–896, 953, 954, and 960) are ethyl vanillin or piperonal derivatives that contain either an ethoxy or a methylene dioxy substituent. They are common components of food or closely related to common components of food and were assigned to structural class II.

Step 2 At current levels of intake, none of the 46 flavouring agents would be expected to saturate the metabolic pathways, and all are predicted to be metabolized to innocuous products.

Step A3 The estimated per capita intakes of 42 of the 46 flavouring agents in this group were below the human intake threshold for their respective classes (i.e., 1800 ΅g/day for structural class I and 540 ΅g/day for structural class II).

These 42 flavouring agents therefore pose no safety concern when used at their current levels.

The estimated intakes of vanillin (No. 889) and methyl salicylate (No. 899), which are in class I, exceed the daily intake threshold of 1800 ΅g/person. The estimated intakes of vanillin are 55 000 ΅g/person per day in Europe and 150 000 ΅g/person per day in the USA, and that of methyl salicylate is 44 000 ΅g/person per day in the USA. The estimated intakes of ethyl vanillin (No. 893) and piperonal (No. 896), in class II, exceed the intake threshold of 540 ΅g/person per day. The estimated intakes of ethyl vanillin are 6200 ΅g/person per day in Europe and 43 000 ΅g/person per day in the USA. For piperonal, the estimated intakes are 1700 ΅g/person per day in Europe and 3200 ΅g/person per day in the USA) (International Organization of the Flavor Industry, 1995; Lucas et al., 1999).

The estimated intakes of these four substances are below their respective ADI values. The daily intakes of vanillin in Europe and the USA, approximately 0.9 and 2.5 mg/kg bw, do not exceed the ADI of 0–10 mg/kg bw for vanillin. The highest estimated daily intakes of ethyl vanillin (0.7 mg/kg bw in the USA) and piperonal (0.05 mg/kg bw in the USA) do not exceed the ADIs of 0–3 mg/kg bw for ethyl vanillin and 0–2.5 mg/kg bw for piperonal. The estimated daily intake of the remaining substance, methyl salicylate, is 0.7 mg/kg bw, which is approximately equal to its ADI of 0–0.5 mg/kg bw.

The estimates of intake based on total annual production include the assumption that only 10% of the population eats these agents. The Committee received a report on the intake of methyl salicylate based on individual dietary records and its use in the USA in baked goods, chewing-gum, hard and soft sweets, and beverages (Edelstein, 2=01). The analysis showed that > 50% of the population would be expected to eat methyl salicylate. Use of this measured proportion of eaters in place of the default assumption of 10% yields an estimated intake of methyl salicylate of 0.1 mg/kg bw, which is below the current ADI of 0–0.5 mg/kg bw.

Step A4 Vanillin (No. 889), methyl salicylate (No. 899), ethyl vanillin (No. 893), and piperonal (No. 896) are not endogenous in humans.

Step A5 The ADI of 0–10 mg/kg bw for vanillin is based on a NOEL of 1000 mg/kg bw per day in a 2-year feeding study in rats (Hagan et al., 1967). This NOEL provides a margin of safety that is about 400 times the per capita intake of vanillin from its current use as a flavouring agent in Europe (0.9 mg/kg bw per day) or in the USA (2.5 mg/kg bw per day).

The ADI of 0–0.5 mg/kg bw for methyl salicylate is based on a NOEL of 50 mg/kg bw per day reported in a 2-year study in dogs (Webb & Hansen, 1963). This NOEL is more than 1000 times greater than the intake of methyl salicylate from its current use as a flavouring agent in Europe (0.008 mg/kg bw per day). This NOEL is also more than 100 times greater than the intake of methyl salicylate in the USA when intake is calculated by using the measured portion of eaters of 50% (0.1 mg/kg bw per day).

A NOEL of 500 mg/kg bw per day of ethyl vanillin was reported in a 14-week feeding study in rats (Hooks et al., 1992). This NOEL is about 700 times greater than the intake of ethyl vanillin from its use as a flavouring agent in Europe (0.1 mg/kg bw per day) or in the USA (0.7 mg/kg bw per day).

A NOEL of 250 mg/kg bw per day for piperonal was reported in a 2-year study in rats (Bar & Griepentrog, 1967). This NOEL is more than 5000 times the intake of piperonal from its use as a flavouring agent in Europe (0.028 mg/kg bw per day) and in the USA (0.05 mg/kg bw per day).

The considerations on intake and other information used to perform the stepwise evaluations of the 46 hydroxy- and alkoxy-substituted benzyl derivatives according to the Procedure are summarized in Table 1.

1.5 Consideration of combined intakes

In the unlikely event that all 36 flavouring agents in structural class I were consumed on a daily basis, the estimated combined intake would exceed the human intake threshold (1800 ΅g/person per day). The estimated combined intake of all class II substances would also exceed the intake threshold (540 ΅g/person per day). However, all 46 substances are expected to be efficiently detoxicated and would not saturate the available detoxication pathways. On the basis of the evaluation of the collective data, combined intake would present no safety concerns.

1.6 Conclusions

The Committee retained the previously established ADIs of 0–10 mg/kg bw for vanillin (No. 889), 0–3 mg/kg bw for ethyl vanillin (No. 893), 0–2.5 mg/kg bw for piperonal (No. 896), and 0–0.5 mg/kg bw for methyl salicylate (No. 899). The Committee noted that the estimated daily intake of 0.7 mg/kg bw of methyl salicylate, based on poundage data, is approximately equal to its ADI of 0–0.5 mg/kg bw within the precision of the intake estimates. The Committee received an analysis of intake based on individual dietary records for methyl salicylate for its potential use in mint-flavoured baked goods, chewing-gum, hard and soft sweets, and beverages. This analysis showed that > 50% of the population would be expected to consume methyl salicylate. The (appropriate) use of this measured proportion of eaters in place of the default assumption of 10% yields an estimated intake of methyl salicylate of 0.1 mg/kg bw, which is less than the current ADI of 0–0.5 mg/kg bw.

On the basis of the available data on metabolism and toxicity, the Committee concluded that none the flavouring agents in this group would present a safety concern when used at their current levels. Other data on toxicity, including the results of studies on genotoxicity and developmental toxicity, were consistent with the results of the safety evaluations conducted according to the Procedure.

2. RELEVANT BACKGROUND INFORMATION

2.1 Explanation

The background information relevant to a safety evaluation of 46 hydroxy- and alkoxy-substituted benzyl derivatives used as flavouring agents are summarized below and in Tables 1 and 2.

2.2 Additional considerations on intake

Production volumes and intake values for each flavouring agent in this group are shown in Table 2.

Twenty-nine of the 46 flavouring agents have been detected as natural components of traditional foods (Maarse et al., 1999; Table 2). Quantitative data on natural occurrence and consumption ratios have been reported for 13 substances in the group. The consumption of eight substances is derived predominantly from their presence in traditional foods (i.e. they have a consumption ratio > 1), whereas para-methoxybenzaldehyde (No. 878), methyl-ortho-methoxybenzoate (No. 880), vanillin (No. 889), methyl salicylate (No. 899), and ethyl salicylate (No. 900) are not consumed primarily from traditional foods (consumption ratio, < 1).

2.3 Biological data

2.3.1 Biochemical data

(a) Absorption, distribution, and excretion

The hydroxy- and alkoxy-substituted benzyl derivatives have been shown to be rapidly absorbed in the gastrointestinal tract, metabolized in the liver to yield benzoic acid derivatives, and excreted primarily in the urine either unchanged or conjugated (Jones et al., 1956; Davison, 1971). Metabolites participate in enterohepatic cycling to some extent, leading to further metabolism by gut bacteria.

In rabbits, 96% of a single oral dose of 400 mg/kg bw 4-hydroxybenzaldehyde (No. 956) was excreted in the urine within 24 h as 4-hydroxybenzoic acid and its glycine, glucuronic acid, and sulfate conjugates (Bray et al., 1952).

In a similar study, groups of four to eight rabbits were given 4-hydroxybenzoic acid (No. 957) at a dose of 100, 250, 500, 1000, or 1500 mg/kg bw by gavage every 3–7 days. Urine was collected continuously and analysed for metabolites. The total urinary recovery of the test material ranged from 84% to 104% . Glucuronic acid and sulfate conjugates were also detected in the urine, at 10–35% and 4–7%, respectively. The concentrations of all the metabolites returned to background values within 24 h after dosing (Bray et al., 1947). In a corresponding study, approximately 94% of 2-hydroxybenzoic acid (No. 958) at a single oral dose of 250 or 500 mg/kg bw given to two groups of four rabbits was excreted unchanged or as the glucuronic acid and sulfate conjugates (Bray et al., 1948).

About 6% of a dose of 52 mg of 2,4-dihydroxybenzaldehyde (No. 908) given by intraperitoneal injection to female albino rats was excreted in the urine as the corresponding hippurate within 24 h (Teuchy et al., 1971).

Three patients being treated for rheumatic fever were given an oral dose of 5330–6000 mg of 2,4-dihydroxybenzoic acid as 1000 mg per dose every 3 h for 2–16 days. The average daily rate of urinary excretion was 43–76%. The average daily excretion of sulfate conjugate per patient was essentially constant throughout the study, but the average daily excretion of glucuronic acid conjugate increased by four- to sixfold over the 16 days (Clarke et al., 1958). In an investigation of the presence of dihydroxybenzoic acid isomers in the urine of 15 persons, only 3,5-dihydroxybenzoic acid was detected (Williams, 1965).

Groups of three or more fasted dogs were given butyl para-hydroxybenzoate (No. 870) at a dose of 1000 mg/kg bw orally or 50 mg/kg bw intravenously. Blood and urine samples were collected at fixed intervals until the concentrations returned to background values within 48 h. The test material was recovered almost entirely as the para-hydroxybenzoic acid conjugate of glucuronic acid, at 48% after the oral dose and 40% after the intravenous dose. Most of the material was excreted between 6 and 30 h after dosing. Although the relatively low rate of recovery with both methods of administration was attributed to incomplete hydrolysis of the ester in the body, incubation of the butyl ester with freshly prepared liver homogenate in vitro showed complete hydrolysis within 30–60 min. In studies with related benzoate esters, such as methyl and ethyl para-hydroxybenzoate, significantly larger amounts of material were recovered (Jones et al., 1956). This finding suggests that increased amounts of the homologous series of alkyl esters may activate other metabolic and excretion pathways. The authors concluded that butyl para-hydroxybenzoate and other alkyl esters are readily absorbed, metabolized, and excreted.

Ten rabbits were each fed 200 mg of veratraldehyde (No. 877) by stomach tube, and urine was collected over the next 24 h. About 70% of the material was recovered in the urine as free corresponding acid (~28%) and its glucuronic acid (~38%) or sulfate (3–7%) conjugate (Sammons & Williams, 1941).

Because of its prevalence and importance as a flavouring agent, vanillin (No. 889) has been the subject of numerous studies on metabolism. Male albino rats were given 100 mg/kg bw of vanillin in a solution of propylene glycol and water by stomach tube; urine and faeces were collected separately for 24-h periods, and bile samples were collected by cannulation of the common bile duct. Only trace amounts of benzoic acid derivatives remained in the urine after the first 24 h, and none remained after 48 h. Free and conjugated forms of vanillic acid and vanillyl alcohol accounted for 94% of the dose in the urine. Vanillin and its primary reduction and oxidation metabolites were also excreted in appreciable amounts in the bile. Bile collected for 5 h from two rats given vanillin at a dose of 100 or 300 mg/kg bw orally contained glucuronide conjugates of vanillin (6%), vanillyl alcohol (8%), and vanillic acid (9%) (Strand & Scheline, 1975).

