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Taking information into account both for putative metabolites and for closely related structural analogues of 3-methoxybutyl acetate highlights the significant extent of the toxicology database that is available to assess the repeat-dose toxicity profile. An understanding of the likely metabolic fate, and hence systemic exposure profiles of these substances, underpins the repeat-dose toxicity profile. 
The repeat-dose toxicity of the structural analogue of 3-methoxybutan-1-ol, namely 3-methoxy-3-methyl-butanol, has been assessed in guideline 90-day studies. The lowest NOEL was 60 mg/kg/day (LEL=200mg/kg/day) and was based on reversible liver/kidney effects (in the absence of histopathological change) and considered by the authors as non-adverse. Although such effects have not been reported in any short-term 3-methoxybutyl acetate study, as this is the lowest NOEL reported for any structural analogues of 3-methoxybutyl acetate considered in this review, it is used albeit as a conservative surrogate NOEL for 3-methoxybutyl acetate. Thus the NOEL for 3-methoxybutyl acetate repeat-dose toxicity is considered to be 60 mg/kg/day.

Key value for chemical safety assessment

Additional information


In the absence of significant data on 3-methoxybutyl acetate itself, a weight of evidence approach is proposed to assess repeat-dose toxicity.  After consideration of putative metabolism and kinetics, the case for which is presented in more detail in CSR Section 5.1, this summary discusses repeat-dose toxicity data for 3 -methoxybutyl acetate and its putative metabolite 3-methoxybutan-1-ol and for candidate read-across substances, followed by an overall weight of evidence assessment. It is concluded that there is sufficient weight of evidence to assess 3 -methoxybutyl acetate repeat-dose toxicity and a conservative, surrogate NOEL is considered to be 60 mg/kg/day, based on reversible liver/kidney changes. For 3 -methoxybutyl acetate repeat-dose toxicity, 3-methoxybutan-1-ol, acetate and butane-1,3-diol are putative metabolites of 3 -methoxybutyl acetate, whilst 3-methyl-3-methoxy-1-butanol, n-butyl acetate and n-butanol are structural analogues of 3 -methoxybutyl actetate and considered as read-across candidates.

  1. n-butyl acetate is a close analogue of 3-methoxybutyl acetate and the n-butyl acetate metabolite n-butanol (and acetate) is included primarily as this is analogous initial metabolic step, that is, the cleavage of 3-methoxybutyl acetate to its metabolite 3-methoxybutan-1-ol (and acetate).

  2. Direct information on 3-methoxybutan-1-ol and acetate, the likely major proximate metabolites of 3-methoxybutyl acetate are also included.

  3. 3-methoxy-3-methylbutan-1-ol has been included as it is a close analogue of the major proximate metabolite, namely, 3-methoxybutan-1-ol.

  4. In turn, butane-1,3-diol is a potential metabolite of 3-methoxybutan-1-ol.

  5. No data were found for another putative metabolite 3-methoxybutanoic acid.

Data are available for repeat dose studies on 3 -methoxybutyl acetate and relevant read-across chemicals as summarised below:

3 -Methoxybutyl acetate:

4 weeks; rat, guinea pig, dog, cat inhalation

Embryotoxicity; rabbit gavage

3-Methoxybutan-1-ol (putative metabolite):

4 weeks; rat, guinea pig, dog, cat inhalation

n-Buty lacetate (surrogate for formation of 3-methoxybutan-1-ol from 3 -methoxybutyl acetate):

    13 weeks; rat inhalation

3-methoxy-3-methylbutanol (structural analogue of 3-methoxybutan-1-ol):

28 day; rat gavage

Approx 50 day; reproduction


30 week; rat, mouse dietary

2 year; rat dietary

2 year; dog dietary

2 year; rat 5 -generation study

Read-across to alkoxy derivatives of C2 and C3 carboxylic acids has been considered. However it is known that for these substances small changes in chemical structure of these acids produce significant change in both the intrinsic biological activity and metabolic fate. Hence read-across from these substances to 3-methoxybutan-1 -ol / 3 -methoxybutyl acetate has been considered unreliable. However, it has been reported that both animal data and data from occupational exposures to, for example, ethylene glycol monomethylether EGEE (and its acetate ester EGEEA) indicate that the critical effects are those on blood profile and reproduction. Therefore when examining the database for 3-methoxybutan-1 -ol, 3 -methoxybutyl acetate and the close analogue 3-methoxy-3-methyl-1 -butanol, particular attention was paid to the blood profile in repeat-dose studies and (discussed in CSR Section 5.9) the results of reproductive and developmental toxicity studies. Absence of such effects would indicate that the putative metabolite of 3 -methoxybutan-1-ol, 3 -methoxybutanoic acid, has a bioavailability and/or intrinsic activity that does not lead to the expression of an adverse blood profile and reproductive effects in vivo.

