WHO FOOD ADDITIVES SERIES: 48

SAFETY EVALUATION OF CERTAIN
FOOD ADDITIVES AND CONTAMINANTS

BENZYL DERIVATIVES

First draft prepared by Dr J. Gry¹, Professor A.G. Renwick² and Professor I.G. Sipes^3

1Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration, Ministry of Food, Agriculture and Fisheries, Søborg, Denmark
²Clinical Pharmacology Group, University of Southampton, Southampton, United Kingdom
³ Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, USA

Evaluation

Introduction

Estimated daily intake

Metabolic considerations

Application of the Procedure for the Safety Evaluation of Flavouring Agents

Consideration of combined intakes

Conclusions

Relevant background Information

Explanation

Additional considerations on intake

Biological data

Biochemical data

Absorption, distribution, and excretion

Metabolism

Toxicological studies

Acute toxicity

Short-term studies of toxicity

Long-term studies of toxicity and carcinogenicity

Genotoxicity

Reproductive toxicity

References

1. EVALUATION

1.1 Introduction

The Committee evaluated a group of 37 flavouring agents¹ that consisted of benzyl alcohol (No. 25), benzaldehyde (No. 22), benzoic acid (No. 850), and related substances (see Table 1) by the Procedure for the Safety Evaluation of Flavouring Agents (see Figure 1, Introduction). All members of this group are aromatic primary alcohols, aldehydes, carboxylic acids, or related esters or acetals. The benzene ring may be ring-substituted with alkyl substituents (Nos 863-869).

Table 1. Summary of results of safety evaluations of benzyl derivatives used as flavouring agents^a

Flavouring agent	No.	CAS no. and structure	Step A3^b Does intake exceed the threshold for human intake?	Step A4 Is the substance or are its metabolites endogenous?	Comments	Conclusion based on current intake
Benzyl alcohol^c	25	100-51-6	Yes Europe: 16 000 USA: 17 000	Yes	See note 1.	No safety concern
Benzyl formate	841	104-57-4	No Europe: 41 USA: 51	NR	See note 2.	No safety concern
Benzyl acetate^c	23	140-11-4	No Europe: 1400 USA: 850	NR	See note 2.	No safety
Benzyl propionate	842	122-63-4	No Europe: 49 USA: 99	NR	See note 2.	No safety concern
Benzyl butyrate	843	103-37-7	No Europe: 120 USA: 290	NR	See note 2.	No safety concern
Benzyl isobutyrate	844	103-28-6	No Europe: 15 USA: 21	NR	See note 2.	No safety concern
Benzyl isovalerate	845	103-38-8	No Europe: 14 USA: 19	NR	See note 2.	No safety concern
Benzyl trans-2-methyl-2- butenoate	846	37526-88-8	No Europe: 0.01 USA: 0.03	NR	See note 2.	No safety concern
Benzyl 2,3-dimethyl- crotonate	847	7492-69-5	No Europe: 0.01 USA: 1	NR	See note 2.	No safety concern
Benzyl acetoacetate	848	5396-89-4	No Europe: 0.2 USA: 0.07	NR	See note 2.	No safety concern
Benzyl benzoate^c	24	120-51-4	Yes Europe: 1900 USA: 4200	Yes	See notes	No safety concern
Benzyl phenylacetate	849	102-16-9	No Europe: 5 USA: 57	NR	See note 2.	No safety concern
Benzaldehyde^c	22	100-52-7	Yes Europe: 9300 USA: 36 000	Yes	See note 3.	No safety concern
Benzaldehyde dimethyl acetal	837	1125-88-8	No Europe: 0.2 USA: 0.3	NR	See note 4.	No safety concern
Benzaldehyde glyceryl acetal	838	1319-88-6	No Europe: 16 USA: 300	NR	See note 4.	No safety concern
Benzaldehyde propylene glycol acetate	839	2568-25-4	No Europe: 0.04 USA: 110	NR	See note 4.	No safety concern
Benzoic acid^c,d	850	65-85-0	No Europe: 39 USA: 340	NR	See note 5.	Evaluation not finalized
Methyl benzoate	851	93-58-3	No Europe: 47 USA: 230	NR	See note 6.	No safety concern
Ethyl benzoate	852	93-89-0	No Europe: 110 USA: 110	NR	See note 6.	No safety concern
Propyl benzoate	853	2315-68-6	No Europe: 0.01 USA: 0.3	NR	See note 6.	No safety concern
Hexyl benzoate	854	6789-88-4	No Europe: 380 USA: 1	NR	See note 6.	No safety concern
Isopropyl benzoate	855	939-48-0	No Europe: 0.004 USA: 0.3	NR	See note 6.	No safety concern
Isobutyl benzoate	856	120-50-3	No Europe: 0.4 USA: 1	NR	See note 6.	No safety concern
Isoamyl benzoate	857	94-46-2	No Europe: 110 USA: 33	NR	See note 6.	No safety concern
cis-3-Hexenyl benzoate	858	25152-85-6	No Europe: 8 USA: 0.1	NR	See note 6.	No safety concern
Linalyl benzoate	859	126-64-7	No Europe: 10 USA: 2	NR	See note 6.	No safety concern
Geranyl benzoate	860	94-48-4	No Europe: 4 USA: 0.03	NR	See note 6.	No safety concern
Glyceryl tribenzoate^d	861	614-33-5	No Europe: ND USA: 49	NR	See note 6.	Evaluation not finalized
Propylene glycol dibenzoate^d	862	19224-26-1	No Europe: ND USA: 14	NR	See note 6.	Evaluation not finalized
Methylbenzyl acetate (mixed ortho, meta, and para)	863	29759-11-3	No Europe: ND USA: 3	NR	See note 2.	No safety concern
para-Isopropylbenzyl alcohol	864	536-60-7	No Europe: 0.3 USA: 0.3	NR	See note 1.	No safety concern
4-Ethylbenzaldehyde	865	4748-78-1	No Europe: 0.4 USA: 6	NR	See note 3.	No safety concern
Tolualdehydes (mixed ortho, meta, and para)	866	1333-09-1	No Europe: 260 USA: 1100	NR	See note 3.	No safety concern
Tolualdehyde glyceryl acetal	867	1333-09-1	No Europe: 0.01 USA: 1	NR	See note 4.	No safety concern
Cuminaldehyde	868	122-03-2	No Europe: 130 USA: 1	NR	See note 3.	No safety concern
2,4-Dimethylbenzaldehyde	869	15764-16-6	No Europe: 0.4 USA: 0.1	NR	See note 3.	No safety concern

Flavouring agent	No.	CAS no. and structure	Step A3/B3^b Does intake exceed the threshold for human intake?	Step A4 Is the substance or are its metabolites endogenous?	Step B4 NOEL for substance or structurally related agent?	Comments	Conclusion based on current intake
Benzyl 2-methoxyethyl acetal	840	7492-39-9	No Europe: ND USA: 1	NR	Yes	See note 7.	No safety concern

CAS, Chemical Abstracts Service; ND, no data; NR, not required for evaluation

^a	Step 1: All the flavouring agents in this group are in structural class I, except for benzyl 2-methoxyethyl acetal (No. 840).
	Step 2: All the flavouring agents in this group are predicted to be metabolized to innocuous products.
^b	The threshold for human intake for class I is 1800 µg/day. All values for intake expressed in µg/day.
^c.	A group ADI of 0–5 mg/kg bw was confirmed by the Committee at its forty-sixth meeting (Annex 1, reference 122) and maintained at the present meeting.
^d	Further information is required to determine whether this substance is currently in use as a flavouring agent.

Notes

1.	Benzyl alcohols are oxidized to corresponding acids, which are conjugated with glycine and excreted as hippuric acid.
2.	Benzyl esters are hydrolysed to the corresponding acid and alcohol.
3.	Benzyl aldehydes are oxidized to the corresponding acids.
4.	Benzaldehyde acetals are hydrolysed to yield the aldehyde.
5.	Benzoic acid is conjugated with glycine and excreted as hippuric acid.
6.	Benzoatae esters are hydrolysed to yield corresponding alcohols and acids.
7.	Hydrolysed to acetaldehyde, benzyl alcohol, and 2-methoxyethanol. A two-generation study of reproductive toxicity with methoxyethanol in rats with a NOEL of 6 mg/kg bw per day provides a safety margin > 10 000 (Gulati et al., 1990a,b).