In Sprague-Dawley albino rats, 60% of a dose of vanillin of 100 mg/kg bw in 0.9% NaCl given by intraperitoneal injection was recovered in the 24-h urine. The urinary metabolites included unconjugated vanillic acid, the sulfate and glucuronic acid conjugates of vanillic acid, and conjugates of vanillyl alcohol, vanillin, and catechol. The urinary glycine conjugate of vanillic acid was not found in this study (Wong & Sourkes, 1966).

Three rabbits given vanillin by gavage at 1000 mg/kg bw excreted an average of 83% of the dose in their urine, with 69% of the dose as free and conjugated vanillic acid and 14% as conjugated vanillin (Sammons & Williams, 1941).

An adult person received 100 mg of vanillin dissolved in water, and urine was collected for 24 h. The concentration of vanillic acid in the urine increased from a background value of 0.3 mg per 24 h to 96 mg per 24 h, accounting for about 94% of the dose (Dirscherl & Wirtzfeld, 1964).

After administration of a single dose of 400 mg/kg bw of salicylaldehyde (No. 897) to a fasted rabbit, 75% of the dose was excreted in urine collected over 24 h. Urine analysis revealed mainly ether-soluble acids, 27% and 3% being accounted for by glucuronic acid and sulfate conjugates of vanillic acid, respectively (Bray et al., 1952).

In male rats, 94% of a dose of 150 mg/kg bw of piperonal (No. 896) in propylene glycol administered by gavage was accounted for in the urine within 24 h. No unchanged compound was excreted, and no metabolites were detected in the urine more than 48 h after dosing (Klungsoyr & Scheline, 1984). In a study of the metabolism of the corresponding esters piperonyl acetate (No. 894) and piperonyl isobutyrate (No. 895) given at 100 mg/kg bw by gavage to male rabbits, 70% of the dose of piperonyl acetate and 11% of that of piperonyl isobutyrate was recovered in 72-h urine (Wright & Holder, 1980).

The results of these studies indicate that the hydroxy- and alkoxy-substituted benzyl derivatives are rapidly absorbed, metabolized, and excreted in the urine mainly as sulfate and glucuronic acid conjugates of the corresponding hydroxybenzoic acid derivatives.

(b) Metabolism

(i) Hydrolysis of esters and acetals

Aromatic esters can be expected to be hydrolysed in vivo by the catalytic activity of carboxylesterases or esterases (Heymann, 1980). B-Esterases, the most important of the group, are active in most mammalian tissue (Heymann, 1980; Anders, 1989) but predominate in hepatocytes (Heymann, 1980). Acetals hydrolyse uncatalysed in gastric juice and intestinal fluids to yield the corresponding aldehydes. In vivo, substituted benzyl esters and benzaldehyde acetals are hydrolysed to the corresponding alcohols, aldehydes, and carboxylic acids.

In 3- to 6-month-old male rabbits, 83% of an oral dose of 100 mg/kg bw of piperonyl acetate (No. 894) and 15% of a aimilar dose of piperonyl isobutyrate (No. 895) were hydrolysed and excreted as either free or conjugated piperonylic acid within 72 h; < 1% of piperonyl alcohol was excreted (Wright & Holder, 1980).

An oral dose of methyl salicylate (No. 899) equivalent to 500 mg/kg bw of salicylic acid was dissolved in 2% methyl cellulose and given to male rats. The plasma concentrations measured within 20 min of dosing showed complete hydrolysis of methyl salicylate. In a similar experiment with three fasted male dogs given 320 mg/kg bw of methyl salicylate in capsules, blood drawn 1 h after dosing showed 95% hydrolysis of methyl salicylate to salicylic acid. In six persons, 79% of a dose of 0.42 ml (approximately 500 mg) of methyl salicylate administered in ginger ale was hydrolysed in the blood within 90 min (Davison et al., 1961).

Several experiments were conducted to study the metabolism of esters of para-hydroxybenzoic acid after oral (1000 mg/kg bw) or intravenous (50 mg/kg bw) administration to dogs. The esters were absorbed in the gastrointestinal tract and rapidly hydrolysed by esterases in the liver and kidney. In the case of butyl para-hydroxybenzoate (No. 870), 48% was recovered after oral and 40% after intravenous administration. Liver preparations from dogs injected with 100 mg/kg bw of the methyl, ethyl, or propyl ester showed 100% hydrolysis within 3 min. In the case of the butyl ester, 100% hydrolysis occurred within 30–60 min (Jones et al., 1956).

Benzyl acetate was rapidly hydrolysed to benzyl alcohol in vitro, with a peak alcohol concentration after 4 min. The absence of benzyl acetate in plasma indicates that benzyl acetate is rapidly hydrolysed to benzyl alcohol, which is then rapidly oxidized to benzoic acid (Yuan et al., 1995). In vitro, 90% of benzyl phenylacetate was hydrolysed within 1 h and 100% within 2 h of incubation with a 2% pancreatin solution (Leegwater & van Straten, 1974).

Acetals of benzaldehyde are also readily hydrolysed. Benzaldehyde propylene glycol acetal was 97% hydrolysed after incubation for 5 h with simulated gastric juice and intestinal fluid in vitro (Morgareidge, 1962).

(ii) Metabolism of alcohols, aldehydes, and other derivatives

In general, hydroxy- and alkoxy- derivatives of benzaldehyde and benzyl alcohol are oxidized to the corresponding benzoic acid derivatives and, to a lesser extent, reduced to the corresponding benzyl alcohol derivatives. The resulting hydroxy- and alkoxy-benzoic acid derivatives form sulfate, glucuronic acid, or glycine conjugates, depending mainly on ring substitution. Hydroxy- and methoxy-substituted benzoic acid derivatives such as vanillic acid tend to form sulfate or glucuronic acid conjugates, while methylenedioxy-substituted benzoic acid derivatives such as piperonylic acid form glycine conjugates. Benzoic acid hydroxy- and alkoxy-derivatives undergo decarboxylation and O-demethylation to a minor extent. Protocatechuic acid is a key intermediate formed by O-demethylation of benzoic acid (Wong & Sourkes, 1966; Strand & Scheline, 1975). Benzyl alcohol derivatives may also be reduced in gut microflora to toluene derivatives, especially if a free para-hydroxyl group is present (Strand & Scheline, 1975; see Figure 1).

FIGURE 1

Figure 1. Metabolism of hydroxy- and alkoxy-substituted benzyl derivatives

In a study of the degradation of some methoxylated aromatic compounds by Actinomyces aureus A-94, anisyl alcohol (No. 871) was oxidized to the corresponding anisic acid, then demethylated and hydroxylated to yield protocatechuic acid (3,4-dihydroxybenzoic acid). Additional enzymatic action converted protocatechuic acid to succinic acid via beta-carboxymuconic acid and beta-oxoadipic acid. In the final stage, succinic acid entered the tricarboxylic acid cycle (Tsai et al., 1965).

Analysis of the medium after incubation of anisyl alcohol (No. 871) with rat caecal extract after approximately 46 h showed the presence of anisic acid. O-Demethylation was not observed (Scheline, 1972).

In the investigation of the metabolism of various aromatic aldehydes and alcohols by rat intestinal microflora, the major metabolites were products of reduction. Microflora-mediated metabolic transformations included reduction, dehydroxylation, O-demethylation, and decarboxylation, leading to a variety of derivatives of benzyl alcohol, benzoic acid, and toluene (Scheline, 1972). After incubation of vanillyl alcohol (No. 886) with rat caecal extract, vanillic acid and the toluene derivatives, 4-methylguaiacol and 4-methylcatechol from complete reduction of the alcohol functional group were present (Scheline, 1972). Analysis of urinary metabolites collected over the first 24 h from male rats given vanillyl alcohol by gavage at a dose of 100 or 300 mg/kg bw showed the presence of vanillic acid and traces of vanillyl alcohol and the glycine conjugate of vanillic acid. Smaller quantities of conjugated fractions of vanillin, guaiacol, catechol, 4-methylguaiacol, and 4-methylcatechol were also found. The presence of catechol and 4-methylcatechol indicated that decarboxy-lation and complete reduction of the alcohol function, respectively, occurred in vivo (Strand & Scheline, 1975).

Rabbits were given 2000 mg/kg bw of veratraldehyde (No. 877) by oral gavage, and urine was collected for 24 h. About 70% of the aldehyde was accounted for in urine, mainly as the corresponding acid veratric acid (28%), and its glucuronic acid conjugate (38%). Veratric acid was decarboxylated to a small extent and O-demethylated to yield catechol (Sammons & Williams, 1946). Presumably, veratric acid enters the enterohepatic circulation where gut microflora decarboxylate it to yield catechol (ortho-hydroxyphenol). Formation of catechol after incubation of veratraldehyde with rat caecal extract indicates that this decarboxylation pathway exists in gut bacteria (Scheline, 1972).

Analysis of the metabolites produced 46 h after incubation of anisaldehyde (No. 878) with rat caecal preparations revealed the presence of anisic acid and anisyl alcohol. Anisaldehyde thus undergoes oxidation and reduction in caecal preparations (Scheline, 1972). In rabbits, about 75% of an oral dose of 2000 mg/kg bw of anisaldehyde was excreted as the glucuronic acid conjugate of para-methoxybenzoic acid (anisic acid) within 24 h (Sammons & Williams, 1946).

In groups of four to six male rats, a single oral dose of 100 or 300 mg/kg bw of vanillin (No. 889) was metabolized and excreted in urine as vanillin, vanillic acid, and vanillyl alcohol within the first 24 h. Small amounts of O-demethylated, decarboxylated, and further reduced metabolites were also identified, which included protocatechuic acid (3,4-dihydroxybenzoic acid, product of O-demethylation), guaiacol (ortho-methoxyphenol, product of decarboxylation), vanilloylglycine, catechol (ortho-hydroxyphenol), 4-methylguaiacol (product of alcohol functional group reduction), and 4-methylcatechol (product of reduction and ring hydroxylation). Only traces of vanillic acid derivatives were detected in urine collected between 24 and 48 h, and no metabolites were detected in urine collected between 48 and 96 h. In a similar experiment in rats, more than 94% of a single oral dose of 100 mg/kg bw of vanillin was excreted in urine within 48 h. Between 65 and 70% of the urinary metabolites were oxidation products (Strand & Scheline, 1975).

Most of a dose of 100 mg/kg bw of vanillin administered by intraperitoneal injection to rabbits was excreted in urine 24 h later. About 69% was oxidized to vanillic acid, and 10% was reduced to vanillyl alcohol. More than 10% was excreted as the glucuronic acid conjugate of vanillin (Sammons & Williams, 1941).

In rats, less than 6% of a dose of 52 mg of 2,4-dihydroxybenzoic acid given by intraperitoneal injection was excreted as the corresponding hippurate (Teuchy et al., 1971). In persons given daily oral doses of up to 6000 mg of 2,4-dihydroxybenzoic acid for up to 16 days, the main urinary metabolites were the glucuronic acid and sulfate conjugates (Clarke et al., 1958).