The following sections review the information available for each substance, followed by a discussion of the relevance to 3 -methoxybutyl acetate repeat dose toxicity, before a brief summary/conclusion.

3-methoxybutyl acetate and 3-methoxybutan-1-ol – systemic exposure

Systemic exposure to either substance, following oral administration, is likely to be very similar since 3-methoxybutan-1-ol is the putative, rapidly and extensively formed, proximate metabolite of 3-methoxybutyl acetate (CSR Section 5.1) and hence repeat-dose systemic toxicity of both substances are considered to be very similar.

3-methoxybutyl acetate (substance of interest)

An oral gavage OECD 414 embryotoxicity study in which 20 female Wistar rats received daily doses of 3 -methoxybutyl acetate from day 7 to day 16 of pregnancy has reported (Hoechst, 1997; see CSR Section 5.9) that there was no evidence of either maternal or embryo toxicity at the limit dose of 1000 mg/kg.

Inhalation exposure of groups of 5 rats and 5 guinea pigs, 2 dogs and 2 cats to 1790 ppm (10704 mg/m3) (Hoechst AG, 1964a) has also been reported for 3 -methoxybutyl acetate.  Exposure was maintained for 2 hr/day, 5 days/week, for 4 weeks to this near saturated concentration of 3 -methoxybutyl acetate.  Dogs and cats were reported to have a small amount of salivation and small degree of irritation of the mucous membrane of the eye at the time of inhalation but between exposures were normal. There were no other adverse clinical observations, clinical chemistry, bodyweight, gross pathological or histopathological changes.

A recent inhalation study (Covance, 2021) with up to 6.0 mg/l showed no test item-related adverse effects on mortality, clinical observations (including FOD/MA), food consumption, ophthalmology, hematology, clinical chemistry, bronchiolar lavage, macroscopic pathology, or microscopic pathology were detected. No statistically significant and biologically relevant effects on body weights, body weight gain, or food consumption were noted in the male low- and mid-dose groups or any female group. However, statistically significant decreased body weights (p ≤0.05) were noted in the high-dose males during the last four weeks of treatment and were accompanied by statistically significant decreases in body weight gain and food consumption. Although the males in the high-dose group gained weight throughout the study, and there were no other signs of toxicity, the statistically significant decreases in body weight and body weight gain were considered adverse. In summary, the No-Observed-Adverse-Effect-Level (NOAEL) for male rats exposed to 3-methoxybutyl acetate is 2 mg/L and the Lowest-Observed-Adverse-Effect-Level (LOAEL) is 6 mg/L. The highest dose tested of 6 mg/L (limit dose/concentration) is the NOAEL for female rats.

3-methoxybutan-1-ol (putative major primary metabolite)

Early studies investigating inhalation exposure of groups of 5 rats and 5 guinea pigs, 2 dogs and 2 cats to 1300 and 2430 ppm (5538 and 10351 mg/m3) has been reported for 3 -methoxybutan-1-ol (Hoechst AG, 1964b).  Exposure was maintained for 2 hr/day, 5 days/week, for 4 weeks to these near saturated concentrations of 3 -methoxybutan-1-ol.  There were no adverse clinical observations, clinical chemistry, bodyweight, gross pathological or histopathological changes.

n-butyl acetate, n-butanol and acetate

A recent review of n-butanol (ECETOC, 2003) considered a well conducted and reported repeat-dose inhalation study available for n-butyl acetate (David et al 2001) where the substance was administered at dose levels of 500, 1500 or 3000 ppm (2375, 7125 and 14250 mg/m3) for 6 hours per day, 5 days per week for 13 weeks to male and female rats. The NOEL was reported as 500 ppm, based on reduced food intake, decreased body and organ weights. On histopathological examination, local effects, specific to n-butyl acetate, included degeneration of olfactory epithelium (1500 and 3000 ppm), irritation in the glandular stomach, and necrosis in the non-glandular stomach in female rats (3000 ppm).

The authors report that because of the rapid hydrolysis of n-butyl acetate to n-butanol, the results are important for the risk assessment of both n-butyl acetate and butanol, with any effects of the ubiquitous natural product acetic acid released assumed to be of insignificant toxicological relevance. Similarly, the hydrolysis of 3 -methoxybutyl acetate to 3-methoxybutan-1-ol is likely to be rapid and the equimolar amounts of acetate produced considered of insignificant toxicological relevance as the natural product, acetate, is rapidly removed by primary metabolic processes (e.g. Smith et al, 2007). 

