Japan Flavour and Fragrance Materials Association’s (JFFMA) safety assessment of food-flavouring substances uniquely used in Japan that belong to the class of aliphatic primary alcohols, aldehydes, carboxylic acids, acetals and esters containing additional oxygenated functional groups

ABSTRACT

We performed a safety evaluation using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) of the following four flavouring substances that belong to the class of ‘aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups’ and are uniquely used in Japan: butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate. Although no genotoxicity study data were found in the published literature, none of the four substances had chemical structural alerts predicting genotoxicity. All four substances were categorised as class I by using Cramer’s classification. The estimated daily intake of each of the four substances was determined to be 0.007–2.9 μg/person/day by using the maximised survey-derived intake method and based on the annual production data in Japan in 2001, 2005 and 2010, and was determined to be 0.250–600.0 μg/person/day by using the single-portion exposure technique and based on average-use levels in standard portion sizes of flavoured foods. Both of these estimated daily intake ranges were below the threshold of toxicological concern for class I substances, which is 1800 μg/person/day. Although no information from in vitro and in vivo toxicity studies for the four substances was available, these substances were judged to raise no safety concerns at the current levels of intake.

Graphical Abstract

KEYWORDS:

• Aliphatic primary alcohols
• aldehydes
• carboxylic acids
• acetals
• and esters containing additional oxygenated functional groups
• Cramer decision tree
• food flavours
• Joint FAO/WHO Expert Committee on Food Additives (JECFA)
• safety
• threshold of toxicological concern

Introduction

Flavouring substances used in Japan

Various flavouring substances have been found or developed that improve the taste and aroma of food products but have no nutritional value. The wide variety of these substances enables the food industry to meet the needs of mimicking the ‘naturally occurring’ flavours of foods. According to an annual use survey by the Japan Flavour and Fragrance Materials Association (JFFMA), approximately 3200 flavouring substances have been used commercially in Japan (Someya 2012). Among them, 134 substances are currently approved as designated additives by the Ministry of Health, Labour and Welfare in Japan. The remaining substances have been classified into 18 designated chemical structural groups and approved for use as food additives under the compliance of the Food Sanitation Act of Japan; however, science-based safety evaluation of these substances is lacking.

Safety evaluation procedures

In 1996, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) adopted a novel safety evaluation procedure for examining flavouring substances (WHO 1996). Under this procedure, the safety of many flavouring substances can be efficiently evaluated utilising the safety evaluation data for substances that have similar chemical structures or metabolic pathways, even if the substances under investigation have no safety data. Evaluation protocols similar to the JECFA procedure are used in the United States by the Flavour and Extract Manufacturers Association of the United States (FEMA) Expert Panel and in the European Union by the European Food Safety Authority (EFSA). In addition, the results of the safety evaluations by JECFA, the FEMA Expert Panel and EFSA are now widely utilised in more than 70 countries (Konishi et al. 2014).

Of the approximately 3200 flavouring substances used in Japan, about 2400 have been judged safe under their intended conditions of use for flavouring by JECFA, the FEMA Expert Panel and/or EFSA. However, this leaves about 800 flavouring substances whose safety has not been evaluated by these organisations. Thus, in 2009, the JFFMA established a Safety Re-evaluation Committee composed of expert scientists in the fields of toxicology and food chemistry as well as representative members of the JFFMA to evaluate substances uniquely used in Japan based on the JECFA evaluation procedure (see Figure S1 in the supplemental data online), with the order of priority being determined by the annual volume of production of the substances.

In the present study, using the JECFA evaluation procedure, we evaluated the safety of the following four flavouring substances that are uniquely used in Japan and that based on the classification of flavouring substances by JECFA belong to the class ‘aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups’: butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate. These four substances have unique and characteristic odours and are used with other flavouring substances to improve the flavour of processed foods, such as jams, jellies and soft drinks.

Collecting information for the evaluation procedure

Chemical information for four flavouring substances and their metabolites

The Chemical Abstracts Service (CAS) number, molecular weight and chemical structure of the four flavouring substances to be subjected to toxicity evaluation (butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate) are shown in Table 1. These substances have not been reported to occur naturally, and, thus, all four substances are expected to be ingested only in foods containing them as flavouring agents (JFFMA 2003, 2006, 2011). These four substances are metabolised to various hydrolysis or oxidation products. Table 1 presents the chemical information of these produced substances to be subjected to toxicity evaluation.