In albino male Wistar rats, 94% of an oral dose of 150 mg/kg bw of piperonal (No. 896) and 90% of a dose of 150 mg/kg bw of piperonyl alcohol dissolved in propylene glycol were excreted in the urine within 24 h. The main urinary metabolites of both substances were piperonylic acid (17–20%) and the glycine conjugate of piperonylic acid (71–72%). No unchanged compound was excreted, and no metabolites were detected 48 h after dosing. Less than 0.7% of the dose was accounted for by O-demethylenation to protocatechuyl alcohol, protocatechu-aldehyde and protocatechuic acid (Klungsoyr & Scheline, 1984).

In male Swiss-Webster mice, 87–93% of an oral dose of 0.75 mg/kg bw of [methylene-14C]piperonyl alcohol, piperonal, or piperonylic acid administered in dimethyl sulfoxide (50 ΅l followed by a wash with 100 ΅l) was accounted for in the urine within 48 h. Most was eliminated within the first 12 h. Less than 10% was excreted in the faeces. In all cases, the major metabolite was the glycine conjugate of piperonylic acid. Minor amounts of unchanged piperonylic acid were also present (Kamienski & Casida, 1970).

(iii) Summary

Esters of hydroxy- and alkoxy-substituted benzyl derivatives can be expected to be hydrolysed to the corresponding benzyl alcohol and benzoic acid derivatives, while the acetals are hydrolysed to the parent benzaldehyde derivative. Thus formed, the alcohols and aldehydes are oxidized mainly to benzoic acid derivatives, which are either excreted unchanged or form sulfate, glycine, or glucuronic acid conjugates. Some glucuronic acid conjugates may pass into the bile and enter the enterohepatic circulation, where they are hydrolysed or subjected to the reduction reactions of gut bacteria. Small amounts of hydroxy- and alkoxybenzoic acid derivatives have been reported to undergo reductive decarboxylation in the gut. Other minor metabolic detoxication pathways include O-demethylation and ring hydroxylation.

2.3.2 Toxicological studies

(a) Acute toxicity

The LD50 values after oral administration for 38 of the 46 benzyl derivatives ranged from 520 mg/kg bw to 13 000 mg/kg bw in male and female rats, guinea-pigs, mice, and rabbits (Deichmann & Kitzmiller, 1940; Draize et al., 1948; Sokol, 1952; Giroux et al., 1954; Doull et al., 1962; Jenner et al., 1964; Taylor et al., 1964; Hagan et al., 1965; Fogleman & Margolin, 1970; Fujii et al., 1970; Davison et al., 1961; Weir & Wong, 1971; Moreno, 1973; Sado, 1973; Moreno, 1974; Wohl, 1974; Levenstein, 1975; Grady et al., 1976; Moreno, 1976, 1977; BASF, 1981; Givaudan Corp., 1982; Mondino, 1982; Moreno, 1982; Peano & Berruto, 1982; Mallory et al., 1983; Sterner & Chibanguza, 1983; National Toxicology Program, 1984; Ohsumi et al., 1984; Ohta et al., 1984; Reagan & Becci, 1984; Inouye et al., 1988; Buch, 1989; Hasegawa et al., 1989; Cerven, 1990; Dow Chemical Co., 1992; Dufour, 1994; Sanders & Crowther, 1997). The LD50 values of most of the compounds are > 1000 mg/kg bw (see Table 3).

Table 3. Acute toxicity of hydroxy- and alkoxy-substituted benzyl derivatives

Flavouring agent (No.)

Species

Sex

Route

LD50 (mg/kg bw)

Reference

4-Hydroxybenzaldehyde (956)

Rat

 

Oral

4000

Dow Chemical Co. (1992)

 

Mouse

 

Intraperitoneal

500–1000

Doull et al. (1962)

4-Hydroxybenzoic acid(957)

Mouse

 

Oral

2200

Sokol (1952)

 

Mouse

NR

Intraperitoneal

210

Mathews et al. (1956)

2-Hydroxybenzoic acid(958)

Mouse

 

Oral

908

Sado (1973)

 

Rat

 

Gavage

1100

Hasegawa et al. (1989)

Butyl-para-hydroxy-benzoate (870)

Mouse

NR

Oral

13 000

Sado (1973)

 

Mouse

NR

Oral

> 5000

Sokol (1952)

Anisyl alcohol (871)

Mouse

NR

Oral

1800

Draize et al. (1948)

 

Rat

NR

Oral

1300

Draize et al. (1948)

Anisyl formate (872)

Rat

NR

Oral

1800

Levenstein (1975)

Anisyl acetate (873)

Rat

M/F

Oral

2200

Weir & Wong (1971)

Anisyl propionate (874)

Rat

NR

Oral

3300

Wohl (1974)

Anisyl butyrate (875)

Rat

NR

Oral

3400

Moreno (1976)

Anisyl phenylacetate (876)

Rat

M
F

Gavage

5400
4600

Reagan & Becci (1984)

 

Rat

NR

Oral

> 5000

Moreno (1977)

Veratraldehyde (877)

Rat

NR

Oral

2000

Moreno (1974)

para-Methoxybenzaldehyde (878)

Rat

NR

Oral

3200

BASF (1981)

 

Rat

M,F

Gavage

1500

Jenner et al. (1964)

 

Guinea-

M,F

Gavage

1300

Jenner et al. (1964)

 

Rat

M,F

Gavage

1500

Taylor et al. (1964)

para-Ethoxybenzaldehyde (879)

Rat

NR

Oral

2100

Moreno (1977)

Methyl-ortho-methoxy-benzoate (880)

Rat

NR

Oral

3800

Moreno (1982)

Methyl anisate (884)

Rat

NR

Oral

> 5000

Leventstein (1975)

Ethyl para-anisate (885)

Rat

NR

Oral

2200

Levenstein (1975)

Vanillyl alcohol (886)

Mouse

M

Intraperitoneal

> 640

Fujii et al. (1970)

Vanillin (889)

Mouse

M

Gavage

1000

Inouye et al. (1988)

 

Rabbit

NR

Gavage

2600

Deichmann & Kitzmiller (1940)

 

Rat

M,F

Gavage

1600

Taylor et al. (1964)

 

Rat

M,F

Gavage

1600

Jenner et al. (1964)

 

Guinea- pig

M,F

Gavage

1,400

Jenner et al. (1964)

4-Hydroxy-3-methoxy-benzoic acid (959)

Mouse

NR

Intraperitoneal

> 2700

Ohta et al. (1984)

 

Rat

NR

Intraperitoneal

5000

Anon. (????)

Vanillin isobutyrate (891)

Rat

M,F

Gavage

4800

Mallory et al. (1983)

Salicylaldehyde (897)

Rat

NR

Oral

520

Moreno (1977)

2-Hydroxy-4-methyl-benzaldehyde (898)

Rat

M,F

Gavage

1500

Mondino (1982)

 

Rat

M,F

Oral

1500

Peano & Berruto (1982)

Methyl salicylate (899)

Mouse

M

Gavage

1400

Ohsumi et al. (1984)

 

Rat

NR

Gavage

1200

Giroux et al. (1954)

Methyl salicylate (899)

Rat

M
F

Oral

3000
2600

Givaudan Corp. (1982)

 

Rat

M,F

Gavage

890

Jenner et al. (1964)

 

Mouse

M

Oral

1100

Davison et al. (1961)

 

Guinea-pig

M,F

Gavage

1100

Jenner et al. (1964)

 

Mouse

M,F

Gavage

1400

National Toxicology Program (1984)

Ethyl salicylate (900)

Rat

NR

Oral

1300

Moreno (1976)

Butyl salicylate (901)

Rat

NR

Oral

1800

Levenstein (1975)

Isobutyl salicylate (902)

Rat

NR

Oral

1600

Moreno (1973)

Isoamyl salicylate (903)

Rat

NR

Oral

4100

Moreno (1982)

 

Rat

M,F

Oral

> 5000

Givaudan Corp. (1982)

Benzyl salicylate (904)

Rat

M

Gavage

2200

Fogleman & Margolin (1970)

Phenethyl salicylate (905)

Rat

NR

Oral

> 5000

Moreno (1973)

ortho-Tolyl salicylate (907)

Rat

M,F

Oral

1.8 ml/kg

Sterner & Chibanguza (1983)

2,4-Dihydroxybenzoic acid (908)

Rat

NR

Oral

> 800

Grady et al. (1976)

Vanillyl ethyl ether (887)

Rat

M,F

Oral

> 2000

Dufour (1994)

Vanillyl butyl ether (888)

Rat

M
F

Gavage

5100
4700

Buch (1989)

Ethyl vanillin (893)

Rat

M,F

Gavage

> 2000

Jenner et al. (1964)

 

Rabbit

NR

Gavage

2000

Deichmann & Kitzmiller (1940)

Ethyl vanillin isobutyrate (953)

Rat

M,F

Oral

> 2000

Sanders & Crowther (1997)

Piperonyl acetate (894)

Rat

NR

Oral

2100

Moreno (1973)

Piperonal (896)

Rat

M,F

Gavage

2700

Jenner et al. (1964)

 

Rat

M,F

Gavage

2700

Taylor et al. (1964)

 

Rat

M,F

Gavage

2700

Hagan et al. (1965)

Ethyl vanillin beta-d-gluco-pyranoside (892)

Rat

M,F

Oral

> 5000

Cerven (1990)

M, male; F, female; NR, not reported

(b) Short-term studies of toxicity

Short-term studies of toxicity have been performed with eight of the substances in this group, but most with butyl para-hydoxybenzoate (No. 870), commonly known as butyl paraben, vanillin (No. 889), methyl salicylate (No. 899), ethyl vanillin (No. 893), and piperonal (No. 896). Each of these four substances has been allocated an ADI. The studies with ethyl vanillin were reviewed comprehensively by the Committee at its thirty-fifth and forty-sixth meetings (Annex 1, references 89 and 117), and the results of that review are included below so as to present all relevant data related to the safety of structurally related hydroxy- and alkoxy-benzaldehyde derivatives.

Mice

Butyl-para-hydroxybenzoate (No. 870)

Groups of 10 male and 10 female 8-week-old ICR/jcl mice were maintained on diets containing pelletized butyl-para-hydroxybenzoate at concentrations calculated to provide an average intake of 900, 1900, 3800, 7500, or 15 000 mg/kg bw per day for 6 weeks. Twenty male and 20 female mice of the same age and strain were used as controls and fed the basal diet. Survival, body-weight gain, and histolological end-points were measured. All the animals given 7500 or 15 000 mg/kg bw per day died within the first 2 weeks of treatment. The body-weight gain of those given 1900 or 3800 mg/kg bw per day was less than 10% of that of the control group, whereas the animals at the lowest concentration showed weight gain similar to that of controls. Histological examination showed atrophy of lymphoid tissue and liver degeneration and necrosis in all groups except those at the lowest dose (Inai et al., 1985).

Rats

Butyl-para-hydroxybenzoate (No. 870)

Butyl-para-hydroxybenzoate was dissolved in soya bean oil at a concentration of 100 mg/0.5 ml and administered by oral intubation to rats for 13–15 weeks at concentrations calculated to result in a daily intake of 0, 0.25, or 50 mg/kg bw. Body weight, measured twice weekly, showed no significant difference from controls. Some animals were killed on a predetermined schedule for histological evaluation. There were no sporadic deaths and no significant histological differences from controls. The NOEL was 50 mg/kg bw per day (Ikeda & Yokoi, 1950).