The Committee previously evaluated five members of the group. Benzyl alcohol (No. 25) was evaluated at the twenty-third and forty-sixth meetings (Annex 1, references 50 and 122); benzyl acetate (No. 23) was evaluated at the eleventh, twenty-seventh, twenty-ninth, thirty-first, thirty-fifth, forty-first, and forty-sixth meetings (Annex 1, references 14, 62, 70, 77, 88, 107, and 122); benzyl benzoate (No. 24) was evaluated at the fifteenth and forty-sixth meetings (Annex 1, references 26 and 122); benzaldehyde (No. 22) was evaluated at the eleventh and forty-sixth meetings (Annex 1, references 14 and 122); and benzoic acid (No. 850) was evaluated at the sixth, ninth, seventeenth, twenty-seventh, and forty-sixth meetings (Annex 1, references 6, 11, 32, 62, and 122). At its forty-sixth meeting, the Committee evaluated the five benzyl derivatives as a group and maintained the group ADI of 0–5 mg/kg bw as benzoic acid equivalents (Annex 1, reference 122).

Of the 37 substances in this group, 29 have been reported to occur naturally in foods. They have been detected in a wide variety of fruits, vegetables, meats, cheeses, and wine (CIVO-TNO, 2000).

1.2 Estimated daily intake

The total annual production of the 37 benzyl derivatives in this group is approximately 210 000 kg in Europe (International Organization of the Flavor Industry, 1995) and 460 000 kg in the USA (National Academy of Sciences, 1987; Lucas et al., 1999). Approximately 91% of the total annual production in Europe and 94% of that in the USA is accounted for by three substances in the group: benzyl alcohol (No. 25), benzaldehyde (No. 22), and benzyl benzoate (No. 24). Approximately 31% and 59% of the total annual production in Europe and the USA, respectively, is accounted for by benzaldehyde, 54% and 28% by benzyl alcohol, and 6% and 7% by benzyl benzoate. The estimated daily intake per person in Europe and the USA is 9300 µg and 36 000 µg for benzaldehyde; 16 000 µg and 17 000 µg for benzyl alcohol; and 1900 µg and 4200 µg for benzyl benzoate, respectively. The estimated daily intake of each flavouring agent in the group is reported in Table 2.

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

Substance (No.)	Most recent annual volume (kg)	Intake^a		Annual volume in naturally occurring foods (kg)^b	Consumption ratio^c
		µg/day	µg/kg bw per day
Benzyl alcohol (25)
Europe	110 000	16 000	270		0.06
USA	130 000	17 000	290	7 100	0.05
Benzyl formate (841)
Europe	290	41	0.7
USA	390	51	0.8	12	0.03
Benzyl acetate (23)
Europe	9 500	1 400	23		0.4
USA	6 500	850	14	3 500	0.5
Benzyl propionate (842)
Europe	340	49	0.8	(+)
USA	750	99	1.6		NA
Benzyl butyrate (843)
Europe	850	120	2	+
USA	2 200	290	5		NA
Benzyl isobutyrate (844)
Europe	110	15	0.3	(+)
USA	160	21	0.4		NA
Benzyl isovalerate (845)
Europe	95	14	0.2	+
USA	150	19	0.3		NA
Benzyl trans-2-methyl-2-butenoate (846)
Europe	0.1	0.01	0.0002	+
USA	0.2	0.03	0.0005		NA
Benzyl 2,3-dimethylcrotonate (847)
Europe	0.1	0.01	0.002	–
USA	10	1	0.02		NA
Benzyl acetoacetate (848)
Europe	2	0.2	0.003	(+)
USA	1	0.07	0.001		NA
Benzyl benzoate (24)
Europe	13 000	1 900	32		0.008
USA	32 000	4 200	70	110	0.003
Benzyl phenylacetate (849)
Europe	35	5	0.08	–
USA	440	57	1		NA
Benzaldehyde (22)
Europe	65 000	9 300	160		0.9
USA	270 000	36 000	600	58 000	0.2
Benzaldehyde dimethyl acetal (837)
Europe	1	0.2	0.003	(+)
USA	2	0.3	0.005		NA
Benzaldehyde glyceryl acetal (838)
Europe	110	16	0.3	–
USA	2 300	300	5		NA
Benzaldehyde propylene glycol acetal (839)
Europe	0.3	0.04	0.0007	+
USA	820	110	1.8		NA
Benzoic acid (850)
Europe	280	39	0.6	+
USA	2 600	340	5.7		NA
Methyl benzoate (851)
Europe	330	47	0.8		0.08
USA	1 700	230	3.8	26	0.02
Ethyl benzoate (852)
Europe	790	110	1.8		1.4
USA	790	110	1.8	1 100	1.4
Propyl benzoate (853)
Europe	0.1	0.01	0.0002	(+)
USA	2	0.3	0.005		NA
Hexyl benzoate (854)
Europe	2 600	380	6.3		0.7
USA	6	1	0.02	1 700	280
Isopropyl benzoate (855)
Europe	0.03	0.004	0.00007	+
USA	2	0.3	0.005		NA
Isobutyl benzoate (856)
Europe	3	0.4	0.007	+
USA	6	1	0.02		NA
Isoamyl benzoate (857)
Europe	790	110	1.8		0.3
USA	250	33	0.6	220	0.9
cis-3-Hexenyl benzoate (858)
Europe	55	8	0.13		2.5
USA	1	0.1	0.002	140	140
Linalyl benzoate (859)
Europe	69	10	0.2	+
USA	14	2	0.03		NA
Geranyl benzoate (860)
Europe	28	4	0.07	–
USA	0.2	0.03	0.0005		NA
Glyceryl tribenzoate (861)
Europe	NR	NA	NA	–
USA	370	49	0.8		NA
Propylene glycol dibenzoate (862)
Europe	NR	NA	NA	–
USA	110	14	0.2		NA
Methylbenzyl acetate (mixed ortho, meta, para) (863)
Europe	NR	NA	NA	(+)
USA	20	3	0.05		NA
para-Isopropylbenzyl alcohol (864)
Europe	2	0.3	0.005	+
USA	2	0.3	0.005		NA
4-Ethylbenzaldehyde (865)
Europe	3	0.4	0.007	+
USA	45	6	0.1		NA
Tolualdehydes (mixed ortho, meta, para) (866)
Europe	1 900	260	4.3	+
USA	8 600	1 100	1.8		NA
Tolualdehyde glyceryl acetal (867)
Europe	0.1	0.01	0.0002	–
USA	5	1	0.02		NA
Cuminaldehyde (868)
Europe	940	130	2.2	+
USA	7	1	0.02		NA
2,4-Dimethylbenzaldehyde (869)
Europe	3	0.4	0.007	+
USA	1	0.1	0.002		NA
Benzyl 2-methoxyethyl acetal (840)
Europe	NR	NA	NA	–
USA	10	1	0.02		NA
Total
Europe	210 000
USA	460 000

NA, not applicable; NR, not reported; +, reported to occur naturally in foods (CIVO-TNO, 2000), but quantitative data were not available; -, not reported to occur naturally in foods

^a	Intake expressed as µg/person per day calculated as follows: [(annual volume, kg) x (1 x 10⁹ µg/kg)/ (population x survey correction factor x 365 days)], where population (10%, ‘eaters only’) = 32 x 10⁶ for Europe and 26 x 10⁶ for the USA. The correction factor = 0.6 for Europe and 0.8 for the USA, representing the assumption that only 60% and 80% of the annual volume of the flavour, respectively, was reported in the poundage surveys (International Organization of the Flavor Industry, 1995; Lucas et al., 1999). Intake expressed as µg/kg bw per day calculated as follows: [(µg/person per day)/body weight], where body weight = 60 kg. Slight variations may occur from rounding.
^b	Quantitative data from Stofberg & Grundschober (1987)
^c	Calculated as follows: (annual consumption in food, kg)/(most recently reported volume as a flavouring agent, kg)

In addition to its presence in food and flavours, benzoic acid is endogenous in the human body. Endogenous benzoic acid is formed through the phenylalanine-tyrosine pathway (Annex 1, reference 123).

1.3 Metabolic considerations

In general, aromatic esters are hydrolysed in vivo by the catalytic activity of carboxylesterases, which predominate in hepatocytes. The acetals in the group are anticipated to be hydrolysed readily under acidic conditions. Benzyl esters and acetals are hydrolysed to benzyl alcohol (and carboxylic acids) and to benzaldehyde (and alcohols), respectively, followed by oxidation to yield benzoic acid. Benzoate esters are hydrolysed to benzoic acid (and alcohols).