Table 1. Chemical information on the four flavouring substances and their metabolites.

Flavouring substance	Hydrolysis product	Oxidation product
Name/CAS No./Molecular weight Chemical structure
Butyl butyrylacetate/–/186.25	Butyrylacetic acid/30414-54-1/144.17	Acetoacetic acid/541-50-4/102.09
	Butanol/71-36-3/74.12
Ethyl 2-hydroxy-4-methylpentanoate/10348-47-7/160.21	2-Hydroxy-4-methylpentanoic acid/498-36-2/132.16	α-Keto acids
3-Hydroxyhexanoic acid/10191-24-9/132.16		Acetoacetic acid/541-50-4/102.09
Methyl hydroxyacetate/96-35-5/90.08	Glycolic acid (hydroxyacetic acid)/79-14-1/76.05	Glyoxylic acid/298-12-4/74.04
		Oxalic acid/144-62-7/90.03

Note: CAS No. = Chemical Abstracts Service number.

Predicting genotoxic potential using chemical structure and in silico models

The genotoxic potential of each substance was evaluated by analysing the biological potential of its chemical structure. While the JECFA decision-tree approach does not cover genotoxicity in its evaluation procedure (WHO 1996), the potential of genotoxicity is now evaluated before the steps of the decision tree are undertaken by incorporating structural alerts and other available information on genotoxicity of structurally related chemicals. In the present study, following the JECFA procedure, the structural alerts for chemical substructure groups were examined, as is generally done for chemicals lacking genotoxicity information (Ashby & Tennant 1988, 1991; Tennant et al. 1990). While genotoxicity study data were not available for any of the four substances, there were no structural alerts on their chemical structures. The genotoxic potential of the four substances was also examined using structure–activity relationship (SAR) analysis (Hansch 1969) with in silico prediction software that has been previously applied to the regulatory evaluation of flavouring substances (Cronin et al. 2003; Ono et al. 2012). Three SAR software programs with different algorithms were used for the analysis: DEREK (v. 10.0.2; Lhasa Ltd, Leeds, UK), MultiCASE (v. 1.90; Multicase Inc., Cleveland, OH, USA), and ADMEWORKS (v. 4.0; Fujitsu Kyusyu Systems Ltd, Fukuoka, Japan). All three programs indicated that all four substances had no genotoxic concern. The available genotoxicity information for structurally related substances was also referred to during our evaluation. Based on these results, we concluded that the JECFA evaluation procedure could be applied to these four flavouring substances.

Estimating daily intake of flavouring substances using SPET and MSDI methods

The development of the maximised survey-derived intake (MSDI) procedure was based on disappearance data from periodic surveys of ingredient manufacturers using the volume of ingredients produced during the survey year (Young et al. 2006). Because the production volume varied every survey year, the maximum production volume of each substance was selected from past surveyed data to avoid any underestimation of the intake. According to the annual production data in Japan from 2001, 2005 and 2010, the annual production volumes as flavourings of butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate were within the ranges of 0.2–12.0, 0.030–0.088, 4.8–11.0 and 0.021–0.030 kg year⁻¹ respectively (JFFMA 2003, 2006, 2011). Based on these data, the daily intake of each of the four flavouring substances was calculated using the MSDI method. Daily intakes of butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate were estimated to be 2.9, 0.021, 2.4 and 0.007 μg/person/day respectively (Table 2).

Table 2. Estimated daily intake data of four flavouring substances uniquely used in Japan.

	Annual volume of production (kg year^−1)			Estimated daily intake (μg/person/day)
Flavouring substance	2001	2005	2010	MSDI^a	SPET^b	Name and code of the food group(s) showing the highest use level^c
Butyl butyrylacetate	–	12.0	0.2	2.9	15.0	04.1.2.5 Jams, jellies, marmalades
Ethyl 2-hydroxy-4-methylpentanoate	–	0.088	0.030	0.021	0.900	14.2.6 Distilled spirituous beverages containing more than 15% alcohol
3-Hydroxyhexanoic acid	4.8	10.0	11.0	2.4	600.0	14.1 Non-alcoholic ‘soft’ beverages (includes fruit juices, coffee and tea)
Methyl hydroxyacetate	–	0.021	0.030	0.007	0.250	01.7 Dairy-based desserts

Notes:

^aCalculated by the maximised survey-derived intake (MSDI) method based on the maximum usage volume from annual production data of 2001, 2005 and 2010 in Japan.

