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WHO FOOD ADDITIVES SERIES 46:PHENOL AND PHENOL DERIVATIVES

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

Evaluation

Introduction

Estimated daily intake

Metabolic considerations

Application of the Procedure for the Safety Evaluation of Flavouring Agents

Consideration of combined intakes from use as flavouring agents

Conclusions

Relevant background information

Explanation

Additional considerations on intake

Biological data

Absorption, distribution, and excretion

Toxicological studies

Acute toxicity

Short-term studies of toxicity

Long-term studies of toxicity and carcinogenicity

Genotoxicity

Reproductive toxicity

Special studies: Effects on forestomach and glandular stomach epithelium

References

1. EVALUATION

1.1 Introduction

The Committee evaluated a group of 48 flavouring agents that includes phenol, an ester of phenol, and resorcinol; alkyl-, alkenyl-, or aryl-substituted phenols and their corresponding esters; alkoxy phenols; and phenol derivatives with alkyl side-chains containing a ketone function (see Table 1). The evaluations were conducted according to the Procedure for the Safety Assessment of Flavouring Agents.

2-Phenylphenol (No. 735) was evaluated by the Committee at its eighth meeting, when an ADI of 0-0.2 mg/kg bw was established (Annex 1, reference 8). The 1999

Table 1. Summary of the results of safety evaluations of phenol and 47 phenol derivatives used as flavouring agentsa

Flavouring agent

No.

CAS no. and structure

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

Step A4
Is the substance or are its metabolites endogenous?

Step A5
Adequate NOEL for substance or related substance?

Comments

Conclusion based on current intake

Structural class I

Phenol

690

108-95-2
chemical structure

No
Europe: 6
USA: 1

N/R

N/R

See note 1.

No safety concern

ortho-Cresol

691

95-48-7
chemical structure

No
Europe: 290
USA: 0.1

N/R

N/R

See note 1.

No safety concern

meta-Cresol

692

108-39-4
chemical structure

No
Europe: 0.1
USA: 0.1

N/R

N/R

See note 1.

No safety concern

para-Cresol

693

106-44-5
chemical structure

No
Europe: 1
USA: 1

N/R

N/R

See note 1.

No safety concern

para-Ethylphenol

694

123-07-9
chemical structure

No
Europe: 4
USA: 0.1

N/R

N/R

See note 1.

No safety concern

ortho-Propylphenol

695

644-35-9
chemical structure

No
Europe: 0.1
USA: 1

N/R

N/R

See note 1.

No safety concern

para-Propylphenol

696

645-56-7
chemical structure

No
Europe: 0.1
USA: 0.1

N/R

N/R

See note 1.

No safety concern

2-Isopropylphenol

697

88-69-7
chemical structure

No
Europe: 16
USA: 0.3

N/R

N/R

See note 1.

No safety concern

4-(1,1-Dimethyl)ethyl-phenol

733

98-54-4
chemical structure

No
Europe: 0.01
USA: 0.001

N/R

N/R

See note 1.

No safety concern

Phenyl acetate

734

122-79-2
chemical structure

No
Europe: 0.07
USA: 0.01

N/R

N/R

See note 2.

No safety concern

ortho-Tolyl acetate

698

533-18-6
chemical structure

No
Europe: 0.1
USA: 40

N/R

N/R

See note 2.

No safety concern

para-Tolyl acetate

699

140-39-6
chemical structure

No
Europe: N/D
USA: 70

N/R

N/R

See note 2.

No safety concern

ortho-Tolyl isobutyrate

700

36438-54-7
chemical structure

No
Europe: 0.03
USA: 0.1

N/R

N/R

See note 2.

No safety concern

para-Tolyl isobutyrate

701

103-93-5
chemical structure

No
Europe: 0.04
USA: 0.01

N/R

N/R

See note 2.

No safety concern

para-Tolyl-3-methyl butyrate

702

55066-56-3
chemical structure

No
Europe: 0.4
USA: 0.1

N/R

N/R

See note 2.

No safety concern

para-Tolyl octanoate

703

59558-23-5
chemical structure

No
Europe: 0.03
USA: 1

N/R

N/R

See note 2.

No safety concern

para-Tolyl laurate

704

10024-57-4
chemical structure

No
Europe: N/D
USA: 0.3

N/R

N/R

See note 2.

No safety concern

para-Tolyl phenyl-acetate

705

101-94-0
chemical structure

No
Europe: 0.7
USA: 0.1

N/R

N/R

See note 2.

No safety concern

2,5-Xylenol

706

95-87-4
chemical structure

No
Europe: 1
USA: 0.03

N/R

N/R

See note 1.

No safety concern

2,6-Xylenol

707

576-26-1
chemical structure

No
Europe: 2
USA: 1

N/R

N/R

See note 1.

No safety concern

3,4-Xylenol

708

95-65-8
chemical structure

No
Europe: 7
USA: 1

N/R

N/R

See note 1.

No safety concern

2,3,6-Trimethylphenol

737

2416-94-6
chemical structure

No
Europe: 0.3
USA: 0.3

N/R

N/R

See note 1.

No safety concern

Thymol

709

89-83-8
chemical structure

No
Europe: 59
USA: 160

N/R

N/R

See note 1.

No safety concern

Carvacrol

710

499-75-2
chemical structure

No
Europe: 16
USA: 0.3

N/R

N/R

See note 1.

No safety concern

para-Vinylphenol

711

2628-17-3
chemical structure

No
Europe: 0.1
USA: 6

N/R

N/R

See note 1.

No safety concern

Resorcinol

712

108-46-3
chemical structure

No
Europe: 1
USA: 0.3

N/R

N/R

See notes 1.

No safety concern

Guaiacol

713

90-05-1
chemical structure

No
Europe: 51
USA: 16

N/R

N/R

See note 1.

No safety concern

ortho-(Ethoxymethyl)-phenol

714

20920-83-6
chemical structure

No
Europe: 2
USA: 0.01

N/R

N/R

See note 1.

No safety concern

2-Methoxy-4-methyl-phenol

715

93-51-6
chemical structure

No
Europe: 37
USA: 3

N/R

N/R

See note 1.

No safety concern

4-Ethylguaiacol

716

2785-89-9
chemical structure

No
Europe: 8
USA: 0.4

N/R

N/R

See note 1.

No safety concern

2-Methoxy-4-propyl-phenol

717

6627-88-9
chemical structure

No
Europe: 210
USA: 0.1

N/R

N/R

See note 1.

No safety concern

Guaiacyl phenylacetate

718

613-70-7
chemical structure

No
Europe: 0.01
USA: 0.1

N/R

N/R

See note 2.

No safety concern

Guaiacyl phenylacetate

719

4112-89-4
chemical structure

No
Europe: 0.4
USA: 2

N/R

N/R

See note 2.

No safety concern

Hydroquinone mono-ethyl ether

720

622-62-8
chemical structure

No
Europe: N/D
USA: 0.4

N/R

N/R

See note 1.

No safety concern

2,6-Dimethoxyphenol

721

91-10-1
chemical structure

No
Europe: 6
USA: 12

N/R

N/R

See note 1.

No safety concern

4-Methyl-2,6-dimethoxy-phenol

722

6638-05-7
chemical structure

No
Europe: N/D
USA: 0.04

N/R

N/R

See note 1.

No safety concern

4-Ethyl-2,6-dimethoxy-phenol

723

14059-92-8
chemical structure

No
Europe: N/D
USA: 1

N/R

N/R

See note 1.

No safety concern

4-Propyl-2,6-dimethoxy-phenol

724

6766-82-1
chemical structure

No
Europe: N/D
USA: 0.1

N/R

N/R

See note 1.

No safety concern

2-Methoxy-4-vinyl-phenol

725

7786-61-0
chemical structure

No
Europe: 3
USA: 1

N/R

N/R

See note 1.

No safety concern

4-Allyl-2,6-dimethoxy-phenol

726

6627-88-9
chemical structure

No
Europe: 0.01
USA: 6

N/R

N/R

See note 1.

No safety concern

2-Hydroxyaceto-phenone

727

118-93-4
chemical structure

No
Europe: 0.1
USA: 8

N/R

N/R

See note 1.

No safety concern

Phenyl salicylate

736

118-55-8
chemical structure

No
Europe: 9
USA: 8

N/R

N/R

See note 1

No safety concern

4-(para-Hydroxy-phenyl)-2-butanone

728

5471-51-2
chemical structure

No
Europe: 2800
USA: 3800

N/R

N/R

Yes. The NOEL of 280 mg/kg bw per day in a 13-week study in rats given multiple doses (Gaunt et al., 1970) is > 1000 times the daily intakes of 46 g/kg bw in Europe and 63 g/kg bw in the USA.

No safety concern

Dihydroxyaceto-phenone

729

28631-86 9
chemical structure

No
Europe: 0.01
USA: 0.1

N/R

N/R

See note 1.

No safety concern

Zingerone

730

122-48-5
chemical structure

No
Europe: 40
USA: 83

N/R

N/R

See note 1.

No safety concern

4-(para-Acetoxyphenyl)-2-butanone

731

3572-06-3
chemical structure

No
Europe: N/D
USA: 0.1

N/R

N/R

See note 2.

No safety concern

Vanillylidene acetone

732

1080-12-2

No
Europe: N/D
USA: 0.1

N/R

N/R

See note 1.

No safety concern

Structural class III

2-Phenylphenolc

735

90-43-7
chemical structure

No
Europe: 0.01
USA: 0.01

N/R

N/R

See note 1.

No safety concern

CAS, Chemical Abstracts Service; N/D, no intake data reported; N/R, not required for evaluation because consumption of the substance was determined to be of no safety concern at Step 3 of the Procedure.

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

b The thresholds for human intake are 1800 g/day for structural class I and 90 g/day for class III. All intake values are expressed in g/day.

c An ADI of 0-0.4 mg/kg bw has been established for 2-phenylphenol (FAO, 1999).

Notes:

1. Detoxication of phenol primarily involves conjugation of the hydroxyl group with sulfate and glucuronic acid.

2. Phenyl acetate undergoes rapid hzydrolysis followed by conjugation with sulfate and glucuronic acid.

Joint FAO/WHO Meeting on Pesticide Residues evaluated 2-phenylphenol and established an ADI of 0-0.4 mg/kg bw (FAO, 1999).

Thirty-two of the 48 flavouring agents in this group are natural components of foods. They have been detected in berries, coffee, and meat.

1.2 Estimated daily intake

The total annual volume of production of the 48 phenols considered here is approximately 25 t in Europe (International Organization of the Flavor Industry, 1995) and 32 t in the USA (National Academy of Sciences, 1987; Lucas et al., 1999; Table 2). About 78% of the total annual volume in Europe and 90% of that in the USA is accounted for by 4-(para-hydroxyphenyl)-2-butanone (raspberry ketone, No. 728 and the estimated intake of this substance is 2.8 mg/day in Europe and 3.8 mg/day in the USA.

Other flavouring agents for which the estimated intakes are in the range 37-300 g/day include ortho-cresol (No. 691) (290 m g/day in Europe), ortho- and para-tolyl acetates (Nos 698 and 699) (40 g/day and 70 g/day, respectively, in the USA), 2-methoxy-4-methylphenol (No. 715) (37 g/day in Europe), thymol (No. 709) (59 g/day in Europe and 160 g/day in the USA), guaiacol (No. 713) (51 g/day in Europe), 2-methoxy-4-propylphenol (No. 717) (210 g/day in Europe), and zingerone (No. 730) (40 g/day in Europe and 83 g/day in the USA). The intakes of all other flavouring agents in the group the intake are in the range 0.01-16 g/day. For seven of the flavouring agents used in the USA, the annual production volumes in Europe were not reported.

1.3 Metabolic considerations

Phenol (No. 690) and its derivatives are rapidly absorbed from the gastrointestinal tract and share common pathways of metabolism (Hughes & Hall, 1995). Phenol (No. 690), phenyl acetate (No. 734), resorcinol (No. 712), and alkyl-, alkenyl-, and aryl-substituted phenols and their corresponding esters (Nos 691-697, 698-711, 733, 735, 737) are conjugated with sulfate and glucuronic acid after hydrolysis of the esters and excreted primarily in the urine. Other metabolic pathways, observed mainly at high doses, include ring hydroxylation and side-chain oxidation. Phenols containing alkoxy groups (Nos 713-717, 720-726) and those that contain a ketone function on an alkyl side-chain (Nos 727-732, 736) are also metabolized mainly by conjugation.

Alternative metabolic pathways include dealkylation of alkoxyphenols, reduction of side-chain ketones, side-chain oxidation, and ring hydroxylation. At very high doses (> 500 mg/kg bw), small amounts of para-cresol (No. 693), para-ethylphenol (No. 694), 2-methoxy-4-methyl phenol (No. 715), 2-methoxy-4-propylphenol (No. 717), 2-methoxy-4-vinylphenol (No. 725), and 4-allyl-2,6-dimethoxyphenol (No. 726) are oxidized to reactive quinone methide intermediates (Thompson et al., 1994, 1995a,b; Figure 1). Given the presence of a detoxication pathway (glutathione conjugation) for such quinone methides, the safety of the intermediates potentially formed after high doses of the specified phenol derivatives would not be a concern under the conditions of their use as flavouring agents.

Figure 1

Table 2. Annual volume and intake of phenol and phenol derivatives used as flavouring substances in Europe and the USA

Substance (No.)