Benzyl derivatives have been shown to be absorbed rapidly through the gut, metabolized primarily in the liver, and excreted in the urine as glycine conjugates of benzoic acid derivatives. Once absorbed, benzyl derivatives are oxidized and excreted primarily as the glycine conjugate of benzoic acid (hippurate). At high doses, the formation of the glycine conjugate is limited; when glycine is depleted, free benzoic acid may sequester acetyl coenzyme A or be excreted unchanged or as the glucuronic acid conjugate. Aromatic ring substitution is anticipated to have little influence on the principal pathway of metabolism.

Oxidation of the alcohol or aldehyde group may be accompanied by oxidation of the alkyl side-chain.

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

Step 1. All the benzyl and benzoate esters and acetals of benzaldehyde (or acetaldehyde) are anticipated to hydrolyse readily to yield benzoic acid, benzyl alcohol, benzaldehyde, acetaldehyde, or alkyl-substituted derivatives thereof. The remaining alcohol or acid components formed by hydrolysis are simple aliphatic substances that are either oxidized to polar, excretable metabolites or metabolized in the fatty acid pathway and tricarboxylic acid cycle. In the Procedure (Annex 1, reference 131), all 37 benzyl derivatives were assigned to structural class I (Cramer et al., 1978).

Step 2. At current levels of estimated intake, 36 of the 37 substances in the group are predicted to be metabolized to innocuous products. The evaluation of these substances therefore proceeded via the left-hand side of the decision tree in the Procedure. One compound, benzyl 2-methoxyethyl acetal (No. 840), is not metabolized to innocuous products and was accordingly evaluated via the right-hand side of the scheme.

Step A3. The estimated daily per capita intakes in Europe and the USA of 33 of the flavouring agents in this group are below the human intake threshold for class I (1800 µg/person per day), and these 33 substances were considered to be of no safety concern when used at current levels. The intakes of the remaining three substances are greater than the threshold for human intake for class I: these are benzyl alcohol (16 000 µg/person per day in Europe and 17 000 µg/person per day in the USA), benzyl benzoate (1900 µg/person per day in Europe and 4200 µg/person per day in the USA), and benzaldehyde (9300 µg/person per day in Europe and 36 000 µg/person per day in the USA). Accordingly, the evaluation of these three substances proceeded to step A4.

Step A4. Benzyl alcohol, benzyl benzoate, and benzaldehyde are readily metabo-lized to benzoic acid, which is endogenous in humans.

Step B3. For benzyl 2-methoxyethyl acetal (No. 840), no data on intake were reported for Europe, and an intake of 1 µg/person per day was reported for the USA, which is below the intake threshold for substances in class I.

Step B4. The NOEL for 2-methoxyethanol, a hydrolysis product of benzyl 2-methoxyethyl acetal (No. 840), in a two-generation study of reproductive toxicity in rats (Gulati et al., 1990a,b), of 6 mg/kg bw per day, provides a safety margin greater than 10 000 in relation to the estimated intake in the USA. Therefore, benzyl 2-methoxyethyl acetal does not pose a safety concern at the estimated level of intake.

The stepwise evaluations of the 37 benzyl derivatives used as flavouring agents are summarized in Table 1.

1.5 Consideration of combined intakes

In the unlikely event that all foods containing these flavouring agents, except benzyl 2-methoxyethyl acetal (No. 840), were to be consumed simultaneously on a daily basis, the estimated combined intake would exceed the human intake threshold for class I. However, these flavouring agents are expected to be efficiently detoxicated and would not saturate the available detoxication pathways. On the basis of the evaluation of the collective data, there would be no safety concerns about combined intake. Furthermore, the total combined daily intake per kilogram of body weight of all benzyl derivatives (0.5 mg in Europe and 1 mg in the USA) is less than the group ADI of 0–5 mg/kg bw for benzoic acid, the benzoate salts (calcium, potassium, and sodium), benzaldehyde, benzyl acetate, benzyl alcohol, and benzyl benzoate, expressed as benzoic acid equivalents, which was established by the Committee at its forty-sixth meeting (Annex 1, reference 122). The three benzyl derivatives that account for more than 90% of the total intake of this group of substances in Europe and the USA are benzyl benzoate (No. 24), which is rapidly hydrolysed to benzyl alcohol and benzoic acid (Nielsen & Bundgaard, 1987), benzaldehyde (No. 22), and benzyl alcohol (No. 25), which are are all readily metabolized to benzoic acid, which is endogenous in humans. In the opinion of the Committee, the endogenous concentration of this substance would not give rise to perturbations outside the physiological range. Therefore, these three substances were considered to be of no safety concern of the current levels of intake.

1.6 Conclusions

The Committee concluded that the flavouring agents in the group of benzyl derivatives would not present safety concerns when used at estimated current levels as flavouring agents. No data on toxicity were required in application of the Procedure to 36 of the 37 benzyl derivatives in the group. However, the Committee noted that the available information was consistent with the results of the safety evaluation. The required data on toxicity were available for benzyl 2-methoxyethyl acetal (No. 840).

2. RELEVANT BACKGROUND INFORMATION

2.1 Explanation

This monograph summarizes the key data relevant to the safety evaluation of 37 derivatives of benzyl alcohol, benzaldehyde, or benzoic acid (Table 1). All members of this group are aromatic primary alcohols, aldehydes, carboxylic acids, or their corresponding esters or acetals. The structural features common to this group of substances include an oxygenated functional group bonded directly to a benzene ring which may be ring-substituted with alkyl substituents. The group contains the parent saturated alcohol, benzyl alcohol (No. 25), and related benzyl esters (Nos 23–24 and 841–849); the corresponding aldehyde, benzaldehyde (No. 22), and related acetals (Nos 837–840); and the corresponding parent carboxylic acid, benzoic acid (No. 850), and related benzoate esters (Nos 851–862). The group also includes seven additional structurally related benzyl derivatives (Nos 863–869) containing ring alkyl substituents.

A subgroup of benzyl derivatives comprising benzyl alcohol (No. 25), benzaldehyde (No. 22), benzyl acetate (No. 23), and benzoic acid (No. 850) and salts was the subject of a recent comprehensive review and safety evaluation by the Committee (Annex 1, references 122 and 123). The review and evaluation contain detailed descriptions of most of the studies on specific benzyl derivatives. The following sections give only summaries of the results of studies described in detail in that review. Other relevant studies, including studies on other benzyl derivatives not discussed in the review, are described in detail.

2.2 Additional considerations on intake

The natural occurrence and total annual production of these are discussed in sections 1.1 and 1.2, respectively, and summarized in Table 2.

Quantitative data on natural occurrence were available for 10 substances in the group (CIVO-TNO, 2000), and consumption ratios were calculated (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987). The consumption ratio for the substance for which the reported annual volume of use in Europe is highest (benzyl alcohol, No. 25) is 0.06, and that for the substance with the highest reported annual volume in the USA (benzaldehyde, No. 22) is 0.2. The consumption ratios for benzyl formate (No. 841), benzyl benzoate (No. 24), and methyl benzoate (No. 851) are < 0.1 (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987) (Table 2).

2.3 Biological data

2.3.1 Biochemical data

(a) Absorption, distribution, and excretion

The benzyl derivatives are rapidly absorbed through the gut, metabolized primarily in the liver, and excreted in the urine as glycine conjugates of benzoic acid derivatives (Davison, 1971; Abdo et al., 1985; Temellini, 1993), as discussed by the Committee at its forty-sixth meeting, when it evaluated benzyl alcohol (No. 25), benzyl acetate (No.23), benzaldehyde (No. 22), and benzoic acid (No. 850) (Annex 1, reference 122). After absorption, benzyl derivatives are metabolized and excreted within 24 h as polar metabolites, mainly as urinary hippuric acid (Table 3).