MSDI value (μg/person/day) = annual volume of production (kg) × 10⁹ (μg kg⁻¹)/population of consumers × 0.8 × 365 days. The population of consumers was assumed to be 12 × 10⁶.

^bCalculated using the single-portion exposure technique (SPET) method based on the average-use levels in standard portion sizes of flavoured foods (μg/person/day).

^cAs listed in the Codex General Standard for Food Additives, Codex Stan 192-1995 (FAO 2011) (available at: http://www.fao.org/gsfaonline/docs/CXS_192e.pdf).

The daily intake of each substance was calculated using the single-portion exposure technique (SPET) (WHO 2008) and based on the estimated use (food category) and use-level survey conducted by JFFMA (unpublished information 2013–16) and single portion sizes of each food category from the predefined food categories in the Codex General Standard for Food Additives. As a result, the estimated daily intakes of butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate were calculated as 15.0, 0.900, 600.0 and 0.250 µg/person/day respectively (Table 2).

Because the daily intake volumes estimated by SPET were higher than those determined by the MSDI method, a safety evaluation was conducted using the values calculated by SPET.

Estimating absorption, metabolism and excretion

No information was available on the absorption, metabolism and excretion of the four substances. Therefore, their structural components or structurally related substances were examined for their metabolic fate. Three of four flavouring substances, butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate and methyl hydroxyacetate, are esters, whereas the remaining substance, 3-hydroxyhexanoic acid, is a fatty acid. Hydrolysis of esters mainly occurs in the intestinal tract, blood and liver and is catalysed by carboxylesterases or esterases in most tissues (Heymann 1980; Anders 1989). Acetyl esters are the preferred substrates of carboxylesterases (Heymann 1980), and the presence of a second oxygenated functional group has little effect on the hydrolysis of esters. Evidence for hydrolysis of esters has come from various types of experiments. For example, incubation of aqueous methyl 2-oxo-3-methylpentanoate with a 2% pancreatin solution (pH 7.5) results in virtually complete hydrolysis (> 98%) within 80 min (Leegwater & van Straten 1979). Dibutyl sebacate is also hydrolysed in vitro in a 10% crude pancreatic lipase solution (Smith 1953). The ¹⁴C-tributylacetyl citrate administered by gavage to male Sprague–Dawley rats at a single dose of 70 mg kg⁻¹ body weight (bw) is rapidly absorbed (half-life, 1 h) and partially hydrolysed in the blood. More than 87% of the radiolabelled tributylacetyl citrate is eliminated within 24 h of dosing. At least nine urinary metabolites representing 59–70% of the dose have been detected. Five metabolites have been identified as the partially hydrolysed mono-, di- and trialkylesters of citric acid. Three metabolites representing 25–36% of the dose have been identified in the faeces. Approximately 2% is eliminated as ¹⁴CO₂ (Hiser et al. 1992). With regard to the metabolic fate of fatty acids, they are metabolised after ingestion in organs such as the liver, heart and skeletal muscle to fatty acyl adenylate by fatty acid-coenzyme A ligase, and fatty acyl adenylate reacts with free coenzyme A to give fatty acyl-coenzyme A. The fatty acyl-coenzyme A enters the mitochondrial matrix via the carnitine shuttle to be metabolised to acetyl-coenzyme A by β-oxidation and is completely oxidised to CO₂ and water (Houten & Wanders 2010).