Most recent annual volume (kg)a

Intakeb

Annual volume in naturally occurring foods (kg)d

Consumption ratioe

g/day

g/kg bw per day

para-Cresol equivalents (mg/kg bw per day)c

Phenol (690)

Europe

43

6

0.1

 

41 193

960

USA

5

1

0.01

 

41 193

8 200

ortho-Cresol (691)

Europe

2039

290

5

 

7 451

3.6

USA

0.5

0.1

0.001

 

7 451

15 000

meta-Cresol (692)

Europe

1

0.1

0.002

 

4 534

4 500

USA

0.5

0.1

0.001

 

4 534

9 100

para-Cresol (693)

Europe

8

1

0.02

 

7 995

1 000

USA

8

1

0.02

 

7 995

1 000

para-Ethylphenol (694)

Europe

29

4

0.1

 

10 235

350

USA

1

0.1

0.002

 

10 235

10 000

ortho-Propylphenol (695)

Europe

1

0.1

0.002

 

+

NA

USA

10

1

0.02

 

+

NA

para-Propylphenol (696)

Europe

0.4

0.1

0.001

 

+

NA

USA

0.5

0.1

0.001

 

+

NA

2-Isopropylphenol (697)

Europe

112

16

0.3

 

+

NA

USA

2

0.3

0.004

 

+

NA

4-(1,1-Dimethyl)ethyl phenol (733)

Europe

0.1

0.01

0.0002

 

+

NA

USA

0.1

0.01

0.0002

 

+

NA

Phenyl acetate (734)

Europe

0.1

0.01

0.0002

 

+

NA

USA

0.1

0.01

0.0002

 

+

NA

ortho-Tolyl acetate (698)

Europe

1

0.1

0.002

 

-

NA

USA

300

40

1

 

-

NA

para-Tolyl acetate (699)

Europe

N/D

N/D

N/D

N/D

-

NA

USA

530

70

1

0.7

-

NA

ortho-Tolyl isobutyrate (700)

Europe

0.2

0.03

0.0005

 

-

NA

USA

1

0.1

0.002

 

-

NA

para-Tolyl isobutyrate (701)

Europe

0.3

0.04

0.0007

0.0006

-

NA

USA

0.1

0.01

0.0002

0.0001

-

NA

para-Tolyl 3-methylbutyrate (702)

Europe

3

0.4

0.007

0.003

-

NA

USA

0.9

0.1

0.002

0.001

-

NA

para-Tolyl octanoate (703)

Europe

0.2

0.03

0.0005

 

-

NA

USA

7

1

0.02

 

-

NA

para-Tolyl laurate (704)

Europe

N/D

N/D

N/D

 

-

NA

USA

2

0.3

0.004

 

-

NA

para-Tolyl phenylacetate (705)

Europe

5

0.7

0.01

0.006

-

NA

USA

0.9

0.1

0.002

0.001

-

NA

2,5-Xylenol (706)

Europe

4

1

0.009

 

1 673

420

USA

0.2

0.03

0.0004

 

1 673

8 400

2,6-Xylenol (707)

Europe

14

2

0.03

 

558

40

USA

4

1

0.009

 

558

140

3,4-Xylenol (708)

Europe

47

7

0.1

 

892

19

USA

7

1

0.02

 

892

130

2,3,6-Trimethylphenol (737)

Europe

2

0.3

0.005

 

+

NA

USA

2

0.3

0.004

 

+

NA

Thymol (709)

Europe

415

59

1

 

25 767

62

USA

1 240

160

3

 

25 767

21

Carvacrol (710)

Europe

114

16

0.3

 

80 203

700

USA

2

0.3

0.004

 

80 203

40 000

para-Vinylphenol (711)

Europe

1

0.1

0.002

 

17 745

18 000

USA

43

6

0.1

 

17 745

410

Resorcinol (712)

Europe

10

1

000

 

+

NA

USA

2

0.3

0.004

 

+

NA

Guaiacol (713)

Europe

358

51

1

 

8 184

44

USA

120

16

0.3

 

8 184

68

ortho-(Ethoxymethyl)phenol (714)

Europe

12

2

0.03

 

-

NA

USA

0.1

0.01

0.0002

 

-

NA

2-Methoxy-4-methylphenol (715)

Europe

257

37

1

 

226

0.9

USA

25

3

0.1

 

226

9

4-Ethylguaiacol (716)

Europe

57

8

0.1

 

625 035

11 000

USA

3

0.4

0.007

 

625 035

210 000

2-Methoxy-4-propylphenol (717)

Europe

1 484

210

4

 

38

0

USA

0.9

0.1

0.002

 

38

42

Guaiacyl acetate (718)

Europe

0.1

0.01

0.0002

 

-

NA

USA

0.9

0.1

0.002

 

-

NA

Guaiacyl phenylacetate (719)

Europe

3

0.4

0.007

 

-

NA

USA

15

2

0.03

-

 

NA

Hydroquinone monoethyl ether (720)

Europe

N/D

N/D

N/D

 

-

NA

USA

3

0.4

0.007

 

-

NA

2,6-Dimethoxyphenol (721)

Europe

44

6

0.1

 

+

NA

USA

89

12

0.2

 

+

NA

4-Methyl-2,6-dimethoxyphenol (722)

Europe

N/D

N/D

N/D

 

+

NA

USA

0.3

0.04

0.0007

 

+

NA

4-Ethyl-2,6-dimethoxyphenol(723)

Europe

N/D

N/D

N/D

 

108

NA

USA

11

1

0.02

 

108

0.1

4-Propyl-2,6-dimethoxyphenol (724)

Europe

N/D

N/D

N/D

 

+

NA

USA

0.5

0.1

0.001

 

+

NA

2-Methoxy-4-vinylphenol (725)

Europe

21

3

0.05

 

38 066

1 800

USA

11

1

0.02

 

38 066

3 500

4-Allyl-2,6-dimethoxyphenol (726)

Europe

0.1

0.01

0.0002

 

+

NA

USA

44

6

0.1

 

+

NA

2-Hydroxyacetophenone (727)

Europe

1

0.1

0.002

 

1 912

1 900

USA

0.1

0.01

0.0002

 

1 912

19 000

Phenyl salicylate (736)

Europe

60

9

0.1

 

-

NA

USA

60

8

0.1

 

-

NA

4-(para-Hydroxyphenyl)-2-butanone (728)

Europe

19 494

2800

46

 

111

0.006

USA

28 900

3800

63

 

111

0.004

Dihydroxyacetophenone (729)

Europe

0.1

0.01

0.0002

 

-

NA

USA

1

0.1

0.002

 

-

NA

Zingerone (730)

Europe

278

40

1

 

20

0.072

USA

630

83

1

 

20

0.032

4-(para-Acetoxyphenyl)-2-butanone (731)

Europe

N/D

N/D

N/D

 

-

NA

USA

0.5

0.1

0.001

 

-

NA

Vanillylidene acetone (732)

Europe

N/D

N/D

N/D

 

-

NA

USA

0.9

0.1

0.002

 

-

NA

2-Phenylphenol (735)

Europe

0.1

0.01

0.0002

 

+

NA

USA

0.1

0.01

0.0002

 

+

NA

Total

Europe

25 000

 

 

 

 

 

USA

32 000

 

 

 

 

 

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

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

b Intake (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 (g/kg bw per day) calculated as follows: [(/person per day)/body weight], where body weight = 60 kg. Slight variations may occur from rounding.

c Calculated as follows: (relative molecular mass alcohol/relative molecular mass ester) x daily per capita intake ('eaters only') ester

d Quantitative data from Stofberg & Grundschober (1987)

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

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

Step 1.

In applying the Procedure for the Safety Evaluation of Flavouring Agents (see Figure 1) to the above-mentioned substances, the Committee assigned 47 of the 48 flavouring agents with the lowest toxic potential to structural class I (Cramer et al., 1978). The remaining flavouring agent, 2-phenylphenol (No. 735), was assigned to structural class III.

Step 2.

At current levels of intake, the flavouring agents can tie predicted to be metabolized to innocuous products, and the pathways involved would not be expected to be saturated. The evaluation of these substances therefore proceeded via the left-hand side of the decision-tree.

Step A3.

The estimated daily per capita intake of 46 of the flavouring agents in structural class I is below the threshold for human intake for the class (i.e. 1800 m g) and that of the one agent in structural class III is below the threshold for human intake for that class (i.e. 90 m g). The Committee concluded that the safety of these 47 flavouring agents poses no concern when they are used at their currently estimated levels of intake. The intakes of 4-(para-hydroxyphenyl)-2-butanone (No. 728) are 2800 g/person per day in Europe (International Organization of the Flavor Industry, 1995) and 3800 g/person per day in the USA (Lucas et al., 1999). These levels exceed the threshold for human intake for class I (1800 g/person per day). Accordingly, evaluation of this substance proceeded to step A4.

Step A4.

4-(para-Hydroxyphenyl)-2-butanone (No. 728) does not occur endogenously in humans. Evaluation of this substance therefore proceeded to step A5.

Step A5.

The NOEL of 280 mg/kg bw per day for 4-(para-hydroxyphenyl)-2-butanone (No. 728) in a 13-week study in rats (Gaunt et al., 1970) provides an adequate margin of safety (i.e. > 10 000) at current levels of intake in Europe (46 g/kg bw per day) and in the USA (63 g/kg bw per day).

Table 1 summarizes the evaluation of phenol and 47 phenol derivatives used as flavouring agents.

1.5 Consideration of combined intakes from use as flavouring agents

In the unlikely event that all foods containing the six esters of para-cresol (Nos 699, 701-705) were consumed concurrently with para-cresol (No. 693) on a daily basis, the combined intake of para-cresol equivalents (see Table 2) would not exceed the threshold for human intake for structural class I (1800 g/person per day). In the unlikely event that all foods containing all 48 substances were consumed daily, the estimated combined intake would exceed the human intake threshold for class I but would not saturate the available high-capacity, conjugation pathways involved in the metabolism of these substances (Hagan et al., 1967; Posternak & Linder, 1969; Gaunt et al., 1970; Maasik, 1970; Kociba et al., 1976; National Toxicology Program, 1980; Hirose et al., 1986; Environmental Protection Agency, 1988a,b; Hirose et al., 1989; Maruyama et al., 1991; National Toxicology Program, 1992a,b). Moreover, approximately 78% of the annual volume consumed in Europe and approximately 90% of that consumed in the USA is accounted for by 4-(para-hydroxyphenyl)-2-butanone (No. 728), for which a NOEL providing an adequate margin of safety was available.

1.6 Conclusions

The Committee concluded that the safety of phenol and the 47 derivatives of phenol in this group would not raise concern at the currently estimated levels of intake. Other data on the toxicity of phenol and its derivatives were consistent with the results of the safety evaluation.

The Committee took note of the ADI of 0-0.4 mg/kg bw for 2-phenylphenol (No. 735) established by the 1999 Joint FAO/WHO Meeting on Pesticide Residues (FAO, 1999).

2. RELEVANT BACKGROUND INFORMATION

2.1 Explanation

This monograph summarizes data pertinent to evaluating the safety of phenol and 47 phenol derivatives used as flavouring substances (see Table 1). The group of substances was selected on the basis of the structural criteria that all members possess an aromatic ring containing one (e.g. phenol, No. 690) or more (e.g. resorcinol, No. 712) free hydroxyl groups or are the esters of phenol derivatives (e.g. phenyl acetate, No. 734). The available chemical, metabolic, and toxicological data indicate that phenol and its derivatives fit into four structural categories: phenol, two esters of phenol, and resorcinol (Nos 690, 734, 712, 736); alkyl-, alkenyl-, or aryl-substituted phenols and their corresponding esters (Nos 691-711, 733, 735, and 737); alkoxy phenols (Nos 713-726); and phenol derivatives that contain alkyl side-chains containing a ketone function (Nos 727-732). Substances with multiple functional groups were categorized according to their most reactive functional group.

2.2 Additional considerations on intake

Thirty-two of the phenol derivatives are natural components of foods and are found predominantly in berries, coffee, and meat (Maarse et al., 1996). Quantitative data on natural occurrence and consumption ratios, which have been reported for 20 of the substances in the group, indicate that they are consumed primarily as natural components of food (i.e. consumption ratios > 1) (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987; see Table 2). Consumption ratios of < 1 were calculated for 2-methoxy-4-methylphenol (No. 715), 4-(para-hydroxyphenyl)2-butanone (No. 728), and zingerone (No. 730), as 0.9, 0.004, and 0.03, respectively.

Phenol (No. 690) has a sharp, burning flavour and a distinct sweet, acrid odour that is discernable at a concentration of 3.8 mg/m3 (National Institute for Occupational Safety and Health, 1976).

2.3 Biological data

2.3.1 Absorption, distribution, and excretion

Phenol (No. 690) is a normal constituent of human and animal tissues, blood, urine, faeces, and sweat. Most endogenous phenol arises from the degradation of tyrosine by Escherichia coli in the intestines. Diets with a high protein content promote phenol production (Babich & Davis, 1981). It is thought that para-cresol (No.693), found in the serum of healthy humans at a concentration of 0.23 0.17 mg/100 ml, is formed by the same bacterial mechanisms (Wengle & Hellström, 1972).