Table 3. Metabolism of benzyl derivatives

Flavouring agent (No.)	Species	Route	Dose	24-h urine/ faeces (% dose)	Major urinary metabolite	Minor urinary metabolites	Reference
Benzyl alcohol (25)	Humans	Oral	1.5 g	84	Hippuric acid		Snapper et al. (1925)
	Humans (neonates)	Intravenous, intramuscular	0.007–0.222 mmol/kg bw	82–85	Hippuric acid	Benzoic acid	LeBel et al. (1988)
	Rabbits	Stomach tube	0.5–16 g	52–84	Hippuric acid	Benzoyl glucuronide	Bray et al. (1952)
Benzyl acetate (23)	Humans	Oral	2.0 g	83	Hippuric acid		Snapper et al. (1925)
	Rats	Oral	5, 50, or 500 mg/kg bw	90/0.3–1.3	Hippuric acid	Benzyl alcohol, benzyl mercaptic acid	Abdo et al. (1985)
	Mice	Oral	10, 100, or 1000 mg/kg bw	90/0.3–1.3	Hippuric acid	Benzyl alcohol, benzyl mercaptic acid	Abdo et al. (1985)
	Rats	Gavage	5, 250, or 500 mg/kg bw	70–89/4 (72 h)	Hippuric acid	Benzyl alcohol, benzoic acid, benzyl mercaptic acid	Chidgey & Caldwell (1986)
	Rats	Subcutaneous	0.3 mg		Hippuric acid	Benzyl mercaptic acid	Clapp & Young (1970)
	Rats	Oral	5 or 500 mg/kg bw	78/2	Hippuric acid	Benzyl mercaptic acid, benzyl alcohol, benzoyl glucuronide, benzoic acid	McMahon et al. (1989)
Benzyl benzoate (24)	Humans	Oral	1.0 g	85	Hippuric acid		Snapper et al. (1925)
Benzaldehyde (22)	Rabbits	Stomach tube	0.5–1.5 g	80–98	Hippuric acid	Benzoyl glucuronide	Bray et al. (1951)
	Rabbits	Oral	0.35–0.75 mg/kg bw	82–83	Hippuric acid	Benzoic acid, benzoyl glucuronide, benzoyl glucuronic acid, benzyl mercaptic acid	Laham et al. (1988)
Benzoic acid (850)	Rats	Oral	0.061, 0.61, 6.1, 61, or 305 mg/kg bw	88–89/1–6	Hippuric acid	Benzoic acid, benzoyl glucuronide, 3-hydroxy-3-phenyl propionic acid	Nutley (1990)
	Mice	Intraperitoneal	0.061, 0.61, 6.1, 61, or 305 mg/kg bw	92–98/1–10	Hippuric acid	Benzoic acid, benzoyl glucuronide, 3-hydroxy--phenyl propionic acid	Nutley (1990)
	Various	Oral	1–400 mg/kg bw	50–100	Hippuric acid	Benzoyl glucuronide, ornithic acid	Bridges et al. (1970)

(b) Metabolism

Benzyl esters and acetals are hydrolysed to benzyl alcohol and carboxylic acids and to benzaldehyde and alcohols, respectively. Benzyl alcohol and benzaldehyde are rapidly oxidized to benzoic acid, while benzoate esters are hydrolysed to benzoic acid and alcohols. The benzoic acid derivatives are excreted primarily as glycine conjugates. Benzoic acid is readily conjugated with glycine, primarily in the liver (see Figure 1). After high doses, formation of the glycine conjugate is limited by the concentration of glycine. When glycine is depleted, free benzoic acid may sequester acetyl coenzyme A or be excreted unchanged or as the glucuronic acid conjugate (Diack & Lewis, 1928; Bray et al., 1951; Williams, 1959; Abdo & Wenk, 1995; Abdo et al., 1998). The metabolism, including hydrolysis, of flavouring agents that were not reviewed by the Committee at its forty-sixth meeting is summarized below.

Figure 1. Metabolism of benzyl derivatives

(i) Hydrolysis of esters

In general, aromatic esters are hydrolysed in vivo by the catalytic activity of carboxylesterases, which are found throughout mammalian tissues but predominate in hepatocytes (Heymann, 1980).

Benzyl and benzoate esters in the group of benzyl derivatives are anticipated to be hydrolysed rapidly to yield the corresponding alcohols and carboxylic acids, as shown in several studies in vitro and in vivo. Benzyl acetate (No. 23) is hydrolysed rapidly, both in vitro and in vivo (Heymann, 1980; Yuan et al., 1995; Annex 1, reference 122).

Methyl benzoate (No. 851), ethyl benzoate (No. 852), propyl benzoate (No. 853) and benzyl benzoate (No. 24) are all hydrolysed to benzoic acid and the corresponding alcohol after incubation with human plasma, diluted to 80% with phosphate buffer at pH 7.4 and 37 °C. The half-times were was 108 min for methyl benzoate, 210 min for ethyl benzoate, 46 min for propyl benzoate, and 19 min for benzyl benzoate (Nielsen & Bundgaard, 1987).

Benzyl phenylacetate was hydrolysed to 90% within 1 h and completely hydrolysed within 2 h of incubation with a 2% pancreatin solution (Leegwater & van Straten, 1974).

(ii) Hydrolysis of acetals

Benzaldehyde-related acetals are anticipated to hydrolyse readily to their component alcohols and benzaldehyde under acidic conditions.

Benzaldehyde propylene glycol acetal (No. 839) was 97% hydrolysed after incubation for 5 with simulated gastric juice and intestinal fluid in vitro (Morgareidge, 1962).

(iii) Metabolism of benzyl alcohol, benzoic acid, and related esters

The metabolism of benzyl alcohol (No. 25), benzyl acetate (No. 23), benzaldehyde (No. 22), and benzoic acid (No. 850) was described comprehensively by the Committee at its forty-sixth meeting (Annex 1, reference 122).

(iv) Metabolism of substituted benzyl alcohols and benzaldehydes

Aromatic ring substitution is anticipated to have little or no effect on the principal pathway of metabolism. Oxidation of the alcohol or aldehyde to the benzoic acid derivative may be accompanied by minor side-chain oxidation.

Cuminaldehyde (No. 868) was administered orally at a dose of 2 g to male rabbits, and their urine was collected for 3 days after treatment. Cuminaldehyde underwent both oxidation of the aldehyde function and oxidation of the alkyl side-chain to yield 9-hydroxycuminic acid and 8-hydroxycuminic acid as the major urinary metabolites. Cumyl alcohol and 2-(para-carboxyphenyl)propionic acid were minor metabolites (Ishida et al., 1989).

2.3.2 Toxicological studies

(a) Acute toxicity

LD₅₀ values after oral administration have been reported for 30 of the 37 substances in this group (see Table 4). In rats, the values ranged from 1000 mg/kg bw for benzaldehyde (No. 22) to 12 000 mg/kg bw for hexyl benzoate (No. 854), and in mice they ranged from 1300 mg/kg bw for benzoic acid (No. 850) to 9400 mg/kg bw for linalyl benzoate (No. 859). The LD₅₀ values for rabbits, guinea-pigs, and cats were in a similar range, from 1000 mg/kg bw to > 5000 mg/kg bw, indicating that the the benzyl derivatives in the group have low acute toxicity.