Groups of 12 Wistar rats of each sex were fed a powdered mixture of butyl-para-hydroxybenzoate and dog chow providing a dose of 0, 2000, or 8000 mg/kg bw per day for 12 weeks. Body weight and food intake were measured every 2 weeks, and necropsy and histological examinations were performed at the end of the experiment. Animals found dead before the end of the study were necropsied and the appropriate tissues were fixed for histopathological evaluation. Although there were no effects at the low dose, all the males and many of the females at the high dose died within several weeks of the beginning of treatment. The body weights and motor activity of these animals were decreased, and they had a slower growth rate than controls. The NOEL was 2000 mg/kg bw per day (Matthews et al., 1956).

para-Methoxybenzaldehyde (No. 878) and piperonal (No. 896)

A group of 10 male and 10 female weanling rats received a diet containing a mixture of eugenol (90 mg/kg bw), para-methoxybenzaldehyde (7.3 mg/kg bw), and piperonal (16 mg/kg bw) at a concentration calculated to provide an average daily intake of 110 mg/kg bw for 90 days. Food intake and growth were measured each week. After 12 weeks on the test material, sugar, albumin, and haemoglobin concentrations were measured in the urine of three male and three female animals. No sugar was found; although the urine of male rats contained albumin, the authors considered the finding pathologically insignificant. All surviving animals were killed at 90 days and necropsied, and organs were weighed. Weekly measurements of body-weight gain, food intake, haematological and clinical chemical end-points, and gross and histological examinations showed no significant difference between test and control animals. The NOEL was 7.3 mg/kg bw per day for para-methoxy-benzaldehyde (No. 878) and 16 mg/kg bw per day for piperonal (No. 896) (Trubeck Laboratories Inc., 1958).

Groups of five male and five female 8-week-old rats were fed diets containing para-methoxybenzaldehyde (No. 878) and piperonal (No. 896) at a concentration providing 500 mg/kg bw per day for 16 weeks, and groups of five male and five female 4-week-old rats were fed the same substances at 50 mg/kg bw per day for 28 weeks. One control group of 20 rats was used for each experiment. No gross pathological changes were seen in any group. The NOEL was 500 mg/kg bw per day for both para-methoxybenzaldehyde and piperonal (Food & Drug Administration, 1954).

Groups of five male and five female weanling Osborne-Mendel rats were given diets containing para-methoxybenzaldehyde (No. 878) or piperonal (No. 896) at a concentration providing 50 mg/kg bw per day for 27–28 weeks or 500 mg/kg bw per day for 15 weeks. Ten male and 10 female rats were used as controls. Body weight, food intake, and general condition were noted weekly, and haematological examinations were performed at the end of the study. No effects were seen at either dose. The NOEL was 500 mg/kg bw per day for both para-methoxybenzaldehyde and piperonal (Hagan et al., 1967).

Weanling Osborne-Mendel rats (number per group unspecified) were fed a diet containing methyl salicylate at concentrations calculated to provide an average daily intake of 500 mg/kg bw for 16 weeks and 50 mg/kg bw for 28 weeks. No adverse effects were observed at either dose (Hagan et al., 1965).

Methyl-ortho-methoxybenzoate (No. 880)

Groups of five male and five female Fischer 344 rats, 28 days old, were maintained on diets containing methyl ortho-methoxybenzoate in corn oil at a concentration calculated to provide an average daily intake of 0 or 94 mg/kg bw for 14 days. The animals were observed for deaths twice a day, and body weight and food consumption were measured weekly. No significant differences were found between test and control groups. At necropsy, the liver and kidney were weighed and examined histologically. The only significant result was a decrease in relative kidney weight in males, but the absolute liver and kidney weights were unchanged in treated animals, and no kidney lesions were found; the authors concluded that the change was not related to treatment. The NOEL was 94 mg/kg bw per day (Van Miller & Weaver, 1987).

Vanillin (No. 889) and ethyl vanillin (No. 893)

The protocols used by the Food & Drug Administration and by Hagan et al. to study para-methoxybenzaldehyde and piperonal were also used in studies in which Osborne-Mendel rats were maintained on a diet containing vanillin at a concentration calculated to provide an average daily intake of 500 mg/kg bw for 16 weeks. In the study by Hagan et al., an additional group of 10 rats was given 50 mg/kg bw per day of vanillin for 28 weeks. No effects were seen at either dose in either study (Food & Drug Administration, 1954; Hagan et al., 1967).

Vanillin or ethyl vanillin was dissolved in corn oil and added to the diet of five male weanling Osborne-Mendel rats at concentrations calculated to provide an average daily intake of 1000 or 2500 mg/kg bw for 1 year. Ten male and 10 female rats were fed a diet containing 3% corn oil as a control. Weekly measurements of body weight and food intake and observations of general condition showed no differences between test and control groups. No differences in haematological parameters were seen at necropsy. The NOEL for vanillin and ethyl vanillin was 2500 mg/kg bw per day (Hagan et al., 1967).

Four groups of eight young albino rats were fed vanillin or ethyl vanillin as a 4% solution in milk at an estimated daily intake of either 20 mg/kg bw for 126 days or 64 mg/kg bw for 70 days. In the 70-day study, half the animals were killed and the other half were put on a recovery diet for 8 more weeks. Additionally, 12 rats were given a dose of 300 mg/kg bw of vanillin or ethyl vanillin as a 4% solution in olive oil orally by gavage twice per week for 14 weeks. Observation of appearance, behaviour, and body-weight gain showed a reduced growth rate and myocardial, renal, hepatic, lung, spleen, and stomach injuries at the dose of 64 mg/kg bw (nature not specified) (Deichmann & Kitzmiller, 1940).

Groups of 20 male and 20 female CD Sprague-Dawley rats were given ethyl vanillin at a dose of 0, 500, 1000, or 2000 mg/kg bw per day in the diet for 13 weeks. Food consumption and body weight were recorded weekly; ophthalmoscopy was performed before treatment and at the end of the study; detailed haematological and clinical chemical examinations were carried out at weeks 6 and 13. At termination, all the animals were necropsied and their organs were weighed. A complete histological examination was performed on controls and rats at the highest dose and was extended to animals at the the low and intermediate doses when treatment-related effects were suspected.

Some differences in food intake and body-weight gain were found in comparison with controls, but the authors considered these to be unrelated to treatment because their severity was not dose-related and intragroup variation in feeding patterns was seen among male rats. No treatment-related differences in haematological parameters were seen at week 6 or at termination.

At autopsy, enlarged cervical lymph nodes were seen in males at the intermediate dose and in animals of each sex at the highest dose. In addition, rats of each sex at the highest dose showed a reduction in adipose tissue. The absolute liver weights were similar to those of controls, but the relative liver weights were increased in rats at the intermediate and high doses. An increased incidence of hepatic peribiliary inflammatory change was seen in males and females at the two higher doses and minor bile-duct hyperplasia in a few males at these doses. No changes were observed in the liver parenchyma and no degenerative or inflammatory changes of the bile-duct epithelium were found. The NOEL was 500 mg/kg bw per day (Hooks et al., 1992).

Methyl salicylate (No. 899)

A series of studies was performed by Abbott & Harrisson (1978) to evaluate the effect of methyl salicylate on bone density. Groups of five male and five female rats were maintained on diets containing methyl salicylate at concentrations calculated to provide an average daily intake of 0, 100, 180, 320, 560, or 1000 mg/kg bw for 11 weeks. The groups received the substance at 50% of the final dose for the first 2 weeks, 75% for the third and fourth weeks, and 100% thereafter. Body weight and food consumption were measured weekly. Males at 320 mg/kg bw per day showed decreased weight gain when compared with controls, and males and females at 560 and 1000 mg/kg bw per day showed both decreased weight gain and increased bone density at the metaphyses of the femur, humerus, tibia, and radius. No such changes were observed at lower doses.

In a 12-week study, two groups of five male rats were fed diets containing methyl salicylate at concentrations calculated to provide an average daily intake of 300 or 1000 mg/kg bw. No control animals were used. All the animals at the high dose died during the first 6 weeks of the study, and were found to have bone lesions on full-body X-rays. All animals at the low dose survived to the end of the study, and none had the bone lesions observed at the high dose.

In a second 11-week study to re-evaluate the effects of a group of structurally related substances on bone density, methyl salicylate was administered in the diet to provide a dose of 1000 mg/kg bw per day to 15 rats. Twenty rats served as controls. Whole body X-rays were performed periodically, and body weights were checked. The mortality rate in the experimental group was 20%, whereas all the control animals survived. Changes in bone density similar to those seen in the first study were reported with both methyl salicylate and acetylsalicylic acid, indicating that the bone changes were associated with high doses of ortho-hydroxybenzoic acid derivatives (salicylates).

In a 6-week study, animals were fed a diet ad libitum containing methyl salicylate estimated to provide an average daily intake of 0, 300, or 1000 mg/kg bw. Ten paired feeding groups were given diets providing methyl salicylate at 300 mg/kg bw per day, methyl para-hydroxybenzoate at 1000 mg/kg bw per day, or no treatment. All pair-fed animals received daily rations ad libitum, equal to the mean daily amount of food consumed by the group given 1000 mg/kg bw per day methyl salicylate. The group given 300 mg/kg bw per day had a slightly lower growth rate than the controls, whereas the changes in body weight of the pair-fed groups were similar to those of rats given methyl salicylate at 1000 mg/kg bw per day ad libitum. Deaths occurred among rats at the high dose of methyl salicylate ad libitum and in all members (including the control group) of the pair-fed groups.

A study was undertaken to evaluate the progression of bone change, in which groups of 10 male and 10 female rats were fed diets providing a dose of methyl salicylate of 300, 450, 600, or 1000 mg/kg bw per day for 11 weeks. A fifth group was fed the basic diet and used as controls. Two animals per group were X-rayed weekly and killed a week later until the end of the experiment. The femurs of certain animals were examined histologically. The group at the highest dose began showing bone changes at week 2, and those at 600 mg/kg bw per day group began showing signs at week 5. The X-rays of the remaining groups were normal throughout the 11-week study. Histological analysis showed an increased incidence of cancellous bone in rats at the high dose at week 2 and in those at 600 mg/kg bw per day by week 8. The NOEL was 450 mg/kg bw per day.

Two groups of 10 male and 10 female rats were maintained on a diet designed to result in an average daily intake of 0, 50 or 500 mg/kg bw of methyl salicylate for 17 weeks. Weekly measurements of body weight and of organ weights at necropsy and the results of gross and microscopic examinations showed no adverse effects (Webb & Hansen, 1963).

Three male and three female rats were fed a diet containing methyl salicylate at a concentration resulting in a dose of 0 or 1000 mg/kg bw per day. As each experimental animal died, one control animal was killed at the same time until all of the animals were dead. The first male died at 11 days and two more at 19 days. The first female died at 31 days, the second at 40 days, and the third at 71 days. The last animal to die was examined by X-rays and microscopically. The treatment was not only lethal but increased the bone density in the metaphysis of all bones and induced laboured respiration, and gastric haemorrhages in the glandular stomach (Webb & Hansen, 1963). The effect of salicylates on bone density was also reported in an abstract (Harrisson et al., 1963).