When phenol and its derivatives are ingested as natural or-added components of food, they are rapidly absorbed from the gastrointestinal tract and participate in common pathways of metabolism (Hughes & Hall, 1995). Phenol (No. 690), phenyl acetate (No. 734), and resorcinol (No. 712), alkyl-, alkenyl-, and aryl-substituted phenols and their corresponding esters (Nos 691-712, 718, 719, 733, 735, and 737) are conjugated with sulfate or glucuronic acid and excreted primarily in the urine. The simple esters of substituted phenols undergo hydrolysis prior to conjugation. Other metabolic pathways, observed mainly at high doses, include ring hydroxylation and side-chain oxidation. Phenols containing alkoxy groups (Nos 713-717 and 720-726) and those that contain an alkyl side-chain with a ketone function (Nos 727-732 and 736) are also metabolized mainly by conjugation. Alkoxy phenols may undergo para-hydroxylation before conjugation. Minor metabolites are formed through alternative pathways that include dealkylation of alkoxyphenols, reduction of side-chain ketones, side-chain oxidation, and ring hydroxylation.

At high doses, para-cresol (No. 693), para-ethylphenol (No. 694), 2-methoxy-4-methylphenol (No. 715), 2-methoxy-4-propylphenol (No. 717), 2-methoxy-4-vinylphenol (No. 725), and 4-allyl-2,6-dimethoxyphenol (No. 726) are oxidized to reactive quinone methide intermediates. Quinone methides are detoxified by glutathione; however, at very high concentrations this detoxication pathway may be saturated. Low concentrations (< 0.25 mal/L) of quinone methide intermediates undergo detoxication via glutathione (Thompson et al., 1994, 1995a,b) (see Figure 1).

Phenol (No. 690), phenyl acetate (734), phenyl salicylate (No. 736), and resorcinol (No. 712)

The primary route of metabolism of phenol (No. 690) involves rapid absorption and conjugation of the hydroxyl group with sulfate or glucuronic acid, primarily in the liver (see Figure 1) (Balling et al., 1995). Ring hydroxylation to yield para-hydroxyphenol (i.e. quinol) has also been reported. In human subjects, in whom > 95% of a single oral dose of 0.01 mg/kg bw of phenol was excreted in the urine within 24 h. The principal urinary metabolites included the sulfate (77%) and glucuronic acid (16%) conjugates of phenol. Quinol monosulfate (the sulfate of 1,4-benzenediol) and quinol monoglucuronide were the only minor metabolites present in detectable amounts (Capel et al., 1972). Sulfation also occurs in the human metabolism of related substances: when seven human tissues were incubated with ortho-, meta-, and para-hydroxybiphenyl, the rate of sulfation of each isomer was highest in the liver and lowest in the brain (Pacifici et al., 1991).

Small amounts of ingested phenol (No. 690) are excreted in the free form. A man ingested one lozenge containing 32.5 mg of phenol per lozenge every 2 h for a total of eight doses in one day, and his urine was collected over 72 h after the first dose, fractionated into 8-h collection periods. The concentration of phenol based on the total volume of urine peaked at 270 mg/L during the third collection period, when the concentration of free phenol peaked at 10 mg/L. The concentrations of both total and free urinary phenols returned to baseline levels (7 and 1 mg/L, respectively) 48 h after the final dose (Fishbeck et al., 1975).

The absorption and metabolism of phenol are comparable in rats. Approximately 70-85% of an oral dose of 0.031 mg/kg bw of phenol (No. 690) was excreted in the urine of female rats within 4 h of administration, and only 1-5% remained in the body 72 h after exposure (Hughes & Hall, 1995). Male and female rats given single intravenous injections of phenol at133 mol/kg bw excreted 53% and 44% as phenyl sulfate, respectively, within 3 h of dosing. The difference in sulfate excretion by males and females was thought to result from a slower rate of excretion of phenyl sulfate by females rather than from a difference in the degree of sulfate conjugation. Approximately 42% of the administered dose was excreted in the urine of both males and females within 3 h as phenyl glucuronide (Meerman et al., 1987).

Glucuronic acid conjugation of phenol (No. 690) is thought to be proportional to the dose, while sulfate conjugation depends on the availability of sulfate (Bray et al., 1952). In rats, increasing an intravenous dose of phenol from 0.013 to 0.266 mg/kg bw resulted in an increase in glucuronidation from 28 to 60% of the dose and a decrease in sulfation from 72 to 40% (Weitering et al., 1979). These observations suggest saturation of the sulfation pathway, which would leave more free phenol available to form the glucuronic acid conjugate and undergo ring hydroxylation to yield polyphenols (quinol) (Williams, 1959; Weitering et al., 1979; Kenyon et al., 1995). In another study, 75 mg/kg bw per day of phenol were administered to rats by intraperitoneal injection. Urinary analysis revealed small amounts of N-acetyl-S(2,5-dihydroxyphenyl)-L-cysteine, a product of glutathione conjugation of benzoquinone intermediates (Nerland & Pierce, 1990).-In rats; also, phenol was excreted in the bile, mostly as the glucuronide conjugate. Approximately 5% of-an intraperitoneal dose of 50 mg/kg bw of phenol was excreted in the bile of female Wistar albino rats, phenyl glucuronide predominating (Abou-el-Makarem et al., 1967). In male rats with ligated kidneys, the biliary excretion of-phenol conjugates was three tines that of rats with intact kidneys (Weitering et al., 1979).

A comparable pattern of metabolism is observed in other animal species. Rabbits given a single lethal dose of 500 mg/kg bw of phenol (No. 690) died or were killed 3, 15, 82, 120, 150, or 360 min later. Phenol was found after 3 min in all tissues tested. Conjugated phenol was present mostly in the liver and kidneys, indicating that detoxication had begun. After 15 min, the liver contained the highest concentration of free (64 mg/100 g) and conjugated phenol (0.9 mg/100 g). After 82 min, total (free and conjugated) phenol was approximately equally distributed in the blood, liver, kidneys, and lungs. After 360 min, approximately 65% of the total phenol was present in the conjugated form (Deichmann, 1944). Also in rabbits, phenol given as single doses of 125 and 250 mg/kg bw was excreted in the urine as phenyl glucuronide (70%) and phenyl sulfate (19%), with small amounts of quinol (1,4-benzenediol; 10%), catechol, and hydroxyquinol (4-hydroxyresorcinol) within 24 h (Porteous & Williams, 1949).

Sheep given a single oral dose of 25 mg/kg bw of phenol (No. 690) excreted 49% as phenylglucuronides, 32% as sulfate conjugates, and 12% as phosphate conjugates. Pigs given the same dose of phenol excreted 83% as glucuronic acid conjugates and only 1 % as sulfate conjugates. Rats receiving the same dose excreted 42% as glucuronic acid conjugates and 55% as sulfate conjugates. Phosphate conjugates were absent from the urine of phenol-treated pigs and rats. Sheep, pigs, and rats excreted 80-90% of the dose within 8 h, indicating rapid absorption and urinary elimination in all species (Kao et al., 1979).

Like phenols, polyphenols are conjugated primarily with sulfate or glucuronic acid and excreted. Three rabbits fed single doses of 100 mg/kg bw of resorcinol (No. 712) through a stomach tube excreted 52% of the dose as glucuronic acid conjugates, 14% as sulfate conjugates, and 11 % as free resorcinol (Garton & Williams, 1949). Male and female Fischer 344 rats that had been given resorcinol as a single oral dose of 112 mg/kg bw excreted about 70% in the urine as glucuronic acid conjugates within 24 h. The urine of females contained a greater portion of sulfate conjugates, while males excreted a larger percentage of a sulfate and glucuronide diconjugate. Less than 3% was excreted in the faeces of both males and females. The ratio of metabolic products was not significantly altered by administration of oral doses of 225 mg/kg-bw per day of resorcinol for 5 consecutive days (Kim & Matthews, 1987).

Phenyl salicylate (No. 736) would be expected to be hydrolysed to salicylic acid and phenol. A man was given one 90-mg capsule of phenyl salicylate each hour for 8 h, and his urine was collected for 72 h after the first dose, fractionated into 8-h collection periods. Total urinary phenol showed a peak of 470 mg/kg during the second collection period, when the concentration of free urinary phenol peaked at 25 mg/kg. Approximately 60 h after the first dose, the concentrations of total and free-urinary phenol had returned to-baseline levels,(7 and 1 mg/kg, respectively) (Fishbeck et al., 1975). Methyl salicylate, a related substance; was hydrolysed in rats, dogs; and humans in vivo (Davison et al., 1961).

Alkyl- and alkenyl-substituted phenols and their corresponding esters (Nos 691-711, 733, 735, and 737)

Monoalkyl-substituted phenols are conjugated mainly with sulfate or glucuronic acid in a manner similar to phenol (No. 690). They undergo a small degree of side-chain oxidation or ring hydroxylation, which results in a second hydroxyl group ortho or para to the existing hydroxyl group (Williams, 1959). In rabbits and dogs, a single oral dose of 200 mg/kg bw of ortho- or meta-cresol (Nos 691 and 692) was hydroxylated to a very minor extent (< 1 %), while up to 10% of para-cresol (No. 693) was oxidized on the side-chain and excreted as para-hydroxybenzoic acid (Bray et al., 1950a). In rats, < 0.2% of an oral dose of para-cresol of 100 mg/kg bw was ring hydroxylated and ortho-methylated to yield 4- and 5-methylguaiacol (2-methoxy-4 or 5-methylphenol) (Bakke, 1970; Bakke & Scheline, 1970).

Studies of metabolism in vitro provide evidence that a reactive metabolite of para-substituted alkylphenols is generated at high doses. The cytotoxicity of para-cresol (No. 693) and para-ethylphenol (No. 694) arises from the oxidative formation of reactive quinone methide intermediates (see Figure 1). At doses < 0.25 mmol/L, this reactive intermediate is detoxicated with glutathione, whereas at high concentrations (> 0.25 mmol/L), the quantity of quinone methides exceeds the availability of glutathione (Thompson et al., 1994).

Groups of 10 male B6C3F1 mice were given single doses of 2-phenylphenol (No. 735) at 15 or 800 mg/kg bw by oral gavage. Within 48 h, 84-98% of the administered dose was recovered in the urine of both groups, and 6-11% was recovered in the faeces; less than 1% was found in the tissues and carcass at necropsy. As part of the same study, two male and two female Fischer 344 rats received a single dose of 28 mg/kg bw by oral gavage, and 81 % of the administered dose was recovered in the urine within 12 h. In both species, most of the 2-phenylphenol that was recovered was in the form of glucuronic acid and sulfate conjugates. Glucuronic acid and sulfate conjugates of the para-hydroxylated metabolite, 4-hydroxy-2-phenylphenol, represented minor components of the recovered dose in both species (Bartels et al., 1998). In rats given a single oral dose of 1000 mg/kg bw of 2-phenylphenol, the glutathione conjugate of 4-hydroxy-2phenylphenol (at 4% of the dose) was detected in bile after 6 h. Conversion of 2-phenylphenol to 4-hydroxy-2-phenylphenol was shown to occur via the microsomal mono-oxygenase system in vitro (Nakagawa & Tayama, 1989).

Incorporation of a second alkyl substituent does not significantly alter the sulfate and glucuronic acid conjugation pathway. Doses of 1 g of 2,5-xylenol, 2,6-xylenol, or 3,4-xylenol (2;5-methylphenol, 2,6-methylphenol, and 3,4-methylphenol; Nos 706-708) given to rabbits were excreted as xylenyl sulfates (8-16%) and glucuronides (50-72%); only 1-3% of the dose was excreted unconjugated. Ring hydroxylation was evident in only a small portion of the metabolites of the 2,6-xylenol isomer (Bray et al., 1950b). Thymol (5-methyl-2-isopropylphenol; No. 23) (No. 709) and carvacrol (2-methyl-5-isopropylphenol) (No. 710) are also excreted after conjugation with sulfates and glucuronic acid. Thymoquinol (2-isopropyl-5-methyl-1,4-benzenediol)has been detected as a minor oxidation product of thymol (Williams, 1959). The period over which metabolites were collected was not specified in either study.

The simple esters of these alkyl-substituted phenols (Nos. 698-705) are expected to undergo hydrolysis in vivo, followed by conjugation and excretion as sulfate and glucuronic acid conjugates. ortho-Tolyl acetate (No. 698) was 60% hydrolysed in vitro after incubation with pancreatin for 2 h at 37 C (Grundschober, 1977). para-Vinylphenol (No. 711), a metabolite of styrene, was excreted as the glucuronic acid conjugate of para-vinylphenol or as a mandelic acid derivative formed by oxidation of the vinyl group (Pfäffli et al., 1981).

Alkoxyphenols (Nos 713-726)

Alkoxyphenols are excreted mainly as glucuronic acid or sulfate conjugates. The presence of both methoxy and hydroxy functions activates the ring towards further substitution, which may lead to the formation of polyphenols. In humans, 73% of a 50-mg dose of guaiacol (2-methoxyphenol; No. 713) was excreted as the sulfate and glucuronic acid conjugates within 14 h (Sedivec & Flek, 1970); however, the period of urine collection was not specified. In cats, a single oral dose of 2,6-dimethoxyphenol (No. 721) at 20 or 40 mg/kg bw was excreted mainly as conjugated 2,6-dimethoxy-4-hydroxyphenol after ortho demethylation (90% as disulfate) and as unconjugated (3%) 2,6-dimethoxy-4-hydroxyphenol within 24 h of dosing. Free (5%) and conjugated (2% with glucuronic acid) forms of the parent phenol were also detected (Miller et al., 1976).