Table 4. Acute toxicity of benzyl derivatives

Flavouring agent (No.)	Species	Sex	Route	LD₅₀ (mg/kg bw)	Reference
Benzyl alcohol (25)	Rabbit Rat	NR NR	Oral Oral	1000 2100	Graham & Kuizenga (1945)
Benzyl alcohol (25)	Rat	M,F	Gavage	1200	Jenner et al. (1964)
Benzyl alcohol (25)	Rat	M,F	Oral	1600	Procter & Gamble (1992)
Benzyl alcohol (25)	Rat	NR	Oral	3100	Smyth et al. (1951)
Benzyl alcohol (25)	Mouse	NR	Gavage	1600	Jenner et al. (1964)
Benzyl formate (841)	Rat	M.F	Gavage	< 5000	Shelanski (1971)
Benzyl acetate (23)	Rabbit	NR	Oral	2600	Graham & Kuizenga (1945)
Benzyl acetate (23)	Rat	M,F	Gavage	2500	Jenner et al. (1964)
Benzyl acetate (23)	Rat	NR	Oral	3700	Graham & Kuizenga (1945)
Benzyl propionate (842)	Rat	NR	Oral	3300	Moreno (1973)
Benzyl butyrate (843)	Rat	NR	Oral	1800	Moreno (1973)
Benzl butyrate (843)	Rat	M,F	Gavage	2300	Jenner et al. (1964)
Benzyl isobutyrate (844)	Rat	M,F	Oral	2800	Owen & Meyer (1971)
Benzyl isovalerate (844)	Rat	NR	Oral	> 5000	Moreno (1974)
Benzyl trans-2-methyl-2-butenoate (846)	Rat	NR	Oral	> 5000	Moreno (1979)
Benzyl benzoate (24)	Cat	NR	Oral	2200	Graham & Kuizenga (1945)
Benzyl benzoate (24)	Rabbit	NR	Oral	2000	Draize et al. (1948)
Benzyl benzoate (24)	Rabbit	NR	Oral	1700	Graham & Kuizenga (1945)
Benzyl benzoate (24)	Guinea-pig	NR	Oral	1100	Draize et al. (1948)
Benzyl benzoate (24)	Rat	NR	Oral	1900	Draize et al. (1948)
Benzyl benzoate (24)	Rat	NR	Oral	2800	Graham & Kuizenga (1945)
Benzyl benzoate (24)	Mouse	NR	Oral	1600	Draize et al. (1948)
Benzyl phenylacetate (849)	Rat	M,F	Oral	> 5000	Owen & Meyer (1971)
Benzaldehyde (22)	Guinea-pig	M,F	Gavage	1000	Jenner et al. (1964)
Benzaldehyde (22)	Rat	M,F	Gavage	1300	Jenner et al. (1964)
Benzaldehyde (22)	Rat	NR	Oral	2800	Sporn et al. (1967)
Benzaldehyde (22)	Rat	M,F	Gavage	1300	Taylor et al. (1964)
Benzaldehyde (22)	Mouse	NR	Food	1200	Schafer & Bowles (1985)
Benzalehyde dimethyl acetal (837)	Rat	NR	Oral	1200	Moreno (1977)
Benzaldehyde glyceryl acetal (838)	Rat	NR	Oral	3700	Levenstein (1974)
Benzaldehyde glyceryl acetal (838)	Rat	NR	Oral	2800	Moreno (1980)
Benzaldehyde propylene glycol acetal (839)	Rat	M,F	Gavage	3000	Lewis & Palanker
Benzoic acid (850)	Mouse	NR	Oral	1200	Schafer & Bowles (1985)
Benzoic acid (850)	Mouse	NR	Oral	2000	Sado (1973)
Benzoic acid (850)	Mouse	NR	Intragastric	2000	Shell BV (1982)
Methyl benzoate (851)	Rabbit Rat	NR NR	Oral Oral	2100 2200	Graham & Kuizenga (1945)
Methyl benzoate (851)	Rat	M,F	Gavage	1400	Jenner et al. (1964)
Methyl benzoate (851)	Rat	M,F	Oral	3400	Smyth et al. (1954)
Methyl benzoate (851)	Mouse	NR	Gavage	3300	Jenner et al. (1964)
Ethyl benzoate (852)	Rabbit Rat	NR NR	Oral Oral	2600 2100	Graham & Kuizenga (1945)
Ethyl benzoate (852)	Rat	M,F	Oral	6500	Smyth et al. (1954)
Hexyl benzoate (854)	Rat	NR	Oral	12 000	Smyth et al. (1951)
Isopropyl benzoate (855)	Rat	NR	Oral	3700	Smyth et al. (1951)
Isobutyl benzoate (856)	Rat	M,F	Gavage	3700	Levenstein (1973)
Isoamyl benzoate (857)	Rat	NR	Oral	6300	Wong & Weir (1971)
Linalyl benzoate (859)	Rabbit	NR	Oral	> 5000	Moreno (1973)
Linalyl benzoate (859)	Rat	NR	Oral	> 5000	Moreno (1973)
Linalyl benzoate (859)	Mouse	M	Oral	9400	Hoffman-LaRoche (1967)
Geranyl benzoate (860)	Rat	NR	Oral	> 5000	Moreno (1973)
para-Isopropylbenzyl alcohol (864)	Rat	NR	Oral	1000	Moreno (1973)
4-Ethylbenzaldehyde (865)	Rat	M,F	Oral	2000	Costello (1984)
Tolualdehydes (mixed ortho, meta, para) (866)	Rat	NR	Oral	2200	Moreno (1973)
Cuminaldehyde (868)	Rat	M,F	Gavage	1400	Jenner et al. (1964)
2,4-Dimethylbenzaldehyde (869)	Rat	M,F	Gavage	< 5000	deGroot et al. (1974)

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

(b) Short-term studies of toxicity

The results of short-term toxicological studies have been reported for nine representative benzyl derivatives. Studies with the parent alcohol, benzyl alcohol (No. 25), the corresponding aldehyde, benzaldehyde (No. 22), and benzoic acid (No. 850) were described in detail by the Committee at its forty-sixth meeting (Annex 1, reference 122). Only the results of studies on the flavouring agents in the group that were not reported at the forty-sixth meeting are presented below. The short-term studies of toxicity on the benzyl derivatives are summarized in Table 5.

Table 5. Results of short-term studies of toxicity with benzyl derivatives

Flavouring agent (No.)	Species, sex	No. of test groups^a/ no. per group^b	Route	Length (days)	NOEL (mg/kg bw per day)	Reference
Benzyl alcohol (25)	Rat, M,F Rat, M,F Mouse, M,F Mouse, M,F	10/10 10/5 10/10 10/5	Gavage Gavage Gavage Gavage	13 weeks 16 13 weeks 16	100 130 100 250	National Toxicology Program (1989)
Benzyl acetate (23)	Rat, M,F Rat, M,F Rat, M,F Mouse, M,F Mouse, M Mouse, M,F	10/5 10/10 12/10 10/5 10/10 10/10	Gavage Gavage Oral Gavage Gavage Gavage	14 13 weeks 13 weeks 14 13 weeks 13 weeks	500 250 460 1000 500 < 430	National Toxicology Program (1986) National Toxicology Program (1993) National Toxicology Program (1986) National Toxicology Program (1993)
Benzaldehyde (22)	Rat, M,F Rat, M,F Rat, M,F Mouse, M,F Mouse, M,F	10/5 12/10 1/5 10/5 10/10	Gavage Gavage Oral Gavage Gavage	13 weeks 13 weeks 27-28 weeks 13 weeks 13 weeks	200 200 >50 300 300	Kluwe et al. (1983) National Toxicology Program (1990) Hagan et al. (1967) Kluwe et al. (1983) National Toxicology Program (1990)
Methyl benzoate (851)	Rat, NR	5/5	Intragastric	6 months	0.005	Kravets-Bekker (1970)
Glyceryl-tribenzoate (861)	Rat, M,F	8/15	Oral	90	600	Carson (1972a)
Propylene glycol dibenzoate (862)	Rat, M,F	8/15	Oral	90	2500^c	Carson (1972b)
Tolualdehydes (mixed ortho, meta, para) (866)	Rat, M,F Rat, M,F	NR/15 NR/5	Oral Gavage	90 13 weeks	36^c250	Oser et al. (1965) Brantom et al. (1972)
2,4-Dimethylbenzaldehyde (869)	Rat, M,F	NR/5	Gavage	2 weeks	0.18^c	deGroot et al. (1974)

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

^a	Does not include control groups
^b	Both male and female animals
^c	Study performed with a single or multiple doses that produced no adverse effects, and so the actual NOEL may be higher.

Benzyl butyrate (No. 843): Groups of 12 weanling rats (strain unspecified) of each sex were given a diet containing a mixture of six aromatic esters commonly used in foods for 12 weeks at concentrations calculated to provide an average daily intake of 0 or 100 times the assumed human intake, calculated to be 130 mg/kg bw per day. The agents were incorporated into the diet in the ratio of their use in foods: ethyl benzoate, 0.15 mg/kg; isobutyl benzoate, 25 mg/kg: benzyl acetate, 19 mg/kg; benzyl butyrate, 25 mg/kg; ethyl methylphenylglycidate, 25 mg/kg; and glycidate M-116, 25 mg/kg. The group receiving the ester blend had normal body-weight gain, food consumption, efficiency of food use, appearance, and behaviour. The blood haemoglobin and urine glucose concentrations did not differ significantly between test and control groups. Traces of albumin present in urine specimens from both control and test groups were regarded as not significant. At autopsy, no treatment-related abnormalities were observed, and the weights of the livers and kidneys were within normal limits for both groups. No histopathological examination was performed (Oser, 1957).

Glyceryl tribenzoate (No. 861): Groups of 15 male and 15 female weanling FDRL-weanling rats were maintained on a diet containing glyceryl tribenzoate at concentrations calculated to provide an average daily intake of 0 (control), 120, 600, or 2600 mg/kg bw, for 90 days. Weekly measurement of body weight and food consumption revealed depressed growth rate and efficiency of food use in males at the high dose. Haematological, blood chemical, and urine analyses gave normal values. No difference in the absolute or relative weights of the liver and kidney were found between test and control animals, and there was no evidence of treatment-related gross or histological lesions (Carson, 1972a).

Propylene glycol dibenzoate (No. 862): Propylene glycol dibenzoate was administered to weanling FDRL-rats at a dose of 0 (control), 130, 630, or 2500 mg/kg bw per day for 90 days in a study similar to that described above. No treatment-related effects were reported, even at the highest dose (Carson, 1972b).