Isoamyl salicylate (No. 903)

Isoamyl salicylate was incorporated in the diet of groups of 15 male and 15 female Wistar rats at a concentration of 0, 50, 500, or 5000 mg/kg for 13 weeks. On the basis of data on body weight and food intake, the authors calculated mean intakes of 0, 4.7, 46, and 420 mg/kg bw per day for males and 0, 4.8, 47, and 480 mg/kg bw per day for females throughout treatment. An additional group of five females and five males received only the two higher doses for 2 or 6 weeks. The animals were weighed on days 1, 2, 6, 9, and 13 and then weekly until day 91. Food and water consumption was measured for 24 h before weighing, and urine was collected during the last 2 days of treatment and analysed. At necropsy, organs were weighed and haematological and histopathological examinations were performed. Rat at the highest dose showed increased relative kidney weights, and males at 25 mg/kg bw per day had increased relative and absolute kidney weights, although with no associated histopathological changes. Decreased weight gain and food intake were observed at the highest dose. In a separate paired-feeding study in which two groups of 10 rats were fed either 0 or the highest dose of methyl salicylate for 98 days, decreases in body weight and food consumption were considered by the authors to be due to poor palatability of the diet. The NOEL was 4.7 mg/kg bw per day (Drake et al., 1975).

Rabbits

Vanillin (No. 889) and ethyl vanillin (No. 893)

Vanillin and ethyl vanillin were given as a solution in milk to one rabbit at 240 mg/kg bw per day for 56 days and to two rabbits at the same dose for 126 days. Both substances were also administered as a solution in 10% glycerol, vanillin at a dose of 83 mg/kg bw per day for 14 days or 103 mg/kg bw per day for 61 days and ethyl vanillin at a dose of 15 mg/kg bw per day for 15 days, 15 mg/kg bw per day for 31 days, 32 mg/kg bw per day for 17 days, 41 mg/kg bw per day for 31 days, or 49 mg/kg bw per day for 49 days.

Appearance, behaviour, and body-weight gain were not significantly affected. There were no gross or histopathological alterations in test animals. In rabbits receiving the substances in glycerol solution, anaemia, diarrhoea, and lack of weight gain were observed at the highest dose; no toxic effects were seen at lower doses. Glycerol poisoning, evidenced by restlessness, tremor, convulsions, and coma, was observed in rabbits given 83 mg/kg bw per day of vanillin for 14 days and in those given 15 mg/kg bw per day of ethyl vanillin for 15 days (Deichmann & Kitzmiller, 1940).

Dogs

Methyl salicylate (No. 899)

Four groups of three male and three female purebred beagles were given capsules containing methyl salicylate at a dose of 150, 300, 500, or 800 mg/kg bw per day for 7.5 months. The dogs were given one-half of the dose at the morning feed and the other half in the afternoon for 6 days/week. Two males and four females were fed the same diet without methyl salicylate and served as a controls. After 6.5 months, three dogs at 300 mg/kg bw per day, two at 150 mg/kg bw per day, and all the control animals were killed, and three at 300 mg/kg bw per day were put on a recovery diet. After 7 months, all the remaining animals were killed, except those returned to the normal diet, which were killed after 8 months.

Body weight was measured weekly and haematological examinations were performed at the fifth month on animals at the two lower doses. After sacrifice, the organs were weighed, and gross and histological examinations were performed. All the animals at the highest dose were dead by the second week. Of the groups at 500 mg/kg bw per day, only two survived the duration of the experiment, while the rest died at weeks 2, 3, 5, and 8. The body weights of the groups at the two lower doses were not significantly different from those of controls, and haematological examinations showed normal values. Increased liver and kidney weights were seen at all doses but not in the animals placed on the recovery diet.

In a further experiment with the same protocol, dogs were fed capsules containing methyl salicylate at a dose of 50 (four males, four females), 100, or 170 mg/kg bw per day (six males, six females). All animals were killed at 6 months, except for two animals of each sex at the highest dose and four controls, which were placed on a recovery diet for 2 months. The same measurements were made as in the preceding study, except that only liver and kidney weights were assessed. There were no adverse effects at any dose. The NOEL was 170 mg/kg bw per day (Abbott & Harrisson, 1978).

Six groups of one male and one female dog were given a capsule containing methyl salicylate at a dose of 0, 50, 100, 250, 500, 800, or 1200 mg/kg bw per day on 6 days/week for up to 59 days. The three animals at the highest dose were either killed in extremis or died within the first month of the experiment. At necropsy, one dog at 800 mg/kg bw per day and both at 250 mg/kg bw per day were examined grossly and microscopically. The dogs at the higher dose vomited 3–4 h after each dose throughout the study. Those at 500 mg/kg bw per day had diarrhoea and weakness during the last 3–4 days of the study. Both animals at the highest dose and one at 800 mg/kg bw per day showed marked fatty metamorphosis in the liver (Webb & Hansen, 1963).

(c) Long-term studies of toxicity and carcinogenicity

Seven long-term studies of toxicity were performed with five of the flavouring agents in this group. Two 2-year studies were conducted on methyl salicylate (No. 899) in rats and one in dogs (Packman et al., 1961; Webb & Hansen, 1963).

Mice

Butyl-para-hydroxybenzoate (No. 870)

Three groups of 50 male and 50 female ICR/jcl mice, 8 weeks old, were fed pellets containing butyl-para-hydroxybenzoate at a dose of 0, 225, 450, or 900 mg/kg bw per day for 102 weeks. Intake was measured once a week for the first 30 weeks, once every other week for the following 20 weeks, and then once every 4 weeks until the end of the experiment. Body weight was measured once a week for the first 6 weeks, once every other week for the next 24 weeks, and then every 4 weeks for the duration of the experiment. All animals found dead during the experiment were necropsied, and all those that were still alive at the end of the experiment were killed and necropsied at week 106. The tissues of all animals, regardless of time of death, were examined histologically. The tumour incidence in the experimental groups was not significantly different from that in the control group. The NOEL was 900 mg/kg bw per day (Inai et al., 1985).

Rats

Vanillin (No. 889) and Ethyl vanillin (No. 893)

Vanillin or ethyl vanillin dissolved in propylene glycol was added to the diet of groups of 12 male and 12 female rats at a concentration estimated to provide an average daily intake of 250, 500, or 1000 mg/kg bw, for 2 years. Twenty control rats were fed 3% propylene glycol. Weekly measurements of body weight and food intake and observations of general condition failed to show any differences between test and control groups. Haematological examinations at necropsy showed no effects in any of the animals at any concentration. The NOEL for vanillin and ethyl vanillin was 1000 mg/kg bw per day (Hagan et al., 1967).

Piperonal (No. 896)

Piperonal was incorporated in the diet of male and female rats at a concentration of 0.5 or 1.1% for 2 years. The appearance, behaviour, and weight of the animals under study were noted, and histological examinations were conducted. The authors reported that no specific results were obtained (Bär & Griepentrog, 1967). As the report of this study that was available was incomplete and difficult to read, these data were not considered in detail.

Methyl salicylate (No. 899)

Groups of 25 male and 25 female rats were fed a diet calculated to provide a dose of methyl salicylate of 0, 50, 250, 500, or 1000 (24 males, 26 females) mg/kg bw per day for 2 years. The diet was prepared every 2 weeks, and an additional 10% methyl salicylate was added to each mix in order to account for evaporation. The animals were weighed weekly. Haematological examinations performed at 3, 11, 17, and 22 months on 10 rats from each group showed normal values. Animals in the control group and at the two higher doses were examined histologically. Gross lesions, organ weights, bones, and muscles were assessed upon termination in each group. Half of the rats at the highest dose died after week 8. Only five rats survived past week 20 and one past week 35, finally dying at week 49. Animals at 500 mg/kg bw per day had increased absolute weights of the testes, heart, and kidney and an increased amount of cancellous bone in the metaphysis. Ten animals at 250 mg/kg bw per day showed gross pituitary lesions, and one male and two females had malignant pituitary tumours. The NOEL was 50 mg/kg bw per day (Webb & Hansen, 1963).

Fifty weanling albino rats equally divided by sex were maintained on a dry diet containing methyl salicylate at concentrations calculated to result in an average daily intake of 0, 35, or 100 mg/kg bw for 2 years. Regular evaluation of growth, survival, food consumption, general physical condition, blood and urine parameters, and necropsy revealed no adverse effects associated with administration of the test substance. The NOEL was 100 mg/kg bw per day (Packman et al., 1961). However, these results were reported only in an abstract.

Dogs

Methyl salicylate (No. 899)

Groups of two male and two female beagles were given a capsule containing methyl salicylate at a dose of 0, 50, 150, or 350 mg/kg bw per day on 6 days/week for 2 years. The animals were weighed weekly, and haematological end-points were measured three times before initiation of the experiment and at 2 weeks, 1, 3, and 6 months, 1 year, and 2 years. Microscopy was performed on the three surviving dogs at the highest dose. Organs were weighed at necropsy, and selected tissues were examined histologically. Apart from two animals that died of unrelated diseases, no deaths occurred during the study, and all of the haematological values were normal. The groups at the two higher doses had enlarged livers and enlarged hepatic cells. Animals at 150 mg/kg bw per day showed growth retardation. The NOEL was 50 mg/kg bw per day (Webb & Hansen, 1963).

The results of short- and long-term studies are summarized in Table 4.

Table 4. Results of short-term studies of toxicity with hydroxy- and alkoxy-substituted benzyl derivatives

Flavouring agent (No.)

Species, sex

No. of test groupsa/no. per groupb

Route

Length(days)

NOEL (mg/kg bw per day)

Reference

Butyl-para-hydroxybenzoate (870)

Mouse, M,F

5/20

Oral

6 weeks

900

Inai et al. (1985)

 

Rat

4/5 or 4/9

Gavage

13–15 weeks

50d

Ikeda & Yokoi (1950)

 

Mouse, M,F

3/100

Oral

102 weeks

900d

Inai et al. (1985)

 

Rat, M,F

2/24

Oral

12 weeks

2000

Matthews et al. (1956)

para-Methoxybenzaldehyde (878)

Rat, M,F

1/20

Oral

12 weeks

7.3d,e

Trubeck Laboratories Inc. (1958)

 

Rat, M,F

1/10

Oral

16 weeksf

500

Food & Drug Administration (1954)

 

Rat, M,F

1/10

Oral

28 weeksf

50d

Food & Drug Administration (1954)

 

Rat, M,F

1/10

Oral

27–28 weeks

50d

Hagan et al. (1967)

 

Rat, M,F

1/10

Oral

15 weeks

500d

Hagan et al. (1967)

Methyl-ortho-methoxybenzoate (880)

Rat, M,F

1/10

Oral

14 days

94d

Van Miller & Weaver (1987)

Vanillin (889)

Rat, M,F

1/10

Oral

27–28 weeks

50d

Hagan et al. (1967)

 

Rat, M

2/5

Oral

1 year

2500d

Hagan et al. (1967)

 

Rat, M,F

1/10

Oral

16 weeks

500d

Hagan et al. (1967)

 

Rat, M,F

3/24

Oral

2 years

1000d

Hagan et al. (1967)

 

Rat, M,F

1/10

Oral

28 weeksf

50d

Food & Drug Administration (1954)

 

Rat, M,F

1/10

Oral

16 weeksf

500d

Food & Drug Administration (1954)

 

Rat

2/8

Oral

70 days

64d

Deichmann & Kitzmiller (1940)

 

Rat

2/8

Oral

126 daysg

20d

Deichmann & Kitzmiller (1940)

 

Rabbit

1/3

Oral

56 or 126 days

240d

Deichmann & Kitzmiller (1940)

 