Like the para-alkylphenols, 2-methoxy-4-alkylphenols can form electrophilic quinone methide intermediates at high cellular concentrations. 2-Methoxy-4-methylphenol (No. 715), 2-methoxy-4-propylphenol (No. 717), 2-methoxy-4-vinylphenol (No. 725), and 4-allyl-2,6-dimethoxyphenol (No. 726) were oxidized to quinone methides in rat liver slices in vitro. The half-lives of the quinone methides formed from these substances were 1, 6, 410, and 4300 s, respectively. Hepatocellular toxicity, measured as intracellular potassium leakage, was detected in rat liver slices incubated with each phenol for 6 h. These findings suggest that only quinone methides with half-lives of 10 s to 10 min are reactive enough to be cytotoxic at high concentrations (Thompson et al., 1995a).

Phenol derivatives with an alkyl side-chain containing a ketone function (Nos 727-732)

The presence of a ketone functiona confers the possibility of rapid metabolism like that of related phenols. Zingerone (3-methoxy-4-hydroxybenzylacetone; No. 730) given to rats as a single oral or intraperitoneal dose of 100 mg/kg bw was excreted in the urine mainly as glucuronic acid and sulfate conjugates. Reduction of the ketone to zingerol (12%) was also observed, with lesser ring hydroxylation to 4(3,4-dihydroxyphenyl)-2-butanone (6%) and side-chain oxidation to 4-hydroxy-3-methoxyphenylacetic acid (8%) (Monge et al., 1976).

The closely related 4-(para-hydroxyphenyl)-2-butanone (No. 728), the phenol derivative produced in the highest volume, would be expected to be metabolized by essentially the same pathways.

2.3.2 Toxicological studies

Studies-lasting 90 days and longer-term studies of toxicity and carcinogenicity were available for 12 of the 48 substances in this group and addressed-all four structural categories of phenol derivatives. Five concerned phenol or resorcinol (National Toxicology Program, 1980; Hirose et al., 1986, 1989; Maruyama et al., 1991; National Toxicology Program, 1992b), 12 were conducted with alkyl- or aryl-substituted phenols, and their corresponding esters (Hodge et al., 1952; Hagan et al., 1967; Posternak & Linder, 1969; Maasik, 1970; Hagiwara et al., 1984; Hiraga & Fujii, 1984; Hirose et al., 1986; Environmental Protection Agency, 1988a,b; National Toxicology Program, 1992a; Quast et al., 1997; Wahle et al., 1997), one was carried out with alkoxy phenols (Posternak & Linder, 1969), and two addressed phenol derivatives with an alkyl side-chain containing a ketone function (Gaunt et al., 1970; Kociba et al., 1976).

2-Phenylphenol (No. 735)

2-Phenylphenol (No. 735) was evaluated by the Committee at its eighth meeting, when an ADI of 0-0.2 mg/kg bw was established (Annex 1, reference 7). The 1999 Joint FAO/WHO Meeting on Pesticide Residues evaluated the toxicity of 2-phenylphenol and established an ADI of 0-0.4 mg/kg bw (FAO, 1999). As the available studies on the toxicology of 2-phenylphenol are summarized in the monograph (WHO, 2000), the data on this substance and its sodium salt (sodium ortho-phenylphenate) are not presented here. However, some consideration was given to studies of the genotoxicity of these substances, because the Joint Meeting found that there were unresolved questions with regard to the genotoxic potential of 2-phenylphenol.

2.3.2.1 Acute toxicity

LD50 values in rats treated orally were available for 29 of the 48 substances in this. group. The values ranged from 300 to 5700 mg/kg (Deichmann & Witherup, 1944; Hodge et al., 1952; Jenner et al., 1964; Taylor et al., 1964; Rinehart et al., 1967; Smyth et al., 1969; Gaunt et al., 1970; Denine & Palanker, 1973; Moreno, 1973; Levenstein,1974; McGee, 1974; Levenstein, 1975; Moreno, 1975, 1977, 1978; Sauer & Salem, 1980; Piccirillo et al. 1981; Piccirillo & Hartman, 1982; Klonne et al., 1988; Van den Heuvel, 1990; Schlicht et al., 1992; Berman et al., 1995). LD50 values for mice treated orally were available for 12 substances and ranged from 120 to 3100 mg/kg bw (MacIntosh, 1945; McOmie et al., 1949; Pellmont, 1973; Uzhdavini et al., 1974; Cioli et al., 1980; Moran et al., 1980; Schafer & Bowles, 1985; Hasegawa et al., 1989). Over half of the LD50 values reported are > 1000 mg/kg bw.

2.3.2.2 Short-term studies of toxicity

The usefulness of a number of the short-term studies of toxicity in mice and rats for evaluating the safety of the substances in this group of flavouring agents was

Table 3. Results of short-term studies of toxicity and long-term studies of toxicity and carcinogenicity on phenol and phenol derivatives used as flavouring agents administered orally

No.

Substance

Species; sex

No. test groupsa/ no. per groupb

Route

Duration

NOEL (mg/kg bw per day)

Reference

690

Phenol

Mice; M, F

5/20

Drinking-water

13 weeks

750

National Toxicology Program (1980)

690

Phenol

Mice; M, F

2/100

Drinking-water

103 weeks

< 620

National Toxicology Program (1980)

690

Phenol

Rats; M, F

5/20

Drinking-water

13 weeks

300

National Toxicology Program (1980)

690

Phenol

Rats; M, F

2/100

Drinking-water

103 weeks

250

National Toxicology Program (1980)

691

ortho-Cresol

Mice; M, F

5/40

Food

13 weeks

< 220 (M)
450 (F)

National Toxicology Program (1992a)

691

ortho-Cresol

Rats; M, F

5/40

Food

13 weeks

250

National Toxicology Program (1992a)

691

ortho-Cresol

Rats; M, F

3/60

Gavage

13 weeks

180

Environmental Protection Agency (1988a)

692, 693

meta-Cresol, para-cresolc

Mice; M, F

5/40

Food

13 weeks

440 (M)
220 (F)

National Toxicology Program (1992a)

692, 693

meta-Cresol, para-cresolc

Rats; M, F

5/40

Food

13 weeks

250

National Toxicology Program (1992a)

693

para-Cresol

Rats; M, F

3/60

Gavage

13 weeks

50

Environmental Protection Agency (1988b)

693

para-Cresol

Hamsters, M

1/15

Food

20 weeks

< 1800

Hirose et al. (1986)

694

para-Ethylphenol

Rats; M, F

1/20-32

Food

90 days

0.20c (M)
0.22c (F)

Posternak & Linder (1969)

733

4-(1,1-Dimethyl)ethyl phenol

Hamsters, M

1/15

Food

20 weeks

< 1800

Hirose et al. (1986)

707

2,6-Xylenol

Rats

2/13

Gavage

8 months

0.06

Maasik (1970)

708

3,4-Xylenol

Rats

2/13

Gavage

8 months

0.14

Maasik (1970)

709

Thymol

Rats; M, F

2/10

Food

19 weeks

1000d

Hagan et al. (1967)

735

2-Phenylphenol

Mice; M, F

3/100

Food

96 weeks

1300 (M)
<750 (F)

Hagiwara et al. (1984)

735

2-Phenylphenol

Mice; M, F

3/50

Food

2 years

< 250

Quest et al. (1997)

735

2-Phenylphenol

Rats; M, F

5/22-24

Food

13 weeks

410 (M)
430 (F)

Hiraga & Fujii (1984)

735

2-Phenylphenol

Rats, M

3/20-24

Food

91 weeks

270

Hiraga & Fujii (1984)

735

2-Phenylphenol

Rats; M, F

3/50

Food

2 years

100

(Hodge et al. (1952)

735

2-Phenylphenol

Rats; M, F

3/NR

Food

2 years

39

Wahle et al. (1997)

712

Resorcinol

Mice; M, F

5/20

Gavage

13 weeks

<28 (M)
220 (F).

National Toxicology Program (1992b)

712

Resorcinol

Mice; M, F

2/120

Gavage

104 weeks

220d (M)
110 (F)

National Toxicology Program (1992b)

712

Resorcinol

Rats; M, F

5/20

Gavage

13 weeks

< 32

National Toxicology Program (1992b)

712

Resorcinol

Rats, M

1/16

Food

51 weeks

800d

Hirose et al. (1989)

712

Resorcinol

Rats, F

3/60

Gavage

104 weeks

100

National Toxicology Program (1992b)

712

Resorcinol

Rats, M

2/60

Gavage

104 weeks

110

National Toxicology Program (1992b)

712

Resorcinol

Hamsters, M

1/15

Food

20 weeks

< 300

Hirose et al. (1986)

712

Resorcinol

Hamsters, F

1/15

Food

16 weeks

< 1800

Maruyama et al. (1991)

721

2,6-Dimethoxyphenol

Rats; M, F

1/20-32

Food

90 days

6.0d (M)
6.9 d (F)

Posternak & Linder (1969)

728

4-(para-Hydroxyphenyl)-2-butanone

Rats; M, F

4/30

Food

13 weeks

280 (M)
700 (F)

Gaunt et al. (1970)

M, male; F, female; if not listed, sex was not specified in the report; NR, not reported

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

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

c Administered as a mixture: meta-cresol, 60%; para-cresol, 40%

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

limited by their short duration; they are summarized briefly below. The results of. studies that were 90 days or longer in duration and in which NOELS were identified are described in greater detail and are shown in Table 3.

Phenol (No. 690)

Mice

A study to determine the doses for a subsequent 2-year study was conducted, in which groups of 10 male and 10-female B6C3F1 mice were provided with tap-water containing phenol (No. 690) at a concentration of 0, 100, 300, 1000, 3000,.or 10 000 mg/L for 13 weeks. The concentrations were calculated (Food & Drug Administration, 1993) to provide average intakes of 0, 25, 75, 250, 750, and 2500 mg/kg bw per day. All the mice survived to the end of the study: The mean body-weight gain of animals at the highest dose was depressed; animals in this group rejected the treated water. Microscopic examination revealed no treatment-related differences between treated and control mice. The NOELwas 750 mg/kg bw per day (National Toxicology Program, 1980).

Rats

A study to determine the doses for a subsequent 2-year study was conducted, in which groups of 10 male and 10 female Fischer 344 rats were provided with tapwater containing phenol (No. 690) at a concentration of 0, 100, 300, 1000, 3000, or 10 000 mg/L for 13 weeks. The concentrations were calculated (Food & Drug Administration, 1993) to provide average intakes of 0, 10, 30, 100, 300, or.1000 mg/ kg bw per day,. All the rats lived to the end of the study. Statistically significant decreases in mean body-weight gain were observed in animals of each sex at the highest dose; animals in this group rejected the treated water. No changes attributable to treatment were observed in tissues or organs. The NOEL was 300 mg/kg bw per day (National Toxicology Program, 1980).

ortho-Cresol (No. 691), meta-cresol (No. 692), and para-cresol (No. 693)

Mice

In a 28-day study of toxicity, groups of five male and five female B6C3F1 mice were fed diets containing ortho-cresol (No. 691), meta-cresol (No. 692), para-cresol (No. 693), or meta/para-cresol (60%/40%) at a concentration of 0, 300, 1000, 3000, 10 000, or 30 000 mg/kg, providing average intakes of 0, 62, 200, 580, 1700, or 4700 mg /kg bw per day. Nineteen animals died or were killed when moribund during the 28 days: two males and one female at the highest dose of ortho-cresol, one female at the second highest dose of meta-cresol, two males and two females at the highest dose of meta-cresol, one male at the second highest dose of para-cresol, and all animals at the highest dose of para-cresol; all animals treated with meta/para-cresol lived to the end of the study. Hunched posture, lethargy, and hypothermia were observed in the groups receiving the highest dose of any substance. At the end of the study, significantly decreased body-weight gains were reported for males or females receiving the two higher doses of any substance. A significantly increased relative liver weight over that of controls was reported in females given meta-cresol at the lowest dose, and this effect was observed consistently in animals at the three higher doses of any substance. Significantly increased relative weights of the brain and kidney were also found in groups receiving the two higher doses of any substance, but no microscopic changes were associated with these changes in organ weights. Uterine and ovarian atrophy were seen in several females mainly at the highest doses of ortho-, meta-, or meta/para-cresol. No gross lesions were seen in the organs of treated animals, but renal and hepatic necroses were prevalent in animals receiving the highest dose of para-cresol, all of which died early in the study. Bone-marrow hypocellularity was found in some mice at the highest doses of para- and meta/para-cresol, and mild nasal and respiratory epithelial hyperplasia was found in most of these animals-presumably due to irritation caused by the test substance. Minimal hyperplasia of the oesophagus, forestomach, and/or respiratory squamous-cell epithelium was also reported among animals in these groups (National Toxicology Program, 1992a).