Butyl butyrylacetate is expected to be hydrolysed to butyrylacetic acid and butanol. Butyrylacetic acid and 3-hydroxyhexanoic acid can be oxidised in vivo to acetoacetic acid, which is endogenous in humans and is formed from the condensation of two acetyl-coenzyme A units in the fatty acid pathway. Acetoacetic acid is released from the liver into the bloodstream and transported to peripheral tissues where it is converted to acetyl-coenzyme A and is completely metabolised (Voet & Voet 1990). When endogenous levels are high, acetoacetic acid yields acetone and carbon dioxide through non-enzymatic decarboxylation of endogenous β-keto acids (Voet & Voet 1990). Ethyl 2-hydroxy-4-methylpentanoate is an α-keto ester, is hydrolysed to 2-hydroxy-4-methylpentanoic acid and is expected to be metabolised in the same manner as endogenous α-keto esters formed from oxidative deamination of leucine in vivo (Voet & Voet 1990). Methyl hydroxyacetate is expected to be hydrolysed to glycolic acid (hydroxyacetic acid), with most of the glycolic acid excreted directly in the urine. A small proportion of the glycolic acid is metabolised to glyoxylic acid and oxalic acid (DuPont 1963).

Safety evaluation of the four substances using the JECFA evaluation procedure

Decision-tree approach

Details of the JECFA evaluation procedure are described in a JECFA evaluation report (WHO 1996). A summary of the decision-tree approach for this procedure is shown in Figures S1 and S2 in the supplemental data online.

Step 1

In the first step, each flavouring substance was classified into one of three chemical structure classes (I, II or III) using the Cramer decision tree, as shown in Figure S2 in the supplemental data online (Cramer et al. 1978). Substances in class I have simple chemical structures with known efficient metabolic pathways that suggest low oral toxicity. Substances in class II are less innocuous than class I, but do not contain structural features suggestive of those that produce toxicity, such as substances in class III. Substances in class III have a chemical structure that may cause significant toxicity.

Based on the results of the Cramer decision-tree analysis (See Figure S2 in the supplemental data online) (Cramer et al. 1978), all four substances were assigned to structural class I (Table 3).

Table 3. Safety evaluation of four flavouring substances using the JECFA evaluation procedure: Step 1, Cramer decision-tree approach.

Flavouring substance	Cramer’s decision tree (N, no; Y, yes)	Chemical structural class
Butyl butyrylacetate	Q1N, Q2N, Q3N, Q5N, Q6N, Q7N, Q16N, Q17N, Q19Y, Q20Y, Q21N, Q18N	I
Ethyl 2-hydroxy-4-methylpentanoate
3-Hydroxyhexanoic acid
Methyl hydroxyacetate

Step 2

In the second step, the metabolic fate of each substance was predicted by its chemical structure and applied using a decision-tree approach consisting of two branches (see Figure S1, Step A3, in the supplemental data online). Based on estimates of the metabolism of the structurally related substances or their metabolites determined by the JECFA procedure, all four substances were predicted to go to ‘A’ side of the decision tree.

Step A3

In this step, the estimated daily intake of each substance was compared with the threshold level of human exposure based on Cramer’s three structural classes (Cramer et al. 1978), with estimated threshold levels of 1800, 540 and 90 µg/person/day for structural classes I, II, and III respectively (Munro et al. 1996, 1999).

As mentioned above, the estimated daily intakes of the four flavouring substances were calculated using SPET because these values were higher than those calculated using the MSDI method. The daily intake of the four class I substances were 0.007–600.0 μg/person/day (Table 4), which was below the threshold of toxicological concern applied to structural class I substances, which is 1800 μg/person/day.

Table 4. Safety evaluation of four flavouring substances using the JECFA evaluation procedure: Steps 2 and A3.

Structure class	Flavouring substance	Step 2	Step A3^a	Decision
Class I	Butyl butyrylacetate	Yes	No (MSDI: 2.9, SPET: 15.0)	Substance would not be expected to be a safety concern
	Ethyl 2-hydroxy-4-methylpentanoate		No (MSDI: 0.021, SPET: 0.900)
	3-Hydroxyhexanoic acid		No (MSDI: 2.4, SPET: 600.0)
	Methyl hydroxyacetate		No (MSDI: 0.007, SPET: 0.250)

Note: ^aEstimated threshold level is 1800 μg/person/day for chemical structural class I.

Considering the combined intake from their use as flavouring agents

Because the JECFA considers evaluation of the combined intake to be unnecessary when the highest MSDI value is less than 20 μg/person/day (WHO 2011), the evaluation examining the combined intake of all four substances was omitted here (Table 2).