Rat

1/6

Gavage

14 weeks

300d,h 

Deichmann & Kitzmiller (1940)

 

Rabbit

2/1

Oral

14 or 61 days

83d

Deichmann & Kitzmiller (1940)

Methyl salicylate (899)

Rat, M,F

1/6

Oral

< 71 days

< 1000i

Webb & Hansen (1963)

 

Rat, M

3/10

Oral

6 weeks

< 300

Abbott & Harrisson (1978)

 

Rat, M,F

4/20

Oral

11 weeks

450

Abbott & Harrisson (1978)

Methyl salicylate (899)

Rat, M

2/5

Oral

12 weeks

300

Abbott & Harrisson (1978)

 

Rat, M,F

2/20

Oral

17 weeks

500d

Webb & Hansen (1963)

 

Dog, M,F

6/2

Oral

59 days

250

Webb & Hansen (1963)

 

Rat, M,F

1/15

Oral

11 weeks

< 1000

Abbott & Harrisson (1978)

 

Dog, M,F

4/6

Oral

7.5 months

< 150

Abbott & Harrisson (1978)

 

Dog, M,F

2/8m
1/12

Oral

6 monthsn 

1700

Abbott & Harrisson (1978)

 

Rat, M,F

5/10

Oral

11 weeks

180

Abbott & Harrisson (1978)

 

Rat

NR

Oral

10 weeks

< 560

Harrisson et al. (1963)

 

Rat, M,F

4/50

Oral

2 years

50

Webb & Hansen (1963)

 

Dog, M,F

3/4

Oral

2 years

50

Webb & Hansen (1963)

 

Rat, M,F

2/50

Oral

2 years

100d

Packman et al. (1961)

 

Mouse, M,F

3/40

Gavage

18 weeks

250

National Toxicology Program (1984)

Isoamyl salicylate (903)

Rat, M,F

3/30

Oral

13 weeks

4.7

Drake et al. (1975)

Ethyl vanillin (893)

Rat, M

2/5

Oral

1 year

2500d

Hagan et al. (1967)

 

Rat, M,F

3/24

Oral

2 years

1000d

Hagan et al. (1967)

 

Rat, M,F

3/40

Oral

13 weeks

500

Hooks et al. (1992)

 

Rabbit

1/3

Oral

56 or 126 days

240d

Deichmann & Kitzmiller (1940)

 

Rat

2/8

Oral

70 daysg

64d

Deichmann & Kitzmiller (1940)

 

Rat

2/8

Oral

126 daysg

20d

Deichmann & Kitzmiller (1940)

 

Rat

1/6

Gavage

14 weeks

300d

Deichmann & Kitzmiller (1940)

 

Rabbit

5/1

Oral

15–49 days

41d

Deichmann & Kitzmiller (1940)

Piperonal (896)

Rat, M,F

1/NR

Oral

28 weeks

50d

Hagan et al. (1965)

 

Rat, M,F

1/NR

Oral

16 weeks

500d

Hagan et al. (1965)

 

Rat, M,F

1/10

Oral

15 weeks

500d

Hagan et al. (1967)

Piperonal (896)

Rat, M,F

1/10

Oral

27–28 weeks

50d

Hagan et al. (1967)

 

Rat, M,F

1/20

Oral

12 weeks

16d,e

Trubeck Laboratories Inc. (1958)

 

Rat, M,F

1/10

Oral

16 weeksf

500d

Food & Drug Administration (1954)

 

Rat, M,F

1/10

Oral

28 weeksf

50d

Food & Drug Administration (1954)

 

Rat, M,F

1/20
1/60

Oral

1.5–2 years

2500

Bar & Griepentrog (1967)

M, male; F, female; NR, not reported

a

Does not include control groups

b

Both male and female animals

c

Five rats per group at low dose; nine rats per group at high dose

d

As either a single or multiple doses had no adverse effects, this dose is not a true NOEL but the highest dose tested that had no adverse effects. The actual NOEL would be higher.

e

Rats fed a mixture containing 123 mg/kg bw eugenol, 10 mg/kg bw para-methoxybenzaldehyde, and 22 mg/kg bw piperonal daily

f.

Rats fed either 0.1% for 28 weeks or 1.0% for 16 weeks

g

Low dose for 126 days; high dose for 70 days with an 8-week recovery period for half of the animals

h

Compound administered twice per week

i

As a single dose had adverse effects, the actual NOEL would be lower.

j

Two groups fed 2.0% and 0.6%ad libitum and one group pair fed 0.6%

k

Rats pair-fed 0.6% showed adverse effects, but those fed ad libitum did not.

l

Two animals at 150 mg/kg bw per day and three at 300 mg/kg bw per day killed after 6.5 months, and three animals at 300 mg/kg bw per day discontinued treatment at 6.5 months and recovered for 1.5 months before being killed.

m

Eight dogs at 50 and 100 mg/kg bw per day and 12 dogs at 167 mg/kg bw per day

n

All animals treated for 6 months and then killed, except for two dogs at the high dose treated for 4 months, placed on control diet for 2 months, and killed with the other animals at 6 months

(d) Genotoxicity

Studies of the genotoxicity of these compounds are summarized in Table 5.

Table 5. Results of studies of the genotoxicity of hydroxy- and alkoxy-substituted benzyl derivatives

No.

Flavouring agent

End-point

Test system

Concentration

Results

Reference

In vitro

870

Butyl-para-hydroxy- benzoate

Chromosomal aberration

Chinese hamster fibroblasts

60 mg/ml

Negativeb

Ishidate et al. (1984)

 

 

Reverse mutation (preincubation)

S. typhimurium TA92, TA1535, TA100, TA1537, TA94, TA98, TA2637

1000 mg/platec

Negativeb

Ishidate et al. (1984)

 

 

Reverse mutation

S. typhimurium TA98, TA100

< 1000 mg/plate

Negativeb

Haresaku et al. (1985)

871

Anisyl alcohol

Reverse mutation (plate incorporation)

S. typhimurium TA100

> 500 mg/plate

Negative

Ball et al. (1984)

877

Veratraldehyde

Reverse mutation

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

8000 mg/plate

Negativeb

Nestmann et al. (1980)

 

 

Reverse mutation

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

8000 mg/plate

Negativeb

Douglas et al. (1980)

 

 

Mutation

Saccharomyces cerevisiae D7, XV185-14C

Not reported

Negativec

Nestmann & Lee (1983)

 

 

Reverse mutation (preincubation)

S. typhimurium TA1535, TA98, TA100, TA97, TA1537

> 6666 mg/plate

Negativeb

Mortelmans et al. (1986)

 

 

Reverse mutation

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

1000 mg/plate

Negativeb

Heck et al. (1989)

 

 

Forward mutation

Mouse lymphoma L5178Y cells

1400 mg/mla

Positiveb

Heck et al. (1989)

 

 

Reverse mutation (preincubation)

S. typhimurium TA100, TA102, TA104, TA1538, TA982

Not reported

Negativeb

Dillon et al. (1992)

 

 

Reverse mutation (preincubation)

S. typhimurium TA100, TA102, TA104

33–3300 mg/plate

Negativeb

Dillon et al. (1998)

 

 

Unscheduled DNA synthesis

Rat hepatocytes

100 mg/mla

Negative

Heck et al. (1989)

878

para-Methoxybenzaldehyde

Reverse mutation (preincubation)

S. typhimurium TA92, TA1535, TA100, TA1537, TA94, TA98, TA2637

5000 mg/platea

Negativeb

Ishidate et al. (1984)

 

 

Reverse mutation

S. typhimurium TA98, TA100

> 500 mg/plate

Negativeb

Kasamaki et al. (1982)

 

 

Chromosomal aberration

Chinese hamster fibroblasts

500 mg/mla

Negativec

Ishidate et al. (1984)

 

 

Reverse mutation (preincubation)

S. typhimurium TA102, TA97

> 1000 mg/plate

Negativeb

Fujita & Sasaki (1987)

 

 

Mutation

B. subtilis H17, M45

22 mg/disc

Negativec

Oda et al. (1979)

 

 

Reverse mutation

S. typhimurium TA102

5000 mg/plate

Negativeb

Müller et al. (1993)

 

 

Reverse mutation

S. typhimurium TA100

> 1000 mg/plate

Negative

Rapson et al. (1980)

 

 

Forward mutation

Mouse lymphoma L5178Y cells

> 470 mg/ml
540–780 mg/ml

Negative
Positivec

Wangenheim & Bolscfoldi (1988)

 

 

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537

410 mg/plate

Negativeb

Florin et al. (1980)

 

 

Chromosomal aberration

Chinese hamster B241 cell line

0.0068 mg/ml

Positiveb

Kasamaki et al. (1982)

 

 

Mutation

Phage PM2

1400 mg/ml

Negative

Becker et al. (1996)

 

 

Sister chromatid exchange

Human lymphocytes

> 270

 

 

 

 

DNA alkaline

unwinding Mouse lymphoma L5178Y/TK+/– cells

> 820 mg/ml 960–1100 mg/ml

Negativeb
Positiveb

Garberg et al. (1988)

 

 

Sister chromatid exchange

Chinese hamster ovary K-1 cells

> 14 mg/ml

Negative

Sasaki et al. (1987)

879

para-Ethoxybenzaldehyde

Reverse mutation

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

3600 mg/plate

Negativeb

Wild et al. (1983)

884

Methyl anisate

Mutation

Escherichia coli Sd-4-73

Not reported

Negativec

Szybalski (1958)

886

Vanillyl alcohol

SOS DNA repair

Escherichia coli PQ37

Not reported

Positivec

Ohshima et al. (1989)

889

Vanillin

Reverse mutation

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

10 000 mg/platea

Negativeb

Heck et al. (1989)

 

 

Mutation

B. subtilis H17, M45

21 mg/disc

Negativec

Oda et al. (1979)

 

 

Chromosomal aberration

Chinese hamster fibroblasts

1000 mg/ml

Negativec

Ishidate et al. (1984)

 

 

Reverse mutation

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

5000 mg/plate

Negativeb

Pool & Lin (1982)

 

 

Reverse mutation (preincubation)

S. typhimurium TA1535, TA98, TA100, TA97, TA1537

> 10 000 mg/plate

Negativeb

Mortelmans et al. (1986)

 

 

Mutation

Escherichia coli Sd-4-73

Not reported

Negativec

Szybalski (1958)

 

 

Reverse mutation

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

Not reported

Negativeb

Nagabhushan & Bhide (1985)

 

 

Reverse mutation

S. typhimurium TA92, TA1535, TA100, TA1537, TA94, TA98, TA2637

10 000 mg/platea

Negativeb

Ishidate et al. (1984)

 

 

Reverse mutation

S. typhimurium TA100

> 1000 mg/plate

Negative

Rapson et al. (1980)

 

 

Forward mutation

Mouse lymphoma L5178Y cells

> 1500 mg/mla

Negativeb

Heck et al. (1989)

 

 

Mutation

Escherichia coli CSH26/pYM3, CSH26/pSK1002

> 15 000 mg/ml

Negative

Takahashi et al. (1990)

 

 

Reverse mutation

S. typhimurium TA98, TA100

> 1000 mg/plate

Negativeb

Kasamaki et al. (1982)

 

 

Chromosomal aberration

Chinese hamster B241 cells

> 0.006 mg/ml

Negative

Kasamaki & Urasawa (1985)

 

 

Sister chromatid exchange

Human lymphocytes

0–150 mg/ml

Positive

Jansson et al. (1986)