In a 13-week study of toxicity, groups of 10 male and 10 female B6C3F1.mice were fed diets containing ortho-cresol (No. 691) at a concentration of 0, 1250, 2500, 5000, 10 000, or 20 000 mg/kg or meta/para-cresol (60%/40%) at concentrations of 0, 625, 1250, 2500, 5000, or 10 000 mg/kg. The average intakes were reported to have been 0, 220, 450, 860, 2200, and 6000 mg/kg bw per day of ortho-cresol and 0, 110, 220, 440, 850, and 3200 mg/kg bw per day of meta/para-cresol. All mice lived to the end of the study, when the body weights of males and females receiving 20 000 mg/kg of diet of ortho-cresol were reduced by about 16% and 21%, respectively, and those of males and females receiving 10 000 mg/kg of diet of meta/para-cresol were reduced by about 9% and 7%, respectively, when compared with controls. Females receiving 10 000 mg/kg of diet of ortho-cresol also showed statistically significant decreases in weight gain (9% reduction compared with controls). Necropsy revealed significant increases in the relative weights of the liver in all groups of males given ortho-cresol and in males receiving 5000 or 10 000 mg/ kg of diet of meta/para-cresol. These responses are consistent with the known hepatocellular sensitivity of male B6C3F1 mice (Maronpot & Boorman, 1982; Maronpot et al., 1987). The relative liver weights were also significantly increased in female mice receiving the three higher doses of ortho-cresol or the highest dose of meta/para-cresol. Males given the highest dose of ortho-cresol had significantly increased relative testicular weights and males and females at this dose had increased relative thymus weights. Haematology, clinical chemistry, and urinary analysis revealed normal values. Microscopic examination of liver tissue showed no remarkable changes. Minimal epithelial hyperplasia was seen in the forestomach in 4 of 10 males and 3 of 10 females receiving the highest dose of ortho-cresol. An increased incidence of hyperplasia of the nasal respiratory epithelium was found in males receiving the two higher doses of meta/para-cresol (4/10 at 5000 mg/kg of diet and 10/10 at 10 000 mg/kg of diet) and in females receiving the highest dose of this substance (7/10). Females given 20 000 mg/kg of diet of ortho-cresol had lengthened estrus cycles. No biologically significant changes in reproductive function were noted in animals given meta/para-cresol at any dose (National Toxicology Program, 1992a).

Rats

A 28-day range-finding study was conducted in which groups of five male and five female Fischer 344 rats were fed diets containing ortho-cresol (No. 691), meta-cresol (No: 692), para-cresol (No. 693), or meta/para-cresol (60%/40%) at a concentration of 0, 300, 1000, 3000, 10 000, or 30 000 mg/kg, providing average intakes of each substance of 0; 26, 87, 260, 860, and 2400 mg/kg bw per day. All rats lived to the end of the study. No clinical signs of toxicity were observed in rats given ortho- or meta-cresol, but male and female rats given the highest dose of para- or meta/para-cresol appeared thin, and animals at the highest dose of each cresol isomer had significantly reduced body-weight gain and decreased food consumption. Significantly increased relative liver weights were observed consistently in groups given the two highest doses of each isomer; a similar effect was seen in males given ortho- or meta/para-cresol at 3000 mg/kg of diet and in females given meta/para-cresol at 1000 or 3000 mg/kg of diet or para-cresol at 3000 mg/kg of diet. Significantly increased relative kidney weights were also observed generally at the two highest doses. The relative weights of the brain and testes were increased in animals at the highest doses; probably due to reduced body-weight gain. No gross or microscopic lesions were recorded in rats treated with ortho-cresol. Mild to moderate uterine atrophy was reported in females given the highest dose of meta- or para-cresol. para-Cresol induced bone-marrow hypocellularity in males at the three higher doses and in females at the two higher doses. This effect was also observed in rats given the highest dose of meta/para-cresol. Male and female rats at the three higher doses of this substance had mild epithelial hyperplasia of the nasal cavity, oesophagus, and forestomach. No gross lesions were seen in the organs of any treated animal (National Toxicology Program, 1992a).

A 13-week study was conducted in which ortho-cresol (No. 691) was given to groups of 30 male and 30 female Sprague-Dawley rats at a dose of 0, 50, 180, or 600 mg/kg bw per day by oral gavage in corn oil. Of the animals at the high dose, 19 females and nine males died during the study; no deaths occurred in the other groups. At the end of the study, males at the high dose showed a 10% reduction in body-weight gain when compared with controls. Clinical chemistry, haematology, urinary analyses, and measurements of organ weights revealed no significant changes in treated rats, No gross or histological lesions and no opthalmological changes were found. Congestion, oedema, and oil droplets were found in the lungs of the 28 animals at the high dose that died during the study, but the authors suggested that these findings were related to terminal stress and aspiration of the mixture of ortho-cresol and corn oil during convulsive seizures. The NOEL was 180 mg/kg bw per day (Environmental Protection Agency, 1988a).

Groups of 20 Fischer 344 rats of each sex were fed diets containing ortho-cresol or meta/para-cresol (60%/40%) at a concentration of 0, 1900, 3800, 7500, 15 000, or 30 000 mg/kg for 13 weeks, providing average intakes of 0, 130, 250, 500, 1000, and 2000 mg/kg bw per day. All rats lived to the end of the study except for one female receiving the highest dose of ortho-cresol. At the end of the study, the body weights of males and females at the two higher doses of both substances were significantly lower than those of. controls, except for males given 15 000 mg/kg of diet of ortho-cresol. The relative weights of the liver were increased in males and females at the three higher doses of both isomers. The relative weights of the kidney were increased in animals at the two higher doses of ortho-cresol and in males at the three higher doses and females at the highest dose of meta/para-cresol. The relative weights of the testes were increased in males at the highest dose of ortho-cresol and at the two higherdoses of meta/para-cresol. Substantially increased concentrations of serum bile salts were reported in male and female rats given either isomer, particularly at the two highest doses, throughout the study. Consistent decreases in the activity of 5'-nucleotidase in serum were seen in rats given meta/para-cresol. Mild uterine atrophy was observed at the two higher doses of meta/para-cresol. Minimal to mild hypocellularity of the bone marrow was reported in males. and females at the two higher doses of both isomers, except in males at 15 000 mg/kg of diet of ortho-cresol. All animals receiving meta/para-cresol showed hyperplasia of the olfactory and respiratory epithelium. Both isomers lengthened the estrus cycle of females treated at 7500 or 30 000 mg/kg of diet. The NOEL for both ortho-cresol and meta/para-cresol was 250 mg/kg bw per day (National Toxicology Program, 1992a).

A 13-week study was conducted in which groups of 30 male and 30 female Sprague-Dawley rats were given para-cresol at a dose of 0, 50, 175, or 600 mg/kg bw per day by oral gavage. At the end of the study, the mean body weights of males and females at the highest dose were significantly lower than those of controls. Females at the intermediate and high doses showed statistically significant reductions in erythrocyte count, haemoglobin, and haematocrit, but compensatory responses to the anaemic state were not observed. Females at the highest dose also had elevated liver transaminase activity, but this finding was attributed to large increases in 4/10 animals, including two with chronic hepatic inflammation. Statistically significant increases in serum cholesterol were reported in females at the highest dose and in total protein in males at the two higher doses. Necropsy revealed significantly increased relative kidney weights in males at the intermediate dose and in males and females at the highest dose. Significantly increased relative weights of the liver, heart, testis, and brain were noted in males at the highest dose, while females at the lowest dose showed significantly increased relative spleen weights. Gross examination showed no treatment-related lesions. Histological evaluation revealed a significantly increased incidence of chronic nephropathy in males at the lowest and highest doses. A significantly increased incidence of epithelial metaplasia of the trachea was observed in animals at the highest dose than in controls, but no other treatment-related histological findings were reported. The NOEL was 50 mg/kg bw per day (Environmental Protection Agency, 1988b).

para-Ethylphenol (No. 694)

Groups of 20-32 male and female Charles River CD rats were fed a basal diet supplemented with para-ethylphenol (No. 694) for 90 days, giving average intakes of 0.20 mg/kg bw per day for males and 0.22 mg/kg bw per day for females. A control group was fed the basal diet alone. Weekly measurements of body weight, food consumption, and the calculated efficiency of food use showed no significant differences from controls. Haematological examinations and clinical chemistry performed at weeks 7 and 13 showed no effects. At necropsy, there were no significant changes in liver or kidney weights. Gross and histological examination of a wide range of organs (unspecified in the report) revealed no lesions related to treatment with either substance. The NOEL was 0.20 mg/kg bw per day (Posternak & Linder, 1969).

2,6-Xylenol (No. 707)

Groups of 13 white rats (sex not specified) were given 2,6-xylenol (No. 707) at a dose of 0.06 or 6.0 mg/kg bw per day by stomach tube for 8 months. No effects on weight gain, haemoglobin concentration, or erythrocyte count were seen, but the higher dose had adverse effects on the cells of the liver, spleen, kidneys, and heart. No pathological or histological changes were seen at the lower dose. The NOEL was 0.06 mg/kg bw per day (Maasik, 1970).

3,4-Xylenol (No. 708)

In the study described above, groups of 13 white rats were given 3,4-xylenol (No. 1708) at a dose of 0.14 or 14 mg/kg bw per day by stomach tube for 8 months. Animals given the higher dose showed delayed weight gain and significantly decreased haemoglobin concentration and erythrocyte count. Adrenaline injections were used to study the effects of 3,4-xylenol on the capacity to regulate blood pressure. This capacity was impaired at the higher dose, but it is unclear whether the effect was statistically significant. The high dose had adverse effects on the cells of the liver, spleen, kidneys, and heart, but no pathological or histological changes were seen at the lower dose. The NOEL was 0.14 mg/kg bw per day (Maasik, 1970).

Thymol (No. 709)

Two groups of five male and five female weanling Osborne-Mendel rats received a diet containing thymol (2-isopropyl-5-methylphenol, No. 709) at a concentration of 1000 or 10 000 mg/kg for 19 weeks, calculated (Food & Drug Administration, 1993) to provide average intakes of 50 or 500 mg/kg bw per day. Body weights, food intake, and the general condition of each animal were recorded weekly. At the end of the study, haematological parameters were measured in both groups and animals at the higher dose were necropsied. No effects on growth or haematological parameters were reported, and no macroscopic or microscopic changes were observed in tissues from rats at the higher dose. The NOEL was 1000 mg/kg bw per day (Hagan et al., 1967).

Resorcinol (No. 712)

Mice

Groups of five male and five female B6C3F1 mice were given resorcinol (No. 712) at a dose of 0, 37.5, 75, 150, 300, or 600 mg/kg bw per day by oral gavage on 5 days/week for 17 days. The clinical findings at the three higher doses included prostration and tremors lasting up to 2 h. One male at 300 mg/kg bw per day and five females and four males at 600 mg/kg bw per day died during the study. At necropsy, the organ weights were found to be similar to those of control animals. Microscopic examination of tissues revealed no abnormalities attributable to treatment (National Toxicology Program, 1992b).

Groups of 10 male and 10 female B6C3F1 mice were given resorcinol (No. 712) at a dose 0, 28, 56, 112, 220, or 420 mg/kg bw per day by oral gavage for 13 weeks. Deaths during the study occurred only among animals at the highest dose (seven males and seven females during the first week, one male at week 4, and one female at week 12). Dypnoea, prostration, and tremors were observed in males at the highest dose-within 30 min after treatment. At the end of the study, the two surviving male; at this dose showed a statistically significant decrease in mean body weight. No other significant changes in body weight were observed. Significant decreases in the absolute and relative weights of the adrenal gland were observed in all groups of treated males. Haematology and clinical chemistry showed no effects, and gross and microscopic examinations revealed no lesions related to treatment. The NOEL was 220 mg/kg, bw per day (National Toxicology Program, 1992b).

Rats

Groups of five male and five female Fischer 344/N rats were given resorcinol (No. 712) at a dose of 0, 27.5, 55, 110, 220, or 450 mg/kg bw per day by oral gavage on 5 days/week for 17 days. Males at the two higher doses and females at the four higher doses showed clinical signs including hyperexcitability and tachypnoea lasting up to 2 h. None of the rats died. The final mean body weights of the treated animals were comparable to those of controls. Females at the highest dose had significantly decreased absolute and relative thymus weights, but no other significant changes in organ weights were reported. Microscopic examination of the tissues revealed no abnormalities attributable to treatment (National Toxicology Program, 1992b).

Groups of 10 male and 10 female Fischer 344/N rats were given resorcinol (No 712) at a dose 0, 32, 65, 130, 260, or 520 mg/kg bw per day by oral gavage for 13 weeks. Eight males and 10 females at the highest dose died within the first 4 weeks; the deaths of two males and four females in the group at 260 mg/kg by per day dose during the first week were attributed to accidental administration o 520 mg/kg bw per day. The final mean body weights of the treated animals were comparable to those of controls. At necropsy, statistically significant increases it the absolute and relative weights of the liver were reported for males receiving 130 or 260 mg/kg bw per day and females receiving 65, 130, or 260 mg/kg bw per day of resorcinol. Significantly increased absolute and relative adrenal gland weights were also reported in all surviving treated males. Haematology, clinical chemistry, and gross and microscopic examination revealed no findings related to treatment (National Toxicology Program, 1992b).

2,6-Dimethoxyphenol (No. 721)

Groups of 20-32 male and female Charles River CD rats were fed a basal diet supplemented with 2,6-dimethoxyphenol (No. 721) for 90 days, providing average intakes of 6.0 mg/kg bw per day for males and 6.8 mg/kg bw per day for females. A control group was fed the basal diet alone. Weekly measurements of body weight, food consumption, and calculated efficiency of food use showed no significant differences from controls. Haematological and clinical chemical examinations at weeks 7 and 13 showed no effects. At necropsy, there were no significant changes in liver or kidney weights. Gross and histological examination of a wide range of organs (unspecified in the report) revealed no lesions related to treatment with either substance. The NOEL was 6.0 mg/kg bw per day (Posternak & Linder, 1969).