Assessing toxicological information for the metabolites and for structurally related substances of the four flavouring substances

Because no in vitro or in vivo toxicity data were available for any of the four substances, data from their metabolites and structurally related substances reported by the JECFA (WHO 2000, 2011) were examined (Table 1 and see Table S1 in the supplemental data online). The four flavouring substances had many structurally related substances, and so data were selected based on those showing close structural relation.

Sub-acute toxicity/carcinogenicity

Regarding toxicity studies of the metabolites and structurally related substances of the four flavouring substances, the results on ethyl acetoacetate (Cook et al. 1992), levulinic acid (Tischer et al. 1942), glycolic acid (USEPA 2000) and propylene glycol (Morris et al. 1942; Weil et al. 1971; Gaunt et al. 1972) have been reported in rats and are shown in Table 5.

Table 5. Sub-acute toxicity and carcinogenicity data summary for the metabolites and structurally related substances of four flavouring substances.

Substance	Animals used	Exposure method (experimental period)	Dose (mg kg^−1 bw day^−1)	Toxicological finding (mg kg^−1 bw day^−1)	Reference
Ethyl acetoacetate	Rat (male/female)	Diet (28–29 days)	100, 300, 1000	NOAEL > 1000	Cook et al. (1992)
Levulinic acid	Rat	Diet (16 days)	1000, 2000	NOAEL > 2000	Tischer et al. (1942)
Glycolic acid	Rat (male/female)	Diet (90 days)	150, 300, 600	NOAEL = 150	USEPA (2000)
Propylene glycol	Rat	Diet (2 years)	310, 630, 1300, 2500	No treatment-related adverse effects at all treated groups. NOAEL > 2500	Gaunt et al. (1972)
	Rat	Diet (2 years)	900, 1800	No treatment-related adverse effects at all treated groups. NOAEL > 1800	Morris et al. (1942)
	Dog	Diet (2 years)	2000, 5000	Increased erythrocyte destruction at 5000 mg kg^−1 bw dose. No significant treatment-related effects at 2000 mg kg^−1 bw dose. NOAEL = 2000	Weil et al. (1971)

Note: bw, body weight; NOAEL, no observed adverse effect level.

In one study, Sprague–Dawley rats, 16 males and 16 females/group, were administered ethyl acetoacetate via their diet at 100, 300 or 1000 mg kg⁻¹ bw day⁻¹ for 28 or 29 days. Although proteinaceous aggregates in the urinary bladder of males and calcium deposits in the kidneys of females were found in the group ingesting 1000 mg kg⁻¹ bw day⁻¹, renal function was unimpaired in the treated male and female animals. Thus, the no observed adverse effect level (NOAEL) of ethyl acetoacetate was determined to be > 1000 mg kg⁻¹ bw day⁻¹ (Cook et al. 1992).

In another study, albino rats (three animals/group) were administered levulinic acid at 0, 1000 or 2000 mg kg⁻¹ bw day⁻¹ via their diet for 16 days. The treated animals showed no indication of toxicity at these doses, and the NOAEL of levulinic acid was determined to be > 2000 mg kg⁻¹ bw day⁻¹ (Tischer et al. 1942).

In a study of rats administered glycolic acid at 0, 150, 300 or 600 mg kg⁻¹ bw day⁻¹ for 90 days, decreases in bw and bw gain were observed at 300 mg kg⁻¹ bw day⁻¹. Thus, the NOAEL of glycolic acid was determined to be 150 mg kg⁻¹ bw day⁻¹ (USEPA 2000).

In a study in which rats received propylene glycol in their diet at a dose of 0, 310, 630, 1300 or 2500 mg kg⁻¹ bw day⁻¹ for 2 years, no treatment-related adverse effects on bw gain or organ weight or on haematological, urinary or clinical chemical endpoints were found. Thus, the NOAEL of propylene glycol in that study was determined to be > 2500 mg kg⁻¹ bw day⁻¹ (Gaunt et al. 1972). In a study in which rats were given propylene glycol as 0%, 2.45% or 4.90% of their dietary intake (equivalent to doses of 0, 900 and 1800 mg kg⁻¹ bw day⁻¹ respectively) for 2 years, no treatment-related adverse effects were found on body growth or histological changes in the examined organs and tissues. Thus, the NOAEL of propylene glycol in that study was determined to be > 1800 mg kg⁻¹ bw day⁻¹ (Morris et al. 1942). In a study in which male and female beagle dogs received propylene glycol in the diet at a dose of 0, 2000 or 5000 mg kg⁻¹ bw day⁻¹ for 2 years, increased erythrocyte destruction was found at a dose of 5000 mg kg⁻¹. However, no significant treatment-related effects on haematological, clinical chemical or urinary endpoints, or on gross or histological appearance were found at a dose of 2000 mg kg⁻¹. Thus, the NOAEL of propylene glycol in that study was determined to be 2000 mg kg⁻¹ bw day⁻¹ (Weil et al. 1971).