 

 

Mitotic gene conversion

S. cerevisiae

10 000 mg/ml

Negative

Rosin (1984)

 

 

Chromosomal aberration

Chinese hamster V79 lung cells

15 000–150 000 mg
300 000 mg

Negativec

Positivec

Tamai et al. (1992)

 

 

Chromosomal aberration

Human lymphocytes

> 610 mg/ml

Negative

Jansson & Zech (1987)

 

 

Chromosomal aberration

Chinese hamster B241 cell line

0.003 mg/ml

Negative

Kasamaki et al. (1982)

 

 

Sister chromatid exchange

Chinese hamster ovary K-1 cells

> 15 mg/ml

Negative

Sasaki et al. (1987)

 

 

Sister chromatid exchange

Human lymphocytes

150–300 mg/ml

Positive

Jansson & Zech (1987)

 

 

Unscheduled DNA synthesis

Rat hepatocytes

500 mg/mla

Negative

Heck et al. (1989)

 

 

SOS DNA repair

Escherichia coli PQ37

Not reported

Positivec

Ohshima et al. (1989)

 

 

Micronucleus formation

Human hepatoma (Hep-G2) cells

50 mg/ml
500 mg/ml

Negative
Positive

Sanyal et al. (1997)

953

Ethyl vanillin iso-butyrate

Reverse mutation

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

> 5000 mg/plate

Negativeb

King & Harnasch (1997)

897

Salicylaldehyde

Mutation

S. typhimurium TA1535/pSK1002

110 mg/ml

Negativeb

Nakamura et al. (1987)

 

 

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537

370 mg/plate

Negativeb

Florin et al. (1980)

 

 

Reverse mutation (preincubation)

S. typhimurium TA98, TA100

Not reported

Negativeb

Sasaki & Endo (1978)

 

 

Sister chromatid exchange

Human lymphocytes

> 61 mg/ml

Negatived

Jansson et al. (1988)

899

Methyl salicylate

Chromosomal aberration

Hamster lung fibroblasts

Not reported

Positivec

Kawachi et al. (1980a,b)

 

 

Mutation

B. subtilis H17, M45

23 mg/disc

Negativec

Oda et al. (1979)

 

 

Chromosomal aberration

Chinese hamster fibroblasts

250 mg/mla

Negativec

Ishidate et al. (1984)

899

Methyl salicylate

Reverse mutation

S. typhimurium TA92, TA1535, TA100, TA1537, TA94, TA98, TA2637

10 000 mg/plate

Negativeb

Ishidate et al. (1984)

 

 

Reverse mutation (preincubation)

S. typhimurium TA1535, TA98, TA100, TA97, TA1537

> 330 mg/plate

Negativeb

Mortelmans et al. (1986)

 

 

Reverse mutation

S. typhimurium TA100, TA98

Not reported

Negativeb

Kawachi et al. (1980a,b)

 

 

Mutation

B. subtilis H17, M45

Not reported

Negativeb

Kawachi et al. (1980a,b)

 

 

Chromosomal aberration

Human embryo fibroblasts

Not reported

Negativec

Kawachi et al. (1980a,b)

 

 

Sister chromatid exchange

Human embryo fibroblasts

Not reported

Negativec

Kawachi et al. (1980a,b)

 

 

Mutation

Silkworm

Not reported

Negativec

Kawachi et al(1980a,b)

888

Vanillyl butyl ether

Reverse mutation

S. typhimurium TA1535, TA100, TA1537, TA98

5000 mg/plate

Negativeb

Watanabe & Morimoto (1989)

 

 

Mutation

Escherichia coli WP2 uvrA

5000 mg/plate

Negativeb

Watanabe & Morimoto (1989)

893

Ethyl vanillin

Reverse mutation

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

> 3600 mg/plate

Negativeb

Wild et al. (1983)

 

 

Mutation

B. subtilis H17, M45

21 mg/disc

Negativec

Oda et al. (1979)

 

 

Chromosomal aberration

Chinese hamster fibroblasts

250 mg/mla

Positivec

Ishidate et al. (1984)

 

 

Reverse mutation (preincubation)

S. typhimurium TA1535, TA98, TA100, TA97, TA1537

> 8000 mg/plate

Negativeb

Mortelmans et al. (1986)

 

 

Reverse mutation

S. typhimurium TA92, TA1535, TA100, TA1537, TA94, TA98, TA2637

10 000 mg/platea

Negativeb

Ishidate et al. (1984)

 

 

Forward mutation

Mouse lymphoma L5178Y cells

> 1000 mg/ml
800 mg/ml

Negatived

Weakly positivec

Heck et al. (1989)

 

 

Reverse mutation (preincubation)

S. typhimurium TA97, TA102

> 1000 mg/plate

Negativeb

Fujita & Sasaki (1987)

 

 

Reverse mutation

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

10 000 mg/plate

Negativeb

Heck et al. (1989)

 

 

Unscheduled DNA synthesis

Rat hepatocytes

200 mg/ml

Negative

Heck et al. (1989)

 

 

Sister chromatid exchange

Human lymphocytes

> 330 mg/ml

Negativec

Jansson et al. (1988)

 

 

Sister chromatid exchange

Chinese hamster ovary K-1 cells

> 17 mg/ml

Negative

Sasaki et al. (1987)

894

Piperonyl acetate

Reverse mutation (preincubation)

S. typhimurium TA1535, TA98, TA100, TA97, TA1537

> 3300 mg/plate

Negativeb

Mortelmans et al. (1986)

 

 

Reverse mutation

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

3600 mg/plate

Negativeb

Wild et al. (1983)

896

Piperonal

Reverse mutation (histidine substitution)

Escherichia coli WP2uvrAtrp–

2400 mg

Negativeb

Sekizawa & Shibamoto (1982)

 

 

Reverse mutation

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

10 000 mg/platea

Negativeb

Heck et al. (1989)

 

 

Reverse mutation

S. typhimurium TA98, TA100

0.05–5000 mg/plate

Negativeb

Kasamaki et al. (1982)

 

 

Reverse mutation

S. typhimurium TA1537, TA1538, TA98, TA100

> 5000 mg/plate

Negativeb

White et al. (1977)

 

 

Mutation

B. subtilis H17, M45

20 mg/disc

Negativec

Oda et al. (1979)

 

 

Reverse mutation

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

2400 mg

Negativeb

Sekizawa & Shibamoto (1982)

 

 

Reverse mutation (preincubation)

S. typhimurium TA1535, TA1537, TA98, TA100

> 10 000 mg/plate

Negativeb

Haworth et al. (1983)

894

Piperonal

Unscheduled DNA synthesis

Rat hepatocytes

500 mg/ml

Positive

Heck et al. (1989)

 

 

Chromosomal aberration

Chinese hamster B241 cell line

0.075 mg/ml

Positive

Kasamaki et al. (1982)

 

 

Chromosomal aberration

Chinese hamster B241 cell line

> 0.15 mg/ml

Negative

Kasamaki et al. (1985)

 

 

Mutation

B. subtilis H17/M45

5000 mg/disc

Positivec

Sekizawa & Shibamoto (1982)

 

 

Forward mutation

Mouse lymphoma L5178Y cells

> 1000 mg/ml

Negativeb

Heck et al. (1989)

In vivo

879

para-Ethoxybenzaldehyde

Sex-linked recessive lethal mutation

Drosophila melanogaster

750 mg/ml

Negative

Wild et al. (1983)

 

 

Micronucleus formation

NMRI mice

> 1000 mg/kg bw

Negative

Wild et al. (1983)

889

Vanillin

Micronucleus formation

Male BDF1 mice

500 mg/kg bw

Negative

Inouye et al. (1988)

893

Ethyl vanillin

Sex-linked recessive lethal mutation

D. melanogaster

8300 mg/ml

Negative

Wild et al. (1983)

 

 

Micronucleus formation

Male BDF1 mice

Not reported

Negative

Furukawa et al. (1989)

 

 

Micronucleus formation

NMRI mice

1000 mg/kg bw

Negative

Wild et al. (1983)

894

Piperonyl acetate

Sex-linked recessive lethal mutation

D. melanogaster

4900 mg/ml

Negative

Wild et al. (1983)

 

 

Micronucleus formation

NMRI mice

> 970 mg/kg bw

Negative

Wild et al. (1983)

896

Piperonal

Dominant lethal mutation

ICR/Ha Swiss mice

> 620 mg/kg bw

Negative

Epstein et al. (1972)

 

 

Dominant lethal mutation

ICR/Ha Swiss mice

1000 mg/kg bw

Negative

Epstein et al. (1972)

aHighest dose if result was negative; lowest active dose if result was positive

b Without metabolic activation

c With and without metabolic activation

d With metabolic activation

e Administered by intraperitoneal injection

f Administered by oral gavage

In vitro

The hydroxy- and alkoxy-substituted benzyl derivatives were not mutagenic in standard assays for reverse mutation with plate incorporation and/or preincubation in Salmonella typhimurium strains TA92, TA94, TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538, and TA2637, at concentrations ranging up to those that are cytotoxic or at maximum test concentrations recommended by ICH/OECD, in the absence and presence of metabolic activation (S9) (White et al., 1977; Sasaki & Endo, 1978; Douglas et al., 1980; Florin et al., 1980; Kawachi et al., 1980a,b; Nestmann et al., 1980; Rapson et al., 1980; Kasamaki et al., 1982; Pool & Lin, 1982; Sekizawa & Shibamoto, 1982; Haworth et al., 1983; Wild et al., 1983; Ball et al., 1984; Ishidate et al., 1984; Haresaku et al., 1985; Nagabhushan & Bhide, 1985; Mortelmans et al., 1986; Fujita & Sasaki, 1987; Heck et al., 1989; Watanabe & Morimoto, 1989; Dillon et al., 1992; Müller et al., 1993; King & Harnasch, 1997; Dillon et al., 1998). An assay for mutation in S. typhimurium strain TA1535/pSK1002, in which umu gene expression was the end-point, gave negative results with salicylaldehyde (No. 897) (Nakamura et al., 1987). Assays for mutation or DNA repair in Escherichia coli strains WP2 uvrA, WP2s, CSH26/pYM3, CSH26/pSK1002, PQ37, and Sd-4-73 with methyl anisate (No. 884), vanillyl alcohol (No. 886), vanillin (No. 889), vanillyl butyl ether (No. 888), and piperonal (No. 896) (Szybalski, 1958; Sekizawa & Shibamoto, 1982; Ohshima et al., 1989; Watanabe & Morimoto, 1989; Takahashi et al., 1990), and Saccharomyces cerevisiae strains D3, D4, D7, and XV185-14C with veratraldehyde (No. 877) (Nestmann & Lee, 1983) also gave negative results.

Mixed results were obtained with the hydroxy- and alkoxy-substituted benzyl derivatives in the assay for DNA repair in Bacillus subtilis strains H17 and M45 for rec mutation, both positive and negative results being reported for piperonal (No. 896) and negative results for para-methoxybenzaldehyde (No. 878), vanillin (No. 889), ethyl vanillin (No. 893), and methyl salicylate (No. 899) (Oda et al., 1979; Kawachi et al., 1980a,b; Sekizawa & Shibamoto, 1982). Some of the differences in the results were apparently laboratory-specific. Oda et al. (1979) reported only negative results with some of the same compounds; however, the studies were reported in Japanese with English abstracts and could not be fully evaluated for methodological or other differences. It was not clear whether cytotoxicity was a factor in the results. No mutations were observed in silkworms treated with methyl-salicylate (No. 899) (Kawachi et al., 1980a,b).