4-(para-Hydroxyphenyl)-2-butanone (No. 728)

Groups of 15 male and 15 female SPF-derived CFE rats were given diet; containing 4-(para-hydroxyphenyl)-2-butanone (No. 728) at a concentration of 0, 0.1, 0.2, 0.4, or 1.0% for 13 weeks. The average intakes, calculated on the basis of data on body weights and food consumption, were 0, 70, 140, 280, and 700 mg/kg bw per day. No changes in appearance, behaviour, or food intake were noted at any time during the study. A slight but statistically significant (p < 0.05) reduction in weight gain was observed in male rats at the highest dose from week 5 onwards, but the decrease in weight was not associated with decreased food consumption. No significant differences were found in absolute organ weights. In males, the relative weights of the liver and kidney were increased at the two higher doses and those of the adrenal gland at the highest dose. The increases in relative organ weights may have been due to the decrease in body weight. Urinary analyses showed ketones in the urine of all treated animals at 7 and 13 weeks. The authors reported that this effect appeared within 12 h in rats fed a diet containing 1% 4-(para-hydroxyphenyl)-2-butanone for-7 days, and disappeared within 9 h when the rats were returned to normal diet. The ketonuria, which was possibly due to metabolites present in the urine, was considered not to be a toxic effect. Histopathological examination of rats at the highest dose showed no effect of treatment on organs of the digestive, reproductive, circulatory, or central nervous systems (Gaunt et al.,1970). The NOEL of 280 mg/kg bw per day is 10 000 times greater than the estimated daily per capita intake of 4-(para-hydroxyphenyl)-2-butanone in Europe (46 g/kg bw per day) and the USA (63 g/kg bw per day) from use as a flavouring substance.

Phenyl salicylate (No. 736)

A study in which three beagle dogs (sex not specified) were given phenyl salicylate (No. 736) at a dose of 250 or 500 mg/kg bw per day in a capsule for 51 days was reported as an abstract. All the treated animals showed decreased appetite, body weight, and activity and dark urine and faeces. The serum activities of alanine and aspartate aminotransferases were elevated. In a subsequent study, three beagle dogs were given phenyl salicylate at a dose of 125 mg/kg bw per day in a capsule for 51 days. At this dose, phenol excretion was markedly elevated. Haematology and tests for liver and kidney function, performed regularly, showed no effects. At necropsy, no gross or microscopic abnormalities were observed, but the usefulness of this study is limited by deficiencies in reporting (Kociba et al., 1976).

2.3.2.4 Long-term studies of toxicity and carcinogenicity

Phenol (No. 690)

Mice

Groups of 50 male and 50 female B6C3F1 mice were given tap-water containing phenol (No. 690) at a concentration of 0, 2500, or 5000 mg/L for 103 weeks, calculated (Food & Drug Administration, 1993) to provide average intakes of 0, 620, or 1200 mg/kg bw per day. The animals were observed twice daily and weighed every 2 weeks during the first 12 weeks, monthly thereafter, and at the end of the study. Water consumption was recorded weekly. Gross and histological examinations were performed on all major organs and tissues, including those of animals found dead or killed moribund during the study. No clinical signs of toxicity were seen. Although the mean body weights were significantly reduced at most times in all treated groups, the food consumption did not differ significantly from that of controls. Water consumption was depressed by 25% in the low-dose group and 40-50% in the high-dose group compared with controls. Neoplasms were observed in both control and treated animals but were of similar number and type to those observed in ageing control B6C3F1 mice in previous studies. Phenol was considered not to be carcinogenic, and there was no histological evidence of toxicity (National Toxicology Program; 1980).

Rats

Groups of 50 male and 50 female Fischer 344 rats were given tap-water containing phenol (No. 690) at a concentration of 0; 2500; or 5000 mg/L for 103 weeks calculated (Food & Drug Administration, 1993) to provide average intakes of 0, 250, or 500 mg/kg bw per day. The same protocol of observations and necropsy as used in the study in mice described above was followed. There was no significant difference in the rate of survival between treated and control rats. Animals at the high dose had lower mean body weights than controls, although the food consumption was comparable. The water consumption of rats at the low and high doses was depressed by 20% and 10% of that of controls, respectively. No clinical signs of toxicity were observed. Significantly increased incidences of leukaemias, lymphomas, and phaeochromocytomas of the adrenal medulla were observed only in males at the low dose. However, none of the tumours were clearly associated with exposure to phenol. The NOEL was 250 mg/kg bw per day (National Toxicology Program, 1980).

Resorcinol (No. 712)

Mice

Groups of 60 male and 60 female B6C3F1 mice were given resorcinol (No. 712) at a dose of 0, 110, or 220 mg/kg bw per day in deionized water by oral gavage on 5 days/week for 104 weeks. All animals were observed twice daily, and clinical signs were noted every 4 weeks. Body weights were measured weekly through week 13 and every 4 weeks thereafter until the last 3 months, when they were recorded every 2 weeks. Ten mice of each sex per dose were selected for interim evaluations of organ weights, haematological and clinical chemical parameters, and histology. Necropsies were performed on all remaining animals at the end of the study. Complete histological examinations were conducted on all control and high-dose mice. For animals at the low dose, only tissues containing gross lesions were examined microscopically at both the interim and terminal evaluations.

The mean body weights of all treated males and females at the low dose were comparable to those of controls throughout the study; however, the mean body weights of females at the high dose were 10-15% lower than those of controls from week 85 through week 104. Recumbence and tremors were common among all treated mice for a short period after dosing. No treatment-related effects were reported at the 15-month interim evaluation. The survival rate of treated mice at termination was similar to that of controls. Some animals examined at 6, 12, 18, and 24 months had mouse hepatitis virus, but no clinical or histological evidence of disease was detected. The incidences of neoplastic and non-neoplastic lesions were not statistically or biologically increased in any group of mice. The clinical signs (ataxia, recumbency and tremors) observed in all treated mice suggest an effect on the central nervous system; however, there were no associated morphological findings. Similar clinical signs were observed in rats in the bioassay described below (National Toxicology Program, 1992a).

Rats

Groups of 60 male and 60 female Fischer 344/N rats were given resorcinol (No. 712) at a dose of 0, 110, or 220 mg/kg bw per day in deionized water by oral gavage on 5 days/week for 103 weeks. Some rats were given one or two extra doses during week 104. As 16 females at the high dose died after 22 weeks of treatment, four further groups of 60 females were given doses of 0, 50, 100, or 150 mg/kg bw per day. The same protocol of observations and evaluations as used in the study in mice described above was followed. Ten rats of each sex per dose were used for interim evaluations of organ weights, haematology, clinical chemistry, and histology at 15 months. As several males at the high dose died prematurely, the remaining animals in the interim group of males at this dose were not killed as originally planned. Instead, 10 males at the high dose in the 2-year study group that either died or were killed when moribund during weeks 62 and 67 were used for the interim histological evaluation. Organ weighings, haematology, and clinical chemistry were not performed in this group of male rats.

At the 15-month interim evaluation, significantly reduced terminal body weights were found for high-dose males (220 mg/kg bw per day) and females (150 mg/kg bw per day). Significantly increased relative weights of the brain were found in males at the low dose and of the liver in females at the high dose, which were associated with decreased body weights. Haematology and clinical chemistry revealed no treatment-related effects (assessed in female rats only). No treatment-related neoplastic or non-neoplastic lesions were found in any group.

The mean body weights of males given 220 mg/kg bw per day and females given 150 mg/kg bw per day were significantly lower than those of controls throughout the 2-year study, whereas the mean body weights of animals at the low dose were comparable to those of controls. Haematology and clinical chemistry revealed normal values. Ataxia, prostration, salivation, and tremors were observed shortly after treatment in all groups of treated males and in females receiving 100 or 150 mg/kg bw per day. These effects lasted for 0.5-1 h and became more pronounced at the end of each 5-day dosing period. The rate of survival of males and females at the high dose was significantly lower than that of controls. At the end of the study, 57% of control males, 15% of high-dose males, 68% of control females, and 50% of high-dose females were still alive. Some animals examined at 6,12, 18, and 24 months were found to be infected with Sendai virus and rat sialodacryoadenitis virus (which is a type of rat coronavirus); however, no clinical or histological evidence of disease was found. Complete histological examination on all remaining animals at the end of the study revealed no pathological lesions. The incidence of neoplastic or non-neoplastic lesions was not statistically or biologically significantly increased in any treated group. A statistically significant decrease in the incidence of mammary gland fibroadenomas was observed in all three groups of treated females, the lesions occurring in 18% of high-dose females, 24% of mid-dose females, 28% of low-dose females, and 50% of control females. In all groups, the incidences were within the range of historical controls (16-53%). Although clinical signs (ataxia, prostration, salivation, and tremors) suggesting an effect of resorcinol on the central nervous system were observed in male rats at both doses and females at the two higher doses, there were no related morphological findings. Similar clinical signs were observed in mice in the bioassay described above (National Toxicology Program, 1992b).

2.3.2.5 Genotoxicity

In vitro

Negative results were reported in the standard assay for reverse mutation in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 incubated with phenol at concentrations up to 9400 g/plate (Florin et al., 1980; Gocke et al., 1981; Pool & Lin, 1982; Haworth et-al., 1983; Aeschbacher et al.; 1989; Massey et al., 1994); ortho-cresol at up to 5000 g/plate (Douglas et a1., 1980; Florin et al., 1980 Nestmann et al., 1980; Pool & Lin, 1982; Haworth et al., 1983; Massey et al., 1994); meta-cresol and para-cresol at up to 5000 g/plate (Douglas et a1., 1980; Florin et al., 1980; Nestmann et al., 1980; Pool & Lin,.1982;Haworth et al., 1983); para-ethylphenol, 2,5-xylenol, 2,6-xylenol, and 3,4-xylenol at 367 g/plate (Florin et al., 1980); 4-(1,1-dimethyl)ethyl phenol at up to 2000 g/plate (Dean et al., 1985); thymol at up to 1000 g/plate (Florin et al., 1980; Azizan & Blevins, 1995); 2-phenylphenol at up to 200 g/plate (National Toxicology Program, 1986); resorcinol at up to 7700 g/plate (Gocke et al., 1981; Haworth et al., 1983); guaiacol at up to 111 726 g/plate (Douglas et al., 1980; Nestmann et al., 1980; Pool & Lin, 1982; Haworth et al., 1983; Aeschbacher et al., 1989); 2,6-dimethoxyphenol at up to 16 000 g/plate (McMahon et al., 1979; Douglas et al., 1980; Florin et al., 1980; Pool & Lin, 1982); 2-hydroxyacetophenone at 408 g/plate (Florin et al., 1980); and phenyl salicylate at up to 333 g (Zeiger et al., 1987), with and without metabolic activation (see Table 4). However, in an assay with a modified minimal ZLM medium for Escherichia coli, the results varied by bacterial strain (Gocke et al., 1981). At concentrations of 470-9400 g/plate, phenol caused reverse mutation only in strain TA98 in the presence of metabolic activation. Resorcinol was mutagenic at doses of 550-7700 g/plate only in TA1535 without metabolic activation and in TA100 with metabolic activation. The same authors reported negative results in all five S. typhimurium strains with and without metabolic activation in the standard Vogel-Bonner medium, which contains a concentration of citrate and other ions that is two to four times higher (Gocke et al., 1981). Negative results with phenol and resorcinol at doses up to 3333 g/plate were reported in another study when Vogel-Bonner medium was used (Haworth et al., 1983). No mutagenicity was found in E. coli exposed to 2,6-dimethoxyphenol at concentrations up to 1000 g/ml (McMahon et al., 1979).

Forward mutation was not induced in mouse lymphoma L5178YTk+/- cells by phenol at concentrations of 100-3200 g/ml or resorcinol at 125-2000 g/ml without metabolic activation (McGregor et al., 1988). However, positive results were reported with phenol at concentrations of 5-42 g/ml with activation and 178-887 g/ml without activation (Wangenheim & Bolcsfoldi, 1988). Positive results in this assay were also reported with 2-phenylphenol at doses of 0.32-60 g/ml without metabolic activation and 0.32-5 g/ml with activation, but these doses were cytotoxic (National Toxicology Program, 1986).

Sister chromatid exchange was not induced in human lymphocytes by phenol at concentrations up to 188 g/ml, ortho-cresol at up to 54 g/ml, meta-cresol at up to 108.g/ml, para-cresol at up to 54 g/ml, para-ethyl phenol at up to 27 g/ml, 2,6-xylenol at up to 31 g/ml, resorcinol at up to 28 g/ml, 2,6-dimethoxyphenol at up to 77 g/ml (Jansson et al., 1986, 1988) or para-vinylphenol at up to 12 g/ml (Jansson et al., 1988) (see Table 4). Phenol at 19 g/ml also did not induce sister chromatid exchange in human lymphocytes, but positive results were reported when the

Table 4. Results of studies of genotoxicity with phenol and phenol derivatives used as flavouring substances

No.