3-methoxy-3-methyl-1-butanol (close structural analogue of putative major proximate metabolite)

A well-conducted repeat-dose toxicity study according to a Guideline for the 28 days repeat-dose toxicity test in mammalian species (Japan) and done to GLP was reported (RIAS, 2003). Crj:CD(SD)IGS rats (5 animals/sex/dose) were given 3-methyl-3-methoxy-1-butanol by gavage (vehicle: distilled water) at doses of 0, 15, 60, 250 or 1000 mg/kg bw/day. The administration period was 28 days, and a 14 day recovery period after administration was also incorporated, with sacrifices on day 29 (end of the administration period) and day 43 (end of the recovery period). Endpoints monitored included observation of general condition, bodyweight gain, food consumption, urinalysis, haematology, and blood biochemistry. Histopathological examinations of the control and highest dose groups of male and female rats were undertaken. No deaths were found in any group and no changes in general condition, body weight gain, food consumption, haematological findings, necropsy findings and histopathological findings were reported. Decreases in blood chloride in males and females at 1000 mg/kg bw/day and increases in the Albumin/Globulin (A/G) ratio and inorganic phosphorus in males at 1000 mg/kg bw/day were reported, however, no such differences remained after the recovery period. There was an increase in the relative kidney weight in males at 250 mg/kg bw/day (11%) and 1000 mg/kg bw/day (15%) and in females at 1000 mg/kg bw/day (16%). In addition an increase in relative weight of the liver in males (10%) and females (13%) at 1000 mg/kg bw/day was reported. However by the end of the recovery period most changes had resolved - only the male liver weight increase at 1000 mg/kg bw/day (7%) had not completely resolved. Based on the increase of relative weight of the kidney in males at 250 mg/kg bw/day and higher, and increases of relative weights of the kidney and liver in females at 1000 mg/kg bw/day, the NOELs for repeated dose toxicity were considered to be 60 mg/kg bw/day for males and 250 mg/kg bw/day for females. This is an overall NOEL for this substance considering both this study and the reproduction study, RIAS (2003b). Similarly, the overall LOEL (reversible liver and kidney effects in the absence of histopathological change) is considered to be 200 mgkg bw/day .

Butane 1,3 -diol (potential metabolite of 3-methoxybutan-1-ol)

Butane-1,3-diol has been approved as a substance used in foodstuffs by The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC, 2005). There are several long-term repeat-dose studies reported for butane-1,3-diol during earlier evaluations of its potential use in synthetic diets in several species (Miller & Dymsza, 1967, Scala & Paynter, 1967, Hess et al., 1981.). 

The aim of the series of studies of Miller and Dymsza (1967) was to consider butane-1,3-diol as a synthetic energy source. Male and female rats and mice were fed butane-1,3-diol for up to 30 weeks at dietary incorporation levels of up to 30% (15,000 mg/kg bodyweight). The main effect reported in the 30-week study was an impairment of the utilisation of the diet when the substance was fed at the 30% level. However, at an incorporation level of 20%, (10,000mg/kg bodyweight) no such impairment was reported. Other clinical chemistry examinations, such as quantification of ketone-bodies in urine and serum, revealed no changes after the 30 week feeding period. Food intake and bodyweight gain are reported with reduced intake and bodyweight gain at the higher, relative to the lower dose levels. Little other information of significance with respect to toxicology of butane-1,3-diol is available from this study.

In the studies of Scala and Payntner (1967) butane-1,3-diol was incorporated into the diet of 60 male and female rats (100,000 ppm; 5,000 mg/kg bodyweight) with 30 male and female rats each receiving a control diet. Animals received the test diets for 2 years. Bodyweight, food and test substance consumption and observations for potential pharmacological effects were regularly recorded. Some clinical chemistry examinations of blood and urine were recorded 6 times during the 2-year study. After one year, 10 animals from each group, and at 2 years all surviving animals were autopsied. Representative organs were weighed and sections of brain, pituitary, thyroid, lung, heart, spleen, kidney, adrenal, pancreas, stomach, small and large intestine, urinary bladder, gonads, bone and bone marrow were examined by histopathology.

Scala and Paynter also examined butan-1,3-diol in dogs. Four groups of 4 male and female dogs were fed diets containing butane-1,3-diol at 0, 0.5, 1 or 3% (3% is equivalent to 30,000 ppm; 750 mg/kg bodyweight) for 2 years with an interim sacrifice following 1 year. The authors report that daily or weekly records were taken of appetite, appearance, elimination, signs of pharmacological effect, bodyweight and food and substance consumption. Clinical chemistry examinations of blood and urine were undertaken 8 times during the 2-year study. After 1 year, 2 animals from each group were sacrificed and the remainder sacrificed after 2 years. At both time points, autopsies were performed and representative organs weighed. Samples of brain, pituitary, thyroid, lung, heart, lymph nodes, liver, spleen, kidney, adrenal, pancreas, stomach, large and small intestine, gall bladder, urinary bladder, gonads, bone and bone marrow were examined by histopathology.