Tolualdehydes (mixed ortho, meta, para) (No. 866): Groups of 15 male and 15 female weanling Sprague-Dawley rats were fed a diet containing tolualdehyde (proportions of ortho, meta, and para isomers not given) at concentrations providing an average daily intake of 36 mg/kg bw for males and 43 mg/kg bw for females, for 90 days. Weekly measurements of body weight and food consumption revealed no significant differences between test and control groups. Haematological, blood chemical, and urine analyses showed normal values. No difference in the absolute or relative weights of organs were found between test and control animals, and there was no evidence of treatment-related effects on gross or histological appearance (Oser et al., 1965).

Groups of 15 male and 15 female CFE rats were given tolualdehyde (approximately equal proportions of meta and para isomers) at a dose of 0 (control), 50, 250, or 500 mg/kg bw in corn oil daily by oral intubation for 13 weeks. Additional groups of five rats of each sex were given the compound at a dose of 0, 250, or 500 mg/kg bw per day for 2 or 6 weeks by the same route. Weekly measurements of body weights and food and water intake showed that the females at 500 mg/kg bw per day had significantly lower body weights after 2 weeks. No changes in body weight were observed in other groups at 2, 6, or 13 weeks. Haematological, blood chemical, and urine analyses performed at 2, 6, and 13 weeks showed a transient increase in erythrocyte count, erythrocyte volume fraction, and haemoglobin concentration in males, but only at 2 weeks. At necropsy at week 13, a significant decrease in the absolute or relative weight of the small intestine was found in all treated groups and decreased relative pituitary weights in females at 500 mg/kg bw per day. The magnitude of the change in the mean weight of the small intestine was not dose-related.

In a second part of the study, groups of 30 female rats were given tolualdehyde at a dose of 0 or 500 mg/kg bw per day in corn oil by gavage for 13 weeks. No significant difference in mean absolute or relative weight of the small intestine was found between the control and treated groups in this part of the study or between the control group in this part and the treated females in the first part of the study. The authors noted that the mean weight of the small intestine of control animals in the first part were abnormally high. The changes in organ weight were not associated with treatment-related gross or histological changes (Brantom et al., 1972).

2,4-Dimethylbenzaldehyde (No. 869): Groups of five male and five female rats were given 2,4-dimethylbenzaldehyde at a dose of 0 (control), 0.18, or 1.8 mg/kg bw by stomach tube daily on 6 days per week for 2 weeks. Males at the highest dose had an increased relative liver weight. Histological examination of the liver and kidneys revealed no differences from controls (deGroot et al., 1974).

(c) Long-term studies of toxicity and carcinogenicity

The results of a series of long-term studies of toxicity and carcinogenicity in mice and rats (Kieckebusch & Lang, 1960; Marquardt, 1960; Shtenberg & Ignat’ev, 1970; Toth, 1984; Abdo et al., 1985; National Toxicology Program, 1986, 1989, 1990, 1993) with four representative benzyl derivatives, benzyl alcohol (No. 25), benzaldehyde (No. 22), benzyl acetate (No. 23), and benzoic acid (No. 850) and sodium benzoate were reviewed by the Committee at its forty-sixth meeting (Annex 1, reference 122). At that meeting, the Committee commented that: "Long-term studies in which benzyl acetate, benzyl alcohol, benzaldehyde, benzoic acid and sodium benzoate were administered in the feed or by gavage to mice and rats were available for review by the Committee. No definitive conclusions could be drawn from carcinogenicity studies of sodium benzoate in mice and rats, as the information provided was insufficient for this purpose, and survival rates in the study in rats were too low to allow it to be considered as conclusive. The Committee reviewed the studies evaluated in the previous monographs and an additional study in which benzaldehyde was administered in corn oil by gavage to rats at 200 or 400 mg/kg bw per day for 103 weeks and to mice at 200 or 400 mg/kg bw per day (males), or 300 or 600 mg/kg bw per day (females) for 103 weeks. On the basis of these studies, the Committee concluded that neither benzyl acetate nor benzyl alcohol is carcinogenic. As in the studies in mice and rats given benzyl acetate in corn oil by gavage, increased incidences of pancreatic acinar cell adenomas in rats and of papillomas of the forestomach in mice were noted after administration of benzaldehyde. However, as in its previous review of benzyl acetate, the Committee concluded that the results of studies in which the compound was administered in the diet were more relevant to its safety assessment as a food additive than those in which it was given in corn oil by gavage." The Committee concluded that the data reviewed were sufficient to demonstrate lack of carcinogenic potential.

(d) Genotoxicity

The results of studies of genotoxicity in vitro and in vivo with benzyl alcohol (No. 25), benzyl acetate (No. 23), benzaldehyde (No. 22), and benzoic acid (No. 850) were reviewed by the Committee at its forty-sixth meeting (Annex 1, reference 122). The Committee concluded that: "None of the four compounds was mutagenic in the Ames test, either with or without metabolic activation. The compounds all induced gene mutations in the mouse lymphoma assay at the thymidine kinase locus (benzoic acid was not tested), although the requirement for metabolic activation varied. Some weak clastogenic activity was noted in in vitro assays, but not in in vivo assays."

Further data on these four flavouring agents and the results of studies with eight other benzyl derivatives in the group (Nos 24, 841, 842, 851, 857, 864, 867, and 868) are presented in Table 6. The results of all 20 assays in vitro with these eight benzyl derivatives were negative, except for one for DNA repair in Bacillus subtilis strains H17 and M45 with benzyl formate (Yoo, 1986).