Both negative and positive results were obtained in assays in isolated mammalian cells with some of the hydroxy- and alkoxy-substituted benzyl derivatives. Mixed results were reported with para-methoxybenzaldehyde and vanillin in assays for sister chromatid exchange in several Chinese hamster cell lines and in human lymphocytes (Jansson et al., 1986; Jansson & Zech, 1987; Sasaki et al., 1987; Jansson et al., 1988). Negative results were obtained in this assay with ethyl vanillin (No. 893), salicylaldehyde (No. 897), and methyl salicylate (No. 899) (Kawachi et al., 1980a,b; Sasaki et al., 1987; Jansson et al.., 1988). Similarly, mixed results were obtained in assays for chromosomal aberration in Chinese hamster and human cell lines with para-methoxybenzaldehyde (No. 878), vanillin (No. 889), ethyl vanillin (No. 893), piperonal (No. 896), and methyl salicylate (No. 899) (Kawachi et al., 1980a,b; Kasamaki et al., 1982; Ishidate et al., 1984; Kasamaki & Urasawa, 1985; Jansson & Zech, 1987; Tamai et al., 1992). The results in the assays for sister chromatid exchange and chromosomal aberrations were generally obtained independently of the presence or absence of metabolic activation. Mixed, but mostly positive, results were obtained with veratraldehyde (No. 877), para-methoxy-benzaldehyde (No. 878), and ethyl vanillin (No. 893) in the assay for forward mutation in L5178Y mouse lymphoma cells, both with and without metabolic activation (Garberg et al., 1988; Wangenheim & Bolcsfoldi, 1988; Heck et al., 1989). Vanillin (No. 889) and piperonal (No. 896) were inactive in this assay (Heck et al., 1989). Vanillin weakly induced micronuclei in human Hep-G2 cells, with only a moderate response at the highest concentration tested (Sanyal et al., 1997). No unscheduled DNA synthesis was observed in rat hepatocytes exposed to veratraldehyde (No. 877), vanillin (No. 889), or ethyl vanillin (No. 893) (Heck et al., 1989). Piperonal (No. 896) caused unscheduled DNA synthesis in one test, but the finding could not be confirmed in subsequent tests (Heck et al., 1989), and the result was considered to be questionable.

para-Methoxybenzaldehyde (No. 878) or benzaldehyde alone did not induce strand breaks in supercoiled DNA from the phage PM2, although positive results were reported with both substances in the presence of CuCl2. The finding that the effect depended on the concentration of copper suggests that DNA-damaging species are produced during redox reactions of aromatic (and aliphatic) aldehydes with CuCl2 (Becker et al., 1996).

Numerous assays for anti-mutagenicity have been conducted in vitro with some of the hydroxy- and alkoxy-substituted benzyl derivatives, including evaluations in several sub-mammalian and mammalian cell lines. Anti-mutagenic activity was reported with para-methoxybenzaldehyde (No. 878) and ethyl vanillin (No. 893) (Ohta et al., 1986b; Imanishi et al., 1990; Ohta, 1995). Mixed results were reported with vanillin (No. 889) (Takahashi et al., 1990; Tamai et al., 1992; Sanyal et al., 1997). Analysis of the concentrations, test organisms, and study methods did not provide an explanation for the discrepant results in these studies. No anti-mutagenic effect was observed with piperonal (No. 896) or methyl salicylate (No. 899) (Ohta et al., 1983, 1986a,b).

In vivo

The hydroxy- and alkoxy-substituted benzyl derivatives were inactive in all assays in vivo in mammals given the compounds orally or by intraperitoneal injection at doses that were significant fractions of the reported lethal doses. Micronuclei were not induced by para-ethoxybenzaldehyde (No. 879) at a dose of 1005 mg/kg bw, ethyl vanillin (No. 893) at 1000 mg/kg bw, vanillin (No. 889) at 500 mg/kg bw, or piperonyl acetate (No. 894) at 620 mg/kg bw (Wild et al., 1983; Furukawa et al., 1989). Piperonal (No. 896) administered by intraperitoneal injection at 1000 mg/kg bw caused a slight increase in the number of early fetal deaths as compared with the incidence in control mice; however, the authors reported that the result was not statistically significant, and no similar finding was reported after administration by oral gavage (Epstein et al., 1972).

In assays for sex-linked recessive lethal mutation in fruit flies (Drosophila melanogaster), negative results were obtained with para-ethoxybenzaldehyde (No. 879), ethyl vanillin (No. 893), and piperonyl acetate (No. 894) after feeding at concentrations of 751, 8309, and 4855 ΅g/ml, respectively (Wild et al., 1983). Vanillin (No. 889) induced an anti-mutagenic response in fruit flies, and both vanillin and para-methoxybenzaldehyde (No. 878) were anti-mutagenic in mice (Imanishi et al., 1990; Sasaki et al., 1990; de Andrade et al., 1992). The data on vanillin, including the results in vitro, suggest some anti-mutagenic activity, although the relevance of this finding is questionable and impossible to extrapolate to the low concentrations to which persons are likely to be exposed from its use as a flavour in food.

Conclusion about genotoxic potential

The hydroxy- and alkoxy-substituted benzyl derivatives did not have mutagenic activity in bacterial or other submammalian cellular systems. Mixed results were obtained in an assay for DNA repair in bacteria and in assays for clastogenicity in isolated mammalian cells. These findings probably reflect the known activity of alcohols or aldehydes in biological systems, as they were seen both with and without metabolic activation, and cytotoxicity was often a limitation at high concentrations. Negative results were obtained in tests for genotoxicity in mice and Drosophila in vivo. In a 2-year study in mice, no difference in tumour incidence from that in controls was found in groups fed doses up to 900 mg/kg bw per day of butyl-para-hydroxybenzoate (No. 870) (Inai et al., 1985). The Committee therefore concluded that the hydroxy- and alkoxy-substituted benzyl derivatives do not have genotoxic potential in vivo.

(e) Reproductive toxicity

Veratraldehyde (No. 877), vanillin (No. 889), ethyl vanillin (No. 893), and piperonal (No. 896)

Four groups of 10 virgin Crl CD rats were given veratraldehyde (No. 877), vanillin (No. 889), ethyl vanillin (No. 893), or piperonal (No. 896) by gavage once a day for 7 days before cohabitation and throughout cohabitation (maximum of 7 days), gestation, parturition, and a 4-day post-parturition period. The maternal indices monitored included observation twice daily, body weight, food consumption, duration of gestation, and fertility parameters (mating and fertility index, gestation index, number of offspring per litter). The indices in the offspring included daily observation, clinical signs, gross external malformations, and body weight. The only consistent effects were reduced body-weight gain by dams at the two higher doses, which was accompanied by a statistically significant reduction in food consumption at the high dose. No effects were observed in the offspring at any dose.

Dams given veratraldehyde (No. 877) at 80, 400, or 800 mg/kg bw per day had decreased body weights and body-weight gain at the two lower doses and an increased mortality rate and gross lesions at the highest dose. Animals given 125, 250, or 500 mg/kg bw per day of vanillin (No. 889) showed a slight, non-significant decrease in food consumption and body weight at the intermediate dose and a statistically significant (p < 0.05) decrease in body weight and an increase in the frequency of clinical signs at the highest dose. Animals given 200, 1000, or 2000 mg/kg bw per day of ethyl vanillin (No. 893) had a slight, non-significant increase in body weight and a statistically significant (p < 0.05) decrease in food consumption at the low dose, while those at the two higher doses had an increased mortality rate, gross lesions, clinical signs, and depressed body-weight gain and food consumption. Animals given 250, 500, or 1000 mg/kg bw per day of piperonal (No. 896) had a slight, non-significant decrease in body weight at the intermediate dose and a statistically significant (p < 0.05) decrease in body weight, a decrease in food consumption, increased mortality rate, and a decreased fertility index at the highest dose. The only effect on offspring was decreased viability and body-weight gain at the high dose of piperonal. In view of the lack of adverse effects on offspring at all doses and on dams at the low dose of each substance, the authors concluded that the compounds had no reproductive or developmental effects (Vollmuth et al., 1990). However, these results were reported in an abstract and a full report was not available.

Methyl salicylate (No. 899)

Four groups of 11-week-old CD-1 mice (40 males and 40 females in the control group; 20 males and 20 females in the treated groups) were given methyl salicylate at a dose of 0, 100, 250, or 500 mg/kg bw per day by gavage in corn oil. All mice were treated for 1 week before, 14 weeks during, and 3 weeks after cohabitation, for a total treatment period of 18 weeks. Reproductive performance was assessed by observing the number of litters per breeding pair, the number of live pups per litter, the proportion of pups born alive, the sex ratio of pups born alive, and the live pup weight. Body weight was measured at weeks 1, 2, 3, 6, 10, 14, and 18. The only significant result was a slight decrease in the mean number of litters in mice at the highest dose. Two attempts to determine which sex was affected were unsuccessful owing to the poor fertility of the control groups in follow-up experiments. Eleven animals died during the 18 weeks of the study, the deaths being distributed relatively evenly among the treated groups. Gavage trauma was the commonest cause of death, and none of the deaths was considered related to the chemical or dose. The NOEL was 250 mg/kg bw per day (National Toxicology Program, 1984).

2,4-Dihydroxybenzoic acid (No. 908)

Groups of 10 adult female Sprague-Dawley rats were given an injection of 380 mg/kg 2,4-dihydroxybenzoic acid (No. 908) on day 9 of gestation. No differences from controls were seen in the total number of implants, the mean fetal weight, or the numbers of live fetuses, resorptions, or malformed fetuses (Koshakji & Schulert, 1973).

In male and female Sprague-Dawley rats given a subcutaneous injection of 430 mg/kg bw of 2,4-dihydroxybenzoic acid, no change in plasma or serum calcium concentration was found 3 h after treatment. When pregnant female rats were given 430 mg/kg bw of 2,4-dihydroxybenzoic acid subcutaneously and a second dose of 210 mg/kg bw 2 h later, no change in serum calcium was observed after the initial dose, but decreased calcium concentrations and high serum 2,4-dihydroxybenzoic acid concentrations were recorded after the second dose. Females killed on day 21 of gestation showed no significant change in the numbers of litters, implantations, resorptions, or dead fetuses. Increased incidences of malformations (kinky tail) and fetotoxicity (33%) were reported. No statistical analysis was presented to support these claims. The authors concluded that the toxic effect of dihydroxybenzoic acid derivatives was different from that of salicylic acid (Saito et al., 1982).

(f) Observations in humans

Methyl salicylate (No. 899)

Six clinicians conducted a retrospective study of 155 cases of juvenile rheumatic arthritis in which children were treated with a wide variety of salicylates. Periodic X-rays were undertaken to determine whether bone density had changed during treatment. The patients were given doses in the range 100–3240 mg/day, depending on their age and weight, for a duration of treatment varying from several months to intermittent dosage for 14 years. There was no accumulation of cancellous bone (Abbott & Harrisson, 1978).

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ENDNOTES

1 During evaluation of these flavouring agents, the Committee questioned whether one substance in this group (No. 870) was used as a flavouring agent and therefore appropriate to be evaluated by this Procedure. Information to address this question will be sought from the relevant manufacturers.



    See Also:
       Toxicological Abbreviations