Genotoxicity

Several in vivo and in vitro genotoxicity studies have examined either the metabolites or the structurally related substances of four flavouring substances, namely lactic acid, ethyl acetoacetate, 1-hydroxypropan-2-one, L-malic acid, pyruvic acid, prop-1-ene-1,2,3-tricarboxylic acid, ethyl lactate, ethyl pyruvate, diethyl malonate, butyl-O-butyryllactate, ethyl 3-hydroxybutyrate and methyl acetoacetate. The results of these studies are summarised in Table 6.

Table 6. In vivo and in vitro genotoxicity data summary for the metabolites and structurally related substances of four flavouring substances.

Substance	Endpoint	Test object	Dose/concentration	Result	Reference
Lactic acid	Chromosomal aberration	Chinese hamster ovary K1 cells	900–1400 μg ml^−1	Positive^a	Morita et al. (1990)
	Reverse mutation (Ames test)	S. typhimurium TA97, TA98, TA100, TA104	0.5–2.0 μl plate^−1	Negative^a	Al-Ani and Al-Lami (1988)
Propylene glycol	Chromosomal aberration	Hamster lung fibroblasts	32,000 μg ml^−1	Positive^b	Ishidate et al. (1984)
			n.r.	Positive^b	Kawachi et al. (1981)
		Human embryonic lung cells	0.001–0.1 μg ml^−1	Negative	Weir (1974)
		Rat	30–5000 mg kg^−1 bw	Negative
		Mice	0.6–24 mg kg^−1 bw	Negative	Vargova et al. (1980)
	Sister chromatid exchange	Chinese hamster ovary Don-6 cell line	3800–23,000 μg ml^−1	Positive	Sasaki et al. (1980)
		Human fibroblastic cell line HE2144	7600 μg ml^−1	Negative
		Hamster lung fibroblasts	n.r.	Negative^b	Kawachi et al. (1981)
	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538	1–10,000 μg plate^−1	Negative	Clark et al. (1979)
		S. typhimurium TA98, TA100, TA1535, TA1537	100–10,000 μg plate^−1	Negative^a	Haworth et al. (1983)
			230 μg plate^−1	Negative^a	Florin et al. (1980)
		S. typhimurium TA92, TA94, TA98, TA100, TA1535, TA1537	10,000 μg plate^−1	Negative^a	Ishidate et al. (1984)
		S. typhimurium TA100	1000 μmol plate^−1	Negative	Stolzenberg and Hine (1979)
		S. typhimurium TA98, TA100	n.r.	Negative^a	Kawachi et al. (1981)
		S. typhimurium	n.r.	Negative	McCann and Ames (1976)
	Host-mediated mutation	S. typhimurium TA1530 and G46	0.01–0.25 ml	Negative	Weir (1974)
		Saccharomyces cerevisiae	0.01–0.25 ml	Positive
Propylene glycol	Micronucleus formation	Chinese hamster ovary Don-6 cell line	3800–23,000 μg ml^−1	Negative	Sasaki et al. (1980)
		Human fibroblastic cell line HE2144	3800–23,000 μg ml^−1	Negative