Table 6. Results of studies of the genotoxicity of benzyl derivatives

No.	Flavouring agent	End-point	Test system	Concentration	Results	Comments	Reference
In vitro
25	Benzyl alcohol	Reverse mutation	S. typhimurium TA92, TA94, TA98, TA100, TA1535, TA1537 (preincubation)	10 000 µg/plate	Negative	Assay performed with and without S9	Ishidate et al. (1984)
		Reverse mutation	S. typhimurium TA100 (plate incorporation)	1000 µg/plate	Negative	Assay performed without S9	Ball et al. (1984)
		Reverse mutation	S. typhimurium TA98, TA100 (plate incorporation)	NR	Negative	Assay performed without S9	Rogan et al. (1986)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537 (preincubation)	6700 µg/plate	Negative	Assay performed with and without S9	Mortelmans et al. (1986)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537 (plate incorporation)	3 µmol/plate	Negative	Assay performed with and without S9	Florin et al. (1980)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 (plate incorporation)	50 000 µg/plate	Negative	Assay performed with and without S9	Heck et al. (1989)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 (plate incorporation)	5 µl/plate	Negative	Assay performed without S9	Milvy & Garro (1976)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537 (preincubation)	6700 µg/plate	Negative	Assay performed with and without S9; cytotoxicity at highest concentration	National Toxicology Program (1989)
		Mutation	E. coli WP2 uvrA	8 mg/plate	Negative	Japanese article, English summary; use of S9 not reported	Yoo (1986)
		DNA repair	B. subtilis H17, M45	21 µg/disc	Negative	Japanese article, English summary tables	Oda et al. (1979)
		DNA repair	B. subtilis H17, M45	10 µg/disk	Positive	Japanese article, English summary tables; inhibition of growth without S9	Kuroda et al.
		DNA repair	B. subtilis H17, M45	20 µl/disk	Positive	Japanese article, English summary; use of S9 not reported	Yoo (1986)
		Chromosomal aberration	Chinese hamster fibroblasts	1.0 mg/ml	Negative	Assay performed without S9; cells exposed for 48 h	Ishidate et al. (1984)
		Chromosomal aberration	Chinese hamster ovary cells	5000 µg/ml	Equivocal	Assay performed with and without S9; positive results not reproducible; no dose– response relationship	Anderson et al. (1990)
		Chromosomal aberration	Chinese hamster ovary cells	5000 µg/ml	Positive	Assay performed with and without S9; positive results reported only with S9	National Toxicology Program (1989)
		Sister chromatid exchange	Chinese hamster ovary cells	5000 µg/ml	Weakly positive	Dose–response relation- ship at 500–1250 µg/ml without S9 and 500–4000 µg/ml with S9	National Toxicology Program (1989)
		Sister chromatid exchange	Chinese hamster ovary cells	5000 µg/ml	Weakly positive	Assay performed with and without S9; no dose–response relationship; increase at single doses	Anderson et al. (1990)
		Mutation	L5178Y mouse lymphoma cells	5000 µg/ml	Equivocal	Positive and negative responses could not be reproduced; no dose– response relationship	McGregor et al. (1988); Myhr et al. (1990)
		Mutation	L5178Y mouse lympho- ma cells	4500 µg/ml	Positive	Assay performed with and without S9; positive result only without S9	National Toxicology Program (1989)
		Mutation	E. coli WP2 uvrA	NR	Negative	Abstract; methods and test concentrations not reported	Kuroda et al. (1984b)
		Cytotoxicity	Human alveolar tumour cells	0.5 mmol/L	Negative		Waters et al. (1982)
		DNA damage	Human alveolar tumour cells	0.5 mmol/L	Negative		Waters et al. 1982)
		DNA damage	Rat hepatocytes	10 mmol/L	Negative	Cytotoxicity at maximum dose	Storer et al. (1996)
		DNA damage	E. coli P3478	50 µl/disc	Negative	Assay performed with and without S9	Fluck et al. (1976)
841	Benzyl formate	DNA repair	B. subtilis H17, M45	20 µl/disc	Positive	Japanese article, English summary; use of S9 not reported	Yoo (1986)
		Mutation	E. coli WP2 uvrA	4.0 mg/plate	Negative	Japanese article, English summary; use of S9 not reported	Yoo (1986)
23	Benzyl acetate	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537 (preincubation)	10 mg/plate	Negative	Assay performed with and without S9	Mortelmans et al. (1986)
		Reverse mutation	S. typhimurium TA98, TA100 (preincubation and plate incorporation)	5000 µg/plate	Negative	Assay performed with and without S9; cytotoxicity at three higher concentrations	Schunk et al. (1986)
		Reverse mutation^a	S. typhimurium TA98, TA100, TA1535, TA1537	3 µmol/plate	Negative	Assay performed with and without S9	Florin et al. (1980)
		DNA repair	B. subtilis H17, M45	21 µg/disc	Negative	Assay performed without S9 Japanese article, English summary tables	Oda et al. (1979)
		DNA repair	B. subtilis H17, M45	20 µl/disc	Positive	Japanese article, English summary; use of S9 not reported	Yoo (1986)
		Mutation	E. coli WP2 uvrA	2.0 mg/plate	Negative	Japanese article, English summary; use of S9 not reported	Yoo (1986)
		Mutation	Mouse lymphoma L5178Y cells	500 µg/ml	Positive	Assay performed with and without S9; positive results only with S9	Caspary et al. (1988)
		Mutation	Human lymphoblast TK6 cells	1500 µg/ml	Positive	Assay performed with and without S9; positive results only with S9	Caspary et al. (1988)
		Mutation	Mouse lymphoma L5178Y cells	1600 µL/mL	Positive	Assay performed without S9; cytotoxicity at maximum (1988) concentration	McGregor et al.
		Mutation	Mouse lymphoma L5178Y cells	NR	Positive	Assay performed with and without S9; positive result only with S9	Rudd et al. (1983)
		Chromosomal aberration	Chinese hamster ovary cells	5000 µg/ml	Negative	Assay performed with and without S9	Galloway et al. (1987)
		Chromosomal aberration	Chinese hamster lung fibroblasts	2.4 mg/ml	Negative	Assay performed with and without S9; cytotoxicity at maximum concentration	Matsuoka et al. (1996)
		Sister chromatid exchange	Chinese hamster ovary cells	5000 µg/ml	Negative	Assay performed with and without S9	Galloway et al. (1987)
		Unscheduled DNA synthesis	Rat hepatocytes	NR	Negative	Abstract; methods and test concentrations not reported	Mirsalis et al. (1983)
842	Benzyl propionate	DNA repair	B. subtilis H17, M45	21 µg/disc	Negative	Assay performed without S9 Japanese article, English summary tables	Oda et al. (1979)
24	Benzyl benzoate	Reverse mutation^a	S. typhimurium TA98, TA100, TA1535, TA1537	3 µmol/plate	Negative	Assay performed with and without S9	Florin et al. (1980)
		Reverse mutation	S. typhimurium TA98, TA100 (preincubation and plate incorporation)	5000 µg/plate	Negative	Assay performed with and without S9; cytotoxicity at three higher concentrations	Schunk et al. (1986)
22	Benzaldehyde	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 (plate incorporation)	38 000 ug/plate	Negative	Assay performed with and without S9	Heck et al. (1989)
		Reverse mutation	S. typhimurium TA98, TA100 (plate incorporation)	300 µl/plate	Negative	Assay of urine samples from rats given benzaldehyde by oral gavage, per- formed with and without S9	Rockwell & Raw (1979)
		Reverse mutation	S. typhimurium TA98, TA100 (plate incorporation)	100 µl/plate	Negative	Assay performed with S9	Rockwell & Raw (1979)
		Reverse mutation^a	S. typhimurium TA98, TA100, TA2637	2 mg/plate	Negative	Japanese article, English summary; assay performed with and without S9	Nohmi et al. (1985)
		Reverse mutation^a	S. typhimurium TA98, TA100, TA1535, TA1537	3 µmol/plate	Negative	Assay performed with and without S9	Florin et al. (1980)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537 (preincubation)	1000 µg/plate	Negative	Assay performed with and without S9	Haworth et al. (1983)
		Reverse mutation	S. typhimurium TA100, TA102, TA104	3300 µg/plate	Negative	Assay performed with and without S9	National Toxicology Program (1990)
		Reverse mutation^a	S. typhimurium TA100	1 mg/plate	Negative	Use of S9 not reported	Rapson et al. (1980)
		Reverse mutation	S. typhimurium TA98, TA100 (preincubation)	NR	Negative	Assay performed with and without S9	Sasaki & Endo (1978)
		Reverse mutation	S. typhimurium TA100, TA102, TA104 (preincubation)	NR	Negative	Assay performed with and without S9	Dillon et al. (1992)
		Reverse mutation	S. typhimurium TA100 (preincubation)	2000 nmol/plate	Negative	Assay performed with and without S9	Vamvakas et al. (1989)
		Reverse mutation^a	S. typhimurium TA98, TA100	500 µg/plate	Negative	Assay performed with and without S9	Kasamaki et al. (1982)
		DNA repair	B. subtilis H17, M45	21 µg/disc	Negative	Japanese article, English summary; use of S9 not reported	Oda et al. (1979)
		DNA repair	B. subtilis H17, M45	NR	Positive	Assay performed with and without S9; positive result only with S9	Matsui et al. (1989)
		Unscheduled DNA synthesis	Rat hepatocytes	250 µg/ml	Negative	Assay performed without S9	Heck et al. (1989)
		Mutation	Mouse L5178Y lymphoma cells	600 µg/ml	Positive	Positive only with S9	Heck et al. (1989)
		Mutation	Mouse L5178Y lymphoma cells	800 µg/ml	Positive	Assay performed without S9; significant increase in mutant fraction at close to toxic doses	McGregor et al. (1991)
		Chromosomal aberrations	Chinese hamster cells	1.2 mg/ml	Positive	Japanese article, English summary; positive results without S9; weakly positive results with S9; cytotoxicity at two higher concentrations	Sofuni et al. (1985)
22	Benzaldehyde	Chromosomal aberrations	Chinese hamster ovary cells	1600 µg/ml	Negative	Assay performed with and without S9	Galloway et al. (1987)
		Chromosomal aberrations	Chinese hamster cells	50 nmol/L	Positive	Assay performed with and without S9	Kasamaki et al. (1982)
		Sister chromatid exchange	Chinese hamster ovary cells	1600 µg/ml	Positive	Assay performed with and without S9	Galloway et al.
		Sister chromatid exchange	Chinese hamster ovary cells	1000 µmol/L	Negative	Assay performed without S9; cytotoxicity at highest concentration	Sasaki et al. (1989)
		Sister chromatid exchange	Human lymphocytes	2 mmol/L	Positive	Assay performed without S9	Jansson et al. (1988)
850	Benzoic acid	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1538 (plate incorporation)	2500 µg/plate	Negative	Assay performed with and without S9	Anderson & Styles (1978)
		Reverse mutation^a	S. typhimurium TA98, TA100, TA1535, TA1536	3.6 µg/plate	Negative	Assay performed with and without S9	Cotruvo et al. (1977)
		Reverse mutation	S. typhimurium TA97, TA98, TA100, TA1535, TA1537 (preincubation)	10 mg/plate	Negative	Assay performed with and without S9	Zeiger et al. (1988)
		Reverse mutation^a	S. typhimurium TA100	1 mg/plate	Negative	Use of S9 not reported	Rapson et al. (1980)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537 (plate incorporation)	1 mg/plate	Negative	Assay performed with S9	McCann et al. (1975)
		Reverse mutation	S. typhimurium TA92, TA94, TA98, TA100, TA1535, TA1537 (preincubation)	10 mg/plate	Negative	Assay performed with and without S9	Ishidate et al. (1984)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 (plate incorporation)	100 µg/plate	Negative	Assay performed without S9	Milvy & Garro (1976)
		Reverse mutation	S. typhimurium TA1535, TA1537, TA1538 (plate incorporation)	0.5%	Negative	Assay performed with and without S9	Food & Drug Admin istration (1975)
		Mutation (umu gene expression)	S. typhimurium TA1535/ pSK1002	1.7 mg/ml	Negative	Assay performed with and without S9	Nakamura et al. (1987)
		DNA repair	B. subtilis H17, H45	NR	Positive	Abstract; methods and test concentration(s) not reported	Nonaka (1989)
		Mutation	Saccharomyces cerevisiae D3	0.18%	Negative	Assay performed with and without S9	Cotruvo et al. (1977)
		Mutation	S. cerevisiae D4	0.15%	Negative	Assay performed with and without S9	Food & Drug Admin- istration (1975)
		Indirect DNA repair (induction of beta-gala- ctosidase)	E. coli PQ37	400 µg/ml	Negative		Glosnicka & Dziadziuszko (1986)
		Chromosomal aberration	Chinese hamster fibroblasts	1.5 mg/ml	Weakly positive	Total incidence of cells with aberrations, 5–9%; performed without S9	Ishidate et al. (1984)
		Sister chromatid exchange	Human lymphocytes	2.0 mmol/L	Negative	Assay performed without S9	Jansson et al. (1988)
851	Methyl benzoate	Reverse mutation	S. typhimurium TA97, TA98, TA100, TA1535, TA1537 (preincubation)	6700 ug/plate	Negative	Assay performed with and without S9	Zeiger et al. (1992)
		Mutation	E. coli Sd-4-73	NR	Negative	Assay performed without S9	Szybalski (1958)
857	Isoamyl benzoate	Mutation	E. coli Sd-4-73	NR	Negative	Assay performed without	Szybalski (1958) S9
864	Isopropylbenzyl alcohol	Reverse mutation	S. typhimurium TA98, TA100 (plate incorporation)	100 µl/plate	Negative	Assay performed with S9	Rockwell & Raw (1979)
		Reverse mutation	S. typhimurium TA98, TA100 (plate incorporation)	300 µl/plate	Negative	Assay of urine samples from rats given isopropyl- benzyl alcohol by oral gavage; performed with and without S9	Rockwell & Raw (1979)
867	Tolualdehydes (mixed ortho, meta, para)	Reverse mutation	S. typhimurium TA104 (preincubation)	0.8 µmol/plate	Negative	Assay performed with and without S9	Marnett et al. (1985)
		Reverse mutation^a	S. typhimurium TA98, TA100, TA1535, TA1537	3 µmol/plate	Negative	Assay performed with and without S9	Florin et al. (1980)
		Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 (plate incorporation)	19 000 ug/plate	Negative	Assay performed with and without S9	Heck et al. (1989)
		Reverse mutation	S. typhimurium TA98, TA100, TA102 (plate incorporation)	0.8 mmol/plate	Negative	Assay performed with and without S9	Aeschbacher et al. (1989)
		Reverse mutation	S. typhimurium TA97, TA100, TA1535, TA1537 (preincubation)	666 µg/plate	Negative	Assay performed with and without S9	Zeiger et al. (1988)
		Unscheduled DNA synthesis	Rat hepatocytes	1000 µg/ml	Negative	Assay performed without S9	Heck et al. (1989)
		Mutation	Mouse L5178Y lymphoma cells	300 µg/ml	Negative	Assay performed with and without S9	Heck et al. (1989)
868	Cuminaldehyde	Reverse mutation	S. typhimurium TA98, TA100 (plate incorporation)	100 µl/plate	Negative	Assay performed with S9	Rockwell & Raw (1979)
		Reverse mutation	S. typhimurium TA98, TA100 (plate incorporation)	300 µl/plate	Negative	Assay of urine samples from rats given cuminaldehyde by gavage; performed with and without S9	Rockwell & Raw (1979)
		Sister chromatid exchange	Chinese hamster ovary cells	333 µmol/L	Negative	Assay performed without S9; cytotoxicity at maximum concentration	Sasaki et al. (1989)
In vivo
25	Benzyl alcohol	Sex-linked recessive lethal mutation	Drosophila melanogaster	5000 mg/kg\| 8000 mg/kg	Negative Negative	In feed By injection	Foureman et al. (1994)
		Micronucleus formation	Mouse bone-marrow cells	200 mg/kg bw	Negative	By intraperitoneal injection	Hayashi et al. (1988)
		Replicative DNA synthesis	Mouse hepatocytes	NR	Positive	Route of administration not reported	Yoshikawa (1996)
		Sex-linked recessive lethal mutation	D. melanogaster	300 mg/kg 20 000 mg/kg	Negative Negative	In feed By injection	National Toxicology Program (1993); Foureman et al. (1994)
		Sister chromatid exchange	Mouse bone-marrow cells	1700 mg/kg bw	Negative	By intraperitoneal injection	National Toxicology Program (1993)
23	Benzyl acetate	Chromosomal aberration	Mouse bone-marrow cells	1700 mg/kg bw	Negative	By intraperitoneal injection	National Toxicology Program (1993)
		Micronucleus formation	Mouse bone-marrow cells	1300 mg/kg bw	Negative	By intraperitoneal injection	National Toxicology Program (1993); Shelby et al. (1993)
		Micronucleus formation	Mouse erythrocytes	50 000 mg/kg	Negative	By intraperitoneal injection	National Toxicology Program (1993)
		Unscheduled DNA synthesis	Rat hepatocytes	NR	Negative	By oral gavage; abstract; methods and doses not reported	Mirsalis et al. (1983)
		Unscheduled DNA synthesis	Rat hepatocytes	1000 mg/kg bw	Negative	By oral gavage	Mirsalis et al. (1989)
		Unscheduled DNA synthesis	Rat pancreatic cells	1000 mg/kg bw	Negative	By oral gavage	Steinmetz & Mirsalis (1984)
		DNA damage	Rat pancreatic cells	500 mg/kg bw	Negative	By gavage	Longnecker et al. (1990)
		DNA damage	Rate pancreatic cells	0.9%	Negative	In diet	Longnecker et al. (1990)
22	Benzaldehyde	Sex-linked recessive lethal mutation	D. melanogaster	1200 mg/kg 2500 mg/kg	Negative Negative	In feed By injection	Woodruff et al. (1985)