Substance

End-point

Test system

Concentration

Result

Reference

690

Phenol

Reverse mutation

S. typhimurium TA98, TA100, TA102

6.59-6587 g/platea,b

Negative

Aeschbacher et al. (1989)

690

Phenol

Reverse mutation

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

3 g/platea,b

Negative

Florin et al. (1980)

690

Phenol

Reverse mutation

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

0-9400 g/platea,b

Negative

Gocke et al. (1981)

690

Phenol

Reverse mutationc

S. typhimurium TA98

0-9400 g/platea
470-9400 g/plateb

Negative
Positive

Gocke et al. (1981)

690

Phenol

Reverse mutationc

S. typhimurium TA100, TA1535, TA1537, TA1538

0-9400 g/platea,b

Negative

Gocke et al. (1981)

690

Phenol

Reverse mutation

S. typhimurium TA1535, TA1537, TA98, TA100

33-3333 g/platea,b

Negative

Haworth et al. (1983)

690

Phenol

Reverse mutation

S. typhimurium TA98, TA100

5 g/platea,b

Negative

Massey et al. (1994)

690

Phenol

Reverse mutation

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

5000 g/platea,b

Negative

Pool & Lin (1982)

690

Phenol

Forward mutation

Mouse lymphoma cells

100-3200 g/mla

Negative

McGregor et al. (1988)

690

Phenol

Forward mutation

Mouse lymphoma cells

178-887 g/mla
5-42 g/mlb

Positive

Wangenheim & Bolcsfoldi (1988)

690

Phenol

Sister chromatid exchange

Human lymphocytes

0-188 g/ml

Negative

" Jansson et al. (1986)

690

Phenol

Sister chromatid exchange

Human lymphocytes

19 g/ml
94 g/ml

Negative
Positive

Morimoto & Wolff (1980)

690

Phenol

Sister chromatid exchange

Chinese hamster ovary cells

300-400 g/mla
1670-3000 g/mlb

Positive
Weakly positive

Ivett et al. (1989)

690

Phenol

Chromosomal aberration

Chinese hamster ovary cells

600-800 g/mla
2000-3000 g/mlb

Negative
Positive

Ivett et al. (1989)

690

Phenol

DNA strand breaks

Chinese hamster ovary cells

94, 110 g/ml

Negative

Sze et al. (1996)

690

Phenol

Micronucleus formation

Human lymphocytes

94 g/mla

Negative

Robertson et al. (1991)

691

ortho-Cresol

Reverse mutation

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

2.5 l/platea,b

Negative

Douglas et al. (1980)

691

ortho-Cresol

Reverse mutation

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

324 g/platea,b

Negative

Florin et al. (1980)

691

ortho-Cresol

Reverse mutation

S. typhimurium TA 1535, TA1537, TA98, TA100

1-100 g/platea,b

Negative

Haworth et al. (1983)

691

ortho-Cresol

Reverse mutation

S. typhimurium TA98, TA100

5 g/platea,b

Negative

Massey et al. (1994)

691

ortho-Cresol

Reverse mutation

S. typhimurium TA98, TA100, TA7535, TA1537 TA1538

2600 g/platea,b

Negativeb

Nestmann et al. (1980)

691

ortho-Cresol

Reverse mutation

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

5000 g/platea,b

Negative

Pool & Lin (1982)

691

ortho-Cresol

Sister chromatid exchange

Human fibroblasts

86.5-433 g/ml
865 g/ml

Negative
Weakly positive

(Cheng & Kligerman (1984)

691

ortho-Cresol

Sister chromatid exchange

Human lymphocytes

0-54 g/ml

Negative

Jansson et al. (1986)

691

ortho-Cresol

Sister chromatid exchange

Human lymphocytes

0-54 g/ml

Negative

Jansson et al. (1988)

692

meta-Cresol

Reverse mutation

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

2000 g/platea,b

Negative

Douglas et al. (1980)

692

meta-Cresol

Reverse mutation

S. typhimurium TA98, A100, TA1535, TA1537, TA1538

324 g/platea,b

Negative

Florin et al. (1980)

692

meta-Cresol

Reverse mutation

S. typhimurium TA 1535, TA1537, TA98, TA100

3.3-333 g/platea,b

Negatived

Haworth et al. (1983)

692

meta-Cresol

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537 TA1538

2000 g/platea,b

Negatived

Nestmann et al. (1980)

692

mete-Cresol

Reverse mutation

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

5000 g/platea,b

Negative

Pool & Lin (1982)

692

meta-Cresol

Sister chromatid exchange

Human fibroblasts

86.5-865 g/ml

Negative

Cheng & Kligerman (1984)

692

meta-Cresol

Sister chromatid exchange

Human lymphocytes

0-108 g/ml

Negative

Jansson et al. (1986)

692

meta-Cresol

Sister chromatid exchange

Human lymphocytes

0-108 g/ml

Negative

Jansson et al. (1988)

693

para-Cresol

Reverse mutation

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

1000 g/platea,b

Negative

Douglas et al. (1980)

693

para-Cresol

Reverse mutation

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

324 g/platea,b

Negative

Florin et al. (1980)

693

para-Cresol

Reverse mutation

S. typhimurium TA 1535, TA1537, TA98, TA100

3.3-333 g/platea,b

Negative

Haworth et al. (1983)

693

para-Cresol

Reverse mutation

S. typhimurium TA98, TA 100

5 g/platea,b

Negative

Massey et al. (1994)

693

para-Cresol

Reverse mutation

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

1000 g/platea,b

Negatived

Nestmann et al. (1980)

693

para-Cresol

Reverse mutation

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

5000 g/platea,b

Negative

Pool and Lin (1982)

693

para-Cresol

Sister chromatid exchange

Human fibroblasts

86.5-865 g/ml

Negative

Cheng & Kligerman (1984)

693

para-Cresol

Sister chromatid exchange

Human lymphocytes

0-54 g/ml

Negative

Jansson et al. (1986)

693

para-Cresol

Sister chromatid exchange

Human lymphocytes

0-54 g/ml

Negative

Jansson et al. (1988)

694

para-Ethylphenol

Reverse mutation

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

367 g/platea,b

Negative

Florin et al. (1980)

694

para-Ethylphenol

Sister chromatid exchange

Human lymphocytes

0-27 g/ml

Negative

Jansson et al. (1986)

694

para-Ethylphenol

Sister chromatid exchange

Human lymphocytes

0-2.7 /ml

Negative

Jansson et al. (1988)

733

4-(1,1-Dimethyl)-ethyl phenol

Reverse mutation

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

0.2-2000 g/platea,b

Negative

Dean et al. (1985)

733

4-(1,1-Dimethyl)-ethyl phenol

Reverse mutation

E. coli. WP2 and WP2 uvrA

0.2-2000 g/platea,b

Negative

Dean et al. (1985)

733

4-(1,1-Dimethyl)-ethyl phenol

Mitotic gene conversion

S. cerevisiae JD1

0.2-2000 g/platea,b

Negative

Dean et al. (1985)

733

4-(1,1-Dimethyl)-ethyl phenol

Chromosomal aberration

Rat liver cell lines RL1, RL2

Not specifiede

Negative

Dean et al. (1985)

706

2,5-Xylenol

Reverse mutation

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

367 g/platea,b

Negative

Florin et al. (1980)

707

2,6-Xylenol

Reverse mutation

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

367 g/platea,b

Negative

Florin et al. (1980)

707

2,6-Xylenol

Sister chromatid exchange

Human lymphocytes

0-31 g/ml

Negative

Jansson et al. (1986)

707

2,6-Xylenol

Sister chromatid exchange

Human lymphocytes

0-31 g/ml

Negative

Jansson et al. (1988)

708

3,4-Xylenolv

Reverse mutation

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

367 g/platea,b

Negative

Florin et al. (1980)

709

Thymol

Reverse mutation

S. typhimurium TA97, TA98, TA100

1000 g/mla,b

Negative

Azizan & Blevins (1995

709

Thymol

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA7537, TA1538

451 g/platea,b

Negative

Florin et al. (1980)

709

Thymol

Sister chromatid

Syrian hamster embryo cells

0.3-30 g/ml

Positive

Fukuda (1987)

709

Thymol

Unscheduled DNA synthesis

Syrian hamster embryo cells

0.3-10 g/mla
1-10 g/mlb

Negative
Positive

Fukuda (1987)

735

2-Phenyl-phenol

Reverse mutation

S. typhimurium TA98, TA700, TA1537

3.3-200 g/platea,b

Negative

National Toxicology Program (1986)

735

2-Phenyl-phenol

Reverse mutation

S. typhimurium TA1535

3.3-200 g/platea
3.3-200 g/plateb

Positive
Negative

National Toxicology Program (1986)

735

2-Phenyl-phenol

Forward mutation

Mouse lymphoma cells

20-60 g/mla
0.32-5 g/mlb

Positive

National Toxicology Program (1986)

735

2-Phenyl-phenol

Sister chromatid exchange

Chinese hamster embryo cells

14.9-20 g/mla
29.9 g/mla
14.9-29.9 g/mb

Negative
Positive
Negative

National Toxicology Program (1986)

735

2-Phenyl-phenol

Chromosomal aberrations

Chinese hamster embryo cells

60-80 g/mla,b

Negative

National Toxicology Program (1986)

711

para-Vinyl-phenol

Sister chromatid exchange

Human lymphocytes

0-12 g/ml

Negative

Jansson et al. (1988)

712

Resorcinol

Reverse mutationc

S. typhimurium TA1535

550-7700 g/platea
0-7700 g/plateb

Positive
Negative

Gocke et al. (1981)

712

Resorcinol

Reverse mutationc

S. typhimurium TA100

550-7700 g/plateb
0-7700 g/platea

Positive
Negative

Gocke et al. (1981)

712

Resorcinol

Reverse mutations

S. typhimurium TA98, TA1537, TA1538

0-7700 g/platea,b

Negative

Gocke et al. (1981)

712

Resorcinol

Reverse mutation

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

0-7700 g/platea,b

Negative

Gocke et al. (1981)

712

Resorcinol

Reverse mutation

S. typhimurium TA 1535, TA1537, TA98, TA100

33-3333 g/platea,b

Negative

Haworth et al. (1983)

712

Resorcinol

Forward mutation

Mouse lymphoma cells

125-2000 g/mla

Positive

McGregor et al. (1988)

712

Resorcinol

Sister chromatid exchange

Human lymphocytes

0-28 g/ml

Negative

Jansson et al. (1986)

712

Resorcinol

Sister chromatid exchange

Human lymphocytes

0-28 g/ml

Negative

Jansson et al. (1988)

712

Resorcinol

Sister chromatid exchange

Chinese hamster embryo cells

0.6-2.2 g/ml

Negative

Wild et al. (1981)

713

Guaiacol

Reverse mutation

S. typhimurium TA98, TA100, TA102

1-111 726 g/platea,b

Negative

Aeschbacher et al. (1989)

713

Guaiacol

Reverse mutation

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

16 000 g/platea,b

Negative

Douglas et al. (1980)

713

Guaiacol

Reverse mutation

S. typhimurium TA1535, TA1537, TA98, TA100

33-10 000 g/platea,b

Negative

Haworth et al. (1983)

713

Guaiacol

Reverse mutation

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

16 000 g/platea,b

Negative

Nestmann et al. (1980)

713

Guaiacol

Reverse mutation

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

5000 g/platea,b

Negative

Pool & Lin (1982)

713

Guaiacol

Sister chromatid exchange

Human lymphocytes

31 g/ml

Positive

Jansson et al. (1988)

715

2-Methoxy-4-methyl-phenol

Sister chromatid exchange

Human lymphocytes

138 g/ml

Positive

Jansson et al. (1988)

716

4-Ethylguaiacol

Sister chromatid exchange

Human lymphocytes

0-152 g/ml

Negative

Jansson et al. (1988)

721

2,6-Dimethoxy-phenol

Reverse mutation

S. typhimurium TA98, TA700, TA1535, TA1537, TA1538

16 000 g/platea,b

Negative

Douglas et al. (1980)

721

2,6-Dimethoxy-phenol

Reverse mutation

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

463 g/platea,b

Negative

Florin et al. (1980)

721

2,6-Dimethoxy-phenol

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537

0.1-1000 g/mla,b

Negative

McMahon et al. (1979)

721

2,6-Dimethoxy-phenol

Reverse mutation

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

5000 g/platea,b

Negative

Pool & Lin (1982)

721

2,6-Dimethoxy-phenol

Reverse mutation

E. coli

1-1000 g/mla,b

Negative

McMahon et al. (1979)

721

2,6-Dimethoxy-phenol

Sister chromatid exchange

Human lymphocytes

0-77 g/ml

Negative

Jansson et al. (1986)

721

2,6-Diinethoxy-phenol

Sister chromatid exchange

Human lymphocytes

0-77 g/ml

Negative

Jansson et al. (1988)

725

2-Methoxy-4-vinyl-phenol

Sister chromatid exchange

Human lymphocytes

75 g/ml

Positive

Jansson et al. (1988)

727

2-Hydroxyacetophenone

Reverse mutation

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

408 g/platea,b

Negative

Florin et al. (1980)

736

Phenyl salicylate

Reverse mutation

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

3-333 g/platea,b

Negative

Zeiger et al. (1987)

a Without metabolic activation

b With metabolic activation

c ZLM medium used in place of Vogel-Bonner medium

d Presumably non-mutagenic, but solubility did not allow testing in amounts that result in lethality

e Concentrations selected corresponded to 0.5, 0.25, and 0.125 of the concentration that caused 50% growth, inhibition (not specified) as determined in an assay for cytotoxicity.

concentration was increased to 94 g/ml (Morimoto & Wolff, 1980). This concentration greatly exceeds that which induces cytotoxicity (47 g/ml), as estimated by the authors. Genotoxic effects observed in conjunction with cytotoxicity may be artefacts of lysosome breakdown and release of DNase. Such action leads to DNA single- and double-strand breaks, sister chromatid exchange, and chromosomal aberration (Bradley et al., 1987). Sister chromatid exchange was also induced in human lymphocytes incubated with guaiacol at doses up to 31 g/ml or with 2-methoxy-4-methy1phenol (No. 715) at doses up to 138 g (Jansson et al., 1988); however, the cytotoxic concentration was. not established for either substance. No evidence of sister chromatid exchange was found in human fibroblasts exposed to ortho-cresol at concentrations up to 433 g/ml or to meta- or para-cresol at 865 g/ml (Cheng & Kligerman, 1984), or in Chinese hamster ovary cells exposed to resorcinol at 0.6-2 g/ml (Wild et al., 1981). Weakly positive results were reported with ortho-cresol at a concentration of 865 g/ml (Cheng & Kligerman, 1984).