Although the detailed histopathology results of the study were not reported and only summary body and organ weight information was reported, the authors concluded that throughout the 2-year tests in both species, the feeding of butane-1,3-diol caused no discernable toxic effects at any level of dietary incorporation up to 3% (750 mg/kg bodyweight) in dogs and 10% (5,000 mg/kg bodyweight) in rats. All dose conversions are taken from AFC, 2005.

Histopathological examination of the testes, ovaries and pituitary glands revealed no treatment-related changes following 5 successive mating cycles (over 77 weeks) of the F1A generation (Hess et al., 1981).

In humans, short-term metabolic studies indicate that butane-1,3-diol can supply up to 10% of total dietary energy without toxic effects (Tobin et al., 1974; Altschule et al., 1977), consistent with the data from animal studies.


From metabolic considerations (CSR Section 5.1), exposure of mammals to 3-methoxybutyl acetate is likely to lead to its rapid hydrolysis to 3-methoxybutan-1-ol and the natural product acetate. The toxicity of the acetate produced is unlikely to be significant due to its incorporation into high-turnover natural processes, such as the citric acid cycle (Smith et. al., 2007), and hence the systemic exposure and therefore the toxicity of administered 3-methoxybutan-1-ol and 3 -methoxybutylacetate will be very similar, if not indistinguishable.

Systemic 3-methoxybutan-1-ol may be either excreted intact or as a sulphate or glucuronide conjugate or may undergo further metabolism. Two competing metabolic pathways have been reported for closely related compounds including 2-methoxy-ethanol and 2-methoxy-propanol (Miller et al., 1984; Jenkins-Sumner et al., 1995; Carney et al., 2003); these involve either initial oxidation or O-demethylation reactions.

Oxidation of 3-methoxybutan-1 -ol would produce 3-methoxy butanoic acid. Any 3-methoxy butanoic acid formed would be excreted, probably as the glucuronide conjugate.

The alternative pathway involving O-demethylation would give butane-1,3-diol followed by oxidation to 3-hydroxy-butanoic acid. 3-hydroxy-butanoic acid produced by this route would subsequently enter and be removed by the high turnover pathways of primary metabolism.

The results of the repeat dose studies with butane-1,3-diol reveal a substance with very low oral toxicity. This is not too surprising since butane-1,3-diol is itself a natural product and can be catabolised to the ketone body 3-hydroxybutyrate, which is an important substance in primary metabolic processes in animals and humans. The oral LD50 of butane-1,3-diol is reported to be in the order of 20,000 to 30,000 mg/kg. It seems reasonable, therefore, that the addition of butane-1,3-diol to diet has little impact on the well-being of the animal, as natural processes will be utilised to remove excess material from circulation. Indeed, only at the highest dose levels, when presumably these natural removal processes become overwhelmed, will ketosis and any resultant toxicity start to be expressed. Clearly, studies with this chemical as a food additive have resulted in toxicity information being generated at exceedingly high dietary inclusion rates.

3-methoxy-3-methyl-1-butanol, differing from 3-methoxybutan-1-ol only by a single methyl group increased liver and kidney weights, in the absence of histopathology change and some clinical chemistry changes (blood chloride and Albumin/Globulin ratio changes) were reported, but had mainly resolved by the end of a 2-week recovery period. Such changes have not been reported with 3 -methoxybutyl acetate, 3-methoxybutan-1-ol, n-butyl acetate, n-butanol or butane-1,3-diol and may be specific to 3-methoxy-3-methyl-1-butanol.


AFC (2005): Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in contact with Food (AFC) on a request from the Commission related to Flavouring Group Evaluation 10: Aliphatic primary and secondary saturated and unsaturated alcohols, aldehydes, acetals, carboxylic acids and esters containing an additional oxygenated functional group and lactones from chemical groups 9, 13 and 30 (Commission Regulation (EC) No 1565/2000 of 18 July 2000) The EFSA Journal (2005) 246, 1-110.

Altschule, M. D., Werthessen, N. T. & Miller, S. A. (1977) J. Toxicol. Environ. Health., 3, 755

Tobin, R. B. et. al. (1975) Federation Proc., 34, 2171

Covance 2021

Justification for classification or non-classification

According to criteria in Regulation (EC) No.1272/2008, the substance is not classified for repeat dose toxicity.

There is sufficient weight of evidence from data available on structural analogues and also 3 -methoxybutan-1-ol, a metabolite of 3-methoxybutyl acetate, to conclude that 3-methoxybutyl acetate will be of low repeat dose toxicity by the oral and inhalation routes with no indication of serious functional or morphological effects.  Classification is therefore not warranted for 3-methoxybutyl acetate under Dir 67/548/EEC or Regulation (EC) No.1272/2008.