		Mice	0–20,000 mg kg^−1 bw	Negative	Hayashi et al. (1988)
Ethyl acetoacetate	Chromosomal aberration	Chinese hamster cells	2 mg ml^−1 (2000 μg ml^−1)	Negative^a	Ishidate et al. (1984)
	Reverse mutation; pre-incubation protocol	S. typhimurium TA92, TA100, TA1535, TA1537 TA94 and TA98	25 mg plate^−1 (25,000 μg plate^−1)	Negative^a
		S. typhimurium TA97, TA102	0.1–10 mg plate^−1 (100–10,000 μg plate^−1)	Negative^a	Fujita and Sasaki (1987)
	DNA repair test (Rec assay)	B. subtilis; H17, M45	20 μl disk^−1 (20,000 μg disk^−1)	Positive	Yoo (1986)
			10–20 μl ml^−1 (10–20 μg ml^−1)	Negative^a	Kuroda et al. (1984)
			20 μg disk^−1	Negative^a	Oda et al. (1978)
	Gene mutation	E. coli; WP2 uvrA	200–1600 μg plate^−1	Positive^b	Yoo (1986)
1-Hydroxypropan-2-one	Reverse mutation	S. typhimurium TA100	n.r.	Positive^c	Garst et al. (1983)
			20–400 μg plate^−1	Positive^a	Yamaguchi (1982)
			500 μg plate^−1	Positive^a	Yamaguchi and Nakagawa (1983)
		S. typhimurium TA104	68 μmoles (5 μg ml^−1)	Positive^c	Marnett et al. (1985)
L-malic acid	Reverse mutation	S. typhimurium TA97, TA98, TA100, TA104	2000 μg plate^−1	Negative^a	Al-Ani and Al-Lami (1988)
Pyruvic acid	Reverse mutation	S. typhimurium TA98, TA100	10–10,000 μg plate^−1	Negative^a	Bjeldanes and Chew (1979)
		S. typhimurium TA100	200 μg plate^−1	Negative^a	Yamaguchi (1982)
Prop-1-ene-1,2,3-tricarboxylic acid	Reverse mutation	S. typhimurium TA100, TA1535, TA1537, TA98	20,000 μg plate^−1	Negative^a	Andersen and Jensen (1984)
Ethyl lactate	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538	n.r.	Negative^a	Clary et al. (1998)
Ethyl pyruvate	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537	32–20,000 μg plate^−1	Negative^a	Andersen and Jensen (1984)
Diethyl malonate	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537	3 μmol plate^−1 (480 μg plate^−1)	Negative^a	Florin et al. (1980)
Butyl-O-butyryllactate	Micronucleus formation	Mice	1514 mg kg^−1 bw	Negative	Wild et al. (1983)
	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538	0–3600 μg plate^−1	Negative^a
	Sex-linked recessive lethal mutation	Drosophila melanogaster	5400 μg ml^−1	Negative
Ethyl 3-hydroxybutyrate	Reverse mutation	S. typhimurium TA97, TA98, TA100, TA1535	n.r.	Negative	Zeiger and Margolin (2000)
Methyl acetoacetate	Reverse mutation	S. typhimurium TA98, TA100, TA1535, TA1537, TA1538 E. coli WP2 uvrA	1–5000 μg plate^−1	Negative ^c	Shimizu et al. (1985)