NR, not reported; S9, exogenous metabolic activation system consisting of 9000 x g supernatant from rodent liver

^{a Not reported whether plate incorporation or preincubation method used}

A total of 12 benzyl derivatives in the group have been tested for genotoxicity. In view of the mainly negative results in the assays in vitro and the uniformly negative results in well-recognized assays in vivo, the Committee concluded that the group of benzyl derivatives is not genotoxic in vivo.

(e) Reproductive toxicity

At its forty-sixth meeting, the Committtee reviewed a series of studies of developmental and reproductive toxicity with benzyl alcohol (No. 25), benzyl acetate (No. 23), benzyl aldehyde (No. 22), and sodium benzoate (Annex 1, reference 122). The Committee concluded that: "Delayed development and reduced fetal and postnatal pup body weights were observed in developmental toxicity studies in rats, mice, hamsters and rabbits, but only at doses that were toxic to the mother. In a teratogenicity study with sodium benzoate, doses that induced severe maternal toxicity were associated with embryotoxic and fetotoxic effects and fetal malformations. A 4-generation study in rats showed no effect on growth, fertility, lactation or survival". The Committee concluded that the data reviewed were sufficient to demonstrate a lack of teratogenic and reproductive potential. No further studies on reproductive toxicity with benzyl derivatives in the group were available for review by the Committee at its present meeting.

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ENDNOTES

¹During evaluation of these agents, the Committee questioned whether some substances in the group (see footnote to Table 1) were in fact used as flavouring agents and should therefore appropriately be evaluated with the Procedure. Information to address this question will be sought from the relevant manufacturers.

    See Also:
       Toxicological Abbreviations

SAFETY EVALUATION OF CERTAIN FOOD ADDITIVES AND CONTAMINANTS