Sister chromatid exchange was induced by phenol at doses of 300-400 g/ml in Chinese hamster ovary cells without metabolic activation, and weakly positive results were reported at 1670-3000 g/ml with metabolic activation. Cytotoxic effects, including cell cycle delay, were also observed at these doses (Ivett et al., 1989). After a 27-h expression period, sister chromatid exchange was induced in Chinese hamster ovary cells incubated with 2-phenylphenol at 15-30 g/ml without metabolic activation. When the treated cells were allowed a 42-h expression period, however, the frequency of sister chromatid exchange was reduced to the control level. The authors suggested that sister chromatid exchange is induced by 2-phenylphenol in the first and second S-phases, but that the DNA damage caused by the substance is eliminated after a subsequent round of DNA synthesis. There was no evidence of sister chromatid exchange in the presence of metabolic activation (National Toxicology Program, 1986). In an assay to determine the ability of thymol to induce sister chromatid exchange in Siberian hamster embryo cells, positive results were reported with concentrations of 0.3-30 g/ml, but negative results were reported in the absence of metabolic activation (Fukuda, 1987).

No chromosomal aberrations were induced when Chinese hamster ovary cells were incubated with phenol at 600-800 g/ml without metabolic activation (Ivett et al., 1989) or 2-phenylphenol at 60-80 g/ml with and without metabolic activation (National Toxicology Program, 1986). However, positive results were reported when a metabolic activation system was added and the phenol concentration was increased to 2000-3000 g/ml (Ivett et al., 1989). The authors did not determine the cytotoxic threshold or the pH of the test media. Mammalian cells in situ rely on complex regulatory mechanisms to maintain homeostatic conditions, and those in culture are not equipped to respond to environmental changes. The pH of the culture media used in mammalian cell assays must be maintained at approximately 6.8-7.5, because lower values or changes in osmolality due to acidic or ionizing test substances (e.g. phenol) may result in false-positive results, especially with metabolic activation systems. Acidity facilitates the breakdown of the components of such systems into mutagenic agents (Brusick, 1986). Micronuclei were not induced in human lymphocytes exposed to phenol at a concentration of 94 g/ml without metabolic activation (Robertson et al., 1991).

No evidence of DNA strand breaks was found in Chinese hamster ovary cells incubated with phenol at a dose of 94 110 g/ml (Sze et al., 1996). Unscheduled DNA synthesis did not occur in Syrian hamster embryo cells incubated with thymol at 1-10 g/ml without the addition of metabolic activation; however, positive results were reported at the same concentrations when an activation system was added (Fukuda, 1987).

In vivo

The results of assays for genotoxicity in vivo were predominantly negative. The frequency of micronucleated polychromatic erythrocytes was not increased in mice after intraperitoneal injections of phenol of doses of 40-188 mg/kg bw (Barale et al., 1990; Shelby et al., 1993; Marrazzini et a1.; 1994; Chen & Eastmond, 1995 [three doses of 50-100 mg/bw administered 24 h apart]). Negative results were obtained in the same assay after intraperitoneal injections of resorcinol at doses of 55-220 mg/kg bw (Gocke et al., 1981 [two doses of 220 mg/kg bw administered 24 h apart]; Wild et al.; 1981). Increased frequencies of micronuclei were reported in mice given intraperitoneal injections of phenol at 120-180 mg/kg bw (Shelby et al., 1993; Marrazzini et al., 1994; Chen & Eastmond, 1995 [three doses of 160 mg/kg bw administered 24 h apart]) or a single oral dose of 250 mg/kg bw (Karim et al., 1986).

The ability of ortho-cresol (No. 691), meta-cresol (No. 692), and para-cresol (No. 693) to induce sister chromatid exchange was also studied in vivo (Cheng & Kligerman, 1984). Mouse bone-marrow cells, alveolar macrophages, and regenerating liver cells were examined after intraperitoneal administration of ortho- or meta-cresol at 200 mg/kg bw or para-cresol at 75 mg/kg bw. The results were negative.

2-Phenylphenol has been tested in a variety of tests for genetic toxicity, both in vitro and in vivo. Scattered positive results were observed in vitro, primarily in tests for chromosomal aberrations. In vivo, 2-phenylphenol did not induce chromosomal aberrations an rat bone marrow or dominant lethal mutations in mice. No DNA damage or DNA adduct formation was observed in the urinary bladder of male mice treated with [14C]2-phenyl phenol. DNA adducts were found by 32P-postlabelling in the urinary bladder, but only at high doses. This observation does not indicate covalent binding of 2-phenylphenol to DNA. The sodium salt of 2-phenylphenol (sodium ortho-phenylphenate) and its metabolites phenyl hydroquinone and phenylbenzoquinone, although less extensively. tested, show the same profile of responses in tests for genetic toxicity as 2-phenylphenol (WHO, 2000).

Overall, the 48 phenol derivatives in this group of flavouring substances are unlikely to be genotoxic in vivo.

2.3.2.6 Reproductive toxicity

Phenol (No. 690), para-cresol (No. 693), resorcinol (No. 712), and hydroquinone monoethyl ether (No. 720)

In a study to develop a model for quantitative assessment of structure-activity relationships for maternal and developmental toxicity, the activity of 27 substituted phenols, including phenol (No. 690), para-cresol (No. 693), and resorcinol (No. 712), was addressed. Groups of 12-15 timed-pregnant Sprague-Dawley rats were given phenol (No. 690), para-cresol (No. 693), resorcinol (No. 712), or hydroquinone monoethyl ether (No. 720) at a dose of 0, 100, 330, 670, or 1000 mg/kg bw per day by oral intubation on day 11 of gestation. The pregnant animals were observed for clinical signs of toxicity for several hours after dosing, and all were weighed on days 10, 11, 12, 14, 17, and 21 of gestation. The weights and viability of the pups were assessed on days 1, 3 and 6 post partum. Any overt malformations were noted, and dead and moribund pups were necropsied. After weaning of each litter, the dam was killed and implantation scars in the uterus were counted. Perinatal loss of offspring was calculated as the difference between the number of uterine implantation sites and the number of live pups on postnatal day 6. The pups were necropsied after weaning and examined for any previously unnoticed external abnormalities.

Hind limb paralysis or tail malformations were found in 21% and 27% of the offspring of dams given phenol at 670 and 1000 mg/kg bw per day, respectively. Significantly decreased maternal weight was also noted in these groups. Pregnant animals that received doses 330 mg/kg bw per day of para-cresol and 670 mg/kg bw per day of resorcinol had decreased body weights. Decreased maternal and pup weights were observed in the groups given hydroquinone monoethyl ether at doses 670 mg/kg bw per day. No statistically significant perinatal loss was observed at any dose of these substances. (Kavlock, 1990).

Thymol (No. 709)

In a study published in 1933, thymol (No. 709) was extracted from the ether oil of Thimus vulgaris and administered to three pregnant rabbits in gelatinous capsules containing 294-299 mg/kg bw once a day for 7 days, beginning on approximately day 19 of gestation. At the end of the study, all the adult animals were necropsied. Macroscopic evaluations were performed on all organs of the chest and abdomen. Microscopic evaluations were performed on tissues from the liver, kidney, ovary, uterus, and placenta. On day 6 of dosing, one rabbit aborted five dead fetuses, however, this rabbit still received the final dose of thymol on the last day of the study. The fetuses carried by the other two rabbits were all found alive at necropsy. No major lesions were found in the uterus or placenta. The fetal liver and kidney appeared normal. No remarkable changes were found on examination of the livers of the adults, except for some nuclei of parvicellular infiltration in the interlobular zones. The kidneys showed enlargement of the capsule and parvicellular infiltrations and tubular degeneration toward the medullary. A few atresic follicles and enhanced interstitial gland development were found in the ovaries (Savignoni & de Maria, 1933).

2.3.2.7 Special studies: Effects on forestomach and glandular stomach epithelium

para-Cresol (No. 693) and 4-(1,1-dimethyl)ethylphenol (No. 733)

A group of 15 male Syrian hamsters were fed a diet containing 1.5% para-cresol (No. 693) or 4-(1,1-dimethyl)ethyl phenol (No. 733) for 20 weeks, calculated (Food & Drug Administration, 1993) to provide an average intake of 1800 mg/kg bw per day. A control group was fed the basal diet alone. At necropsy, the body and organ weights were measured, and the forestomach, glandular stomach, and urinary bladder were evaluated for hyperplasia and papillomatous lesions. The average body weight of the test animals did not differ significantly from that of the controls. A slight, nonsignificant increase in liver weight was reported in hamsters given 4-(1,1-dimethyl)ethyl phenol, and those given para-cresol had mild to moderate (< 0.5 mm) hyperplasia in the forestomach. A thickening of the epithelium that appeared as a white, keratin-like substance was reported in the anterior and posterior walls along the lesser curvature of and adjacent to the oesophagus. Prominent thickening of the forestomach epithelium, severe (> 0.5 mm) hyperplasia and papillomatous lesions of the forestomach and a significant increase in the labelling index in forestomach epithelium were observed in hamsters given 4-(1,1-dimethyl)ethyl phenol. Marked or significant changes were not reported in the stomach, and no changes were observed in the urinary bladder (Hirose et al., 1986).

Resorcinol (No. 712)

Five young male Fischer 344 rats were fed a basal diet containing 0.8% resorcinol for 8 weeks, calculated (Food & Drug Administration, 1993) to provide an average intake of 800 mg/kg bw per day. Bromodeoxyuridine (BrdU) staining was used to assess cell proliferation and was coupled with histological examination to evaluate the effects of resorcinol on the forestomach and glandular stomach epithelium. No difference was found between treated and control animals (Shibata et al., 1990).

Resorcinol was tested in a study of the effects of dihydroxybenzene derivatives on the induction of tumours of the forestomach and glandular stomach in rats by N-methyl-N'-nitro-N-nitrosoguanidine. Two groups of 16 male Fischer 344 rats, with and without pretreatment with the nitrosamine, were given a diet containing 0.8% resorcinol for 51 weeks. This concentration was calculated (Food & Drug Administration, 1993) to provide an average intake of 800 mg/kg bw per day. A control group of rats was fed the basal diet only. Mild to moderate hyperplasia of the glandular forestomach epithelium was seen in two rats given resorcinol alone, but this incidence was not significantly different from that of the controls. Resorcinol did not induce papillomas, carcinomas, or squamous-cell carcinomas of the forestomach and did not promote tumours induced by N-methyl-N'-nitro-N-nitrosoguanidine (Hirose et al., 1989).

In a study to assess the effects of 13 phenolic compounds, including para-cresol (No. 693) and 4-(1,1-dimethyl)ethyl phenol (No. 733), on the induction of proliferative lesions in the forestomach, glandular stomach, and urinary bladder, 15 male Syrian golden hamsters were given a diet containing 0.25% resorcinol for 20 weeks. This dose was calculated (Food & Drug Administration, 1993) to provide an average intake of 300 mg/kg bw per day. Control animals were fed the basal diet alone. At necropsy, the body and liver weights of the treated animals did not differ significantly from those of controls. Twelve of the 15 hamsters given resorcinol showed mild to moderate hyperplasia of the forestomach mucosa, but the incidence of these effects did not differ significantly from that of the controls (7/12). There was no evidence of neoplastic lesions in the forestomach of hamsters given resorcinol (Hirose et al., 1986).

In a study to assess the effects of catechol antixoxidants on pancreatic carcinogenesis initiated by N-nitrosobis(2-oxopropyl)amine, a diet containing 1.5% resorcinol was fed to 15 female Syrian hamsters for 16 weeks, calculated (Food & Drug Administration, 1993) to provide an intake of 1800 mg/kg bw per day. Control animals were fed the basal diet alone. At necropsy, the pancreas, liver, and stomach were removed for evaluation. Resorcinol weakly inhibited the effects of the nitrosamine. The livers of hamsters given resorcinol alone weighed significantly less than those of controls. Epithelial hyperplasia was seen in the forestomach and glandular, stomach of resorcinol-treated animals, but there was no evidence of neoplastic lesions in the stomach, liver, or pancreas (Maruyama et al.,1991).

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    See Also:
       Toxicological Abbreviations
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