Notes:

^aWith and without S9 metabolic activation.

^bConcentrations used were 10-fold higher than those of spontaneous revertants.

^cWithout S9 metabolic activation.

bw, body weight; n.r., not reported.

Lactic acid displayed a negative result in the reverse mutation assay (Ames test) (Al-Ani & Al-Lami 1988) but a positive result in the chromosome aberration test. However, this positive result was found using non-physiological (low) pH levels (Morita et al. 1990). Lactic acid is an endogenous substance and is well estimated to be metabolised into endogenous products.

Propylene glycol showed positive results only in minor number of in vitro assays, i.e., two chromosomal aberration tests, one sister chromatid exchange assay and one host-mediated mutation test, and negative results in most in vitro assays and all in vivo assays (Weir 1974; McCann & Ames 1976; Clark et al. 1979; Stolzenberg & Hine 1979; Florin et al. 1980; Sasaki et al. 1980; Vargova et al. 1980; Kawachi et al. 1981; Haworth et al. 1983; Ishidate et al. 1984; Hayashi et al. 1988).

Ethyl acetoacetate, an ester of β-keto acid, displayed negative results in both the chromosomal aberration and reverse mutation assays (Ishidate et al. 1984; Fujita & Sasaki 1987). But ethyl acetoacetate also produced negative results in two studies testing DNA repair using rec assays (Oda et al. 1978; Kuroda et al. 1984). However, the results of another DNA repair test were positive, and in that same study a gene mutation assay also provided a positive result (Yoo 1986). Ethyl acetoacetate includes a centre of Michael reactivity, which is recognised by the JECFA as one of the structural alerts for genotoxicity. However, the JECFA concluded that this substance is readily metabolised in vivo (WHO 2000). Thus, esters of β-keto or β-hydroxy acids are hydrolysed to acetoacetic acid or its β-hydroxy or aldehyde precursor. The latter two can be oxidised in vivo to acetoacetic acid, which is an endogenous product in humans and is formed from the condensation of two acetyl-coenzyme A units in the fatty acid pathway. It is released from the liver into the bloodstream and transported to peripheral tissues, where it is converted to acetyl-coenzyme A and is completely metabolised. When endogenous levels are high, β-keto acids may undergo non-enzymatic decarboxylation, which for acetoacetic acid yields acetone and carbon dioxide (Voet & Voet 1990).

Positive results were found using several reverse mutation assays for 1-hydroxypropan-2-one (Yamaguchi 1982; Garst et al. 1983; Yamaguchi & Nakagawa 1983; Marnett et al. 1985). However, 1-hydroxypropan-2-one is an endogenous metabolite of acetone that is endogenously formed from the degradation of body fat and fatty acids, and it occurs at low concentrations in the blood of healthy humans without acetone exposure (Wang et al. 1994). In this major metabolic pathway, the majority of the acetone in blood would be oxidised to 1-hydroxypropan-2-one (WHO 1998). Taking these data together, we determined that the daily intake of 1-hydroxypropan-2-one as flavouring substances does not affect human health.

Pyruvic acid, L-malic acid, prop-1-ene-1,2,3-tricarboxylic acid, ethyl lactate, ethyl pyruvate, diethyl malonate, butyl-O-butyryllactate, ethyl 3-hydroxybutyrate and methyl acetoacetate showed negative results with respect to mutagenicity (Bjeldanes & Chew 1979; Florin et al. 1980; Yamaguchi 1982; Wild et al. 1983; Andersen & Jensen 1984; Shimizu et al. 1985; Al-Ani & Al-Lami 1988; Clary et al. 1998; Zeiger & Margolin 2000).

Based on the results of sub-acute toxicity/carcinogenicity and genotoxicity studies, we concluded that the metabolites of the four flavouring substances as well as substances structurally related to these flavouring substances do not pose a safety concern when used at low levels of intake or as flavouring substances.

Evaluation of substances based on the JECFA procedure

This review presented the key scientific data relevant to a safety evaluation using the JECFA procedure for the safety evaluation of flavouring agents of four flavouring substances that are uniquely used in Japan and belong to the JECFA classification of ‘aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups’. Regarding predicting genotoxicity, none of the four substances had structural alerts, and none showed genotoxic potential based on in silico software analyses. Although no metabolism data were available for any of these four substances, it is well estimated that esters and fatty acids in this group can be metabolised to endogenous products. All four substances were categorised as class I using the Cramer decision tree (Cramer et al. 1978). The estimated daily intake for each substance was within 0.007 and 2.9 µg/person/day as determined by the MSDI method and within 0.250 and 600.0 by SPET, which was below the toxicological threshold of concern for this class (1800 μg/person/day). Thus, it was concluded that none of the four substances raised safety concerns when used for flavouring foods at the current estimated intake levels.

Conclusions

In conclusion, the four flavouring substances, butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate, pose no health risk to humans when used to flavour foods, and the intake of each substance at the present levels of use as a food-flavouring ingredient is safe.

Acknowledgments

The authors thank Professor Takeshi Yamazaki, Department of Food and Health Sciences, Jissen Women’s University, for his expert advice in preparing the manuscript.

Disclosure statement

Kenji Saito, Fumiko Sekiya, Shim-mo Hayashi, Yoshiharu Mirokuji, Hiroyuki Okamura and Shinpei Maruyama are employed by flavour manufacturers whose product lines include flavouring substances. The views and opinions expressed in this article are those of the authors and not necessarily those of their respective employers. Yasuko Hasegawa-Baba, Atsushi Ono, Madoka Nakajima, Masakuni Degawa, Shogo Ozawa, Makoto Shibutani and Tamio Maitani declare that no conflicts of interest exist.

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Additional information

Funding

This work was supported by the Japan Flavour and Fragrance Materials Association.