3-methoxybutyl acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Additional information

In the absence of significant direct information on 3-methoxybutyl acetate for this endpoint a weight of evidence approach is proposed for the reproductive toxicity of 3 -methoxybutyl acetate. After careful consideration of structural analogues of 3 -methoxybutyl acetate, putative metabolism and kinetics, the case for which is presented in more detail in CSR Section 5.1, this summary discusses the candidate data individually followed by an overall weight of evidence discussion. For 3 -methoxybutyl acetate, the candidate read-across substances with information reported are acetate, butane-1,3-diol and 3-methyl-3-methoxy-1-butanol. These candidate substances are related to 3 -methoxybutyl acetate in that they are either structural analogues, putative metabolites, or structural analogues of putative metabolites.

3 -Methoxybutyl acetate (the substance of direct interest)

No data are available on the reproductive toxicity of 3 -methoxybutyl acetate.

3-methoxybutan-1-ol (putative proximate metabolite of 3-methoxybutyl acetate)

No data are available on the reproductive toxicity of 3-methoxybutan-1-ol.

Acetate (putative proximate metabolite)

The recent review of acetic acid for inclusion in Annex I of 91/414/EEC concluded that  “Based on human exposure to orally ingested acetic acid from various foods and the lack of evidence that such exposure is related to fertility problems and developmental deficiencies in humans, neither a new multigeneration study nor any other postnatal evaluation or developmental toxicity study are required” (EU DAR, 2008).

3-methoxy-3-methyl-1-butanol (structural analogue of the putative proximate metabolite, 3-methoxybutan-1-ol)

Relevant data are available on 3-methoxy-3-methyl-1-butanol. This substance is a close structural analogue, with only an additional methyl group, of putative metabolite 3-methoxybutan-1-ol.

In an OECD Guideline 421 reproduction/developmental toxicity test, rats (CrjCD(SD)IGS) were exposed to 3-methoxy-3-methy-1-butanol by oral gavage at 0, 8, 40, 200, or 1000 mg/kg bw/day. The test included a 14 day pre-mating exposure period and a total test duration of 47 days for males and 42-52 days for females. The NOAEL was 40 mg/kg bw/day for males and 200 mg/kg bw/day for females based on liver and kidney weight increases (in the absence of histopathological change). No changes in oestrous cycle, copulation index, fertility index, gestation period, number of corpora lutea, number of implantations, gestation index, delivery index, or delivery and nursing conditions were seen in the parent animals. Moreover, no changes in total number of pups born, number of live newborn pups, sex ratio, live birth index, body weight, morphology or viability index of newborns on day 4 of nursing were seen in the pups.

The authors concluded no-effect-level for reproductive capability of male and female parent animals and for pup development were both 1000 mg/kg/day.

Butane-1,3-diol (a potential metabolite of 3 -methoxybutyl acetate)

Potential developmental and reproductive effects of this substance were evaluated in a complex 2-year, 5-generation study, with dietary incorporation of butane-1,3-diol at up to 24% (Hess et al, 1981). 

The butane-1,3 -diol in this series of experiments was included in the diet at concentrations of 0, 5, 10 and 24% (prepared by substituting butane-1,3-diol for equal amounts by weight of corn starch and dextrose).

The F0 generation comprised of groups of 25 male and 25 female rats (Wistar derived). After exposure to test diets for 28 days, blood and urine samples were taken (10 males and 10 females) for clinical chemistry examination (blood; alkaline phosphatase, glucose, hematocrit, hemoglobin and total and differential leucocyte counts: urine; albumin, glucose, ketones, occult blood, pH, specific gravity and microscopic examination of the sediment).

F0 generation rats were then paired (1 to 1 within each group). Mating was confirmed by a vaginal sperm plug and the day designated day 0 of pregnancy. If mating was not confirmed (7 days) the female was paired with another male animal from the same exposure group (additional 7 days). Females not confirmed pregnant within this time period were discarded.

F0 generation females were fed test diets throughout the mating, gestation and lactation phase of the study and were allowed to deliver naturally. The number of pregnant females, the number of pups born live or dead, the number of pups surviving and their body weights were recorded and this first series of litters was the F1A generation. Selected pups were brought to maturity and after 11 weeks blood and urine samples were collected from 10 rats per sex per group for clinical chemistry analysis, as before. Thereafter 25 males and 25 females per group were paired to produce the F2 generation.

A second series of litters (F1B) was produced by the F0 parents1-2 weeks after weaning of the first litters, with each female mated with a different male and data collected as described above. Ten males per group were reared to sexual maturity and used in a dominant lethal test.

After some 77 weeks, five successive mating cycles were achieved with the F1A rats and the reproductive index was calculated for each litter. For FIA rats, which survived for at least 66 weeks, the gonads and pituitary glands were examined microscopically.

Selected pups of the first litters of the F1A females became the F2A parents, and were used to produce the F3 generation. Each set of F2A parents was mated twice to yield two litters of offspring F3A and F3B. The F3A females were allowed to produce F4A and F4B litters. However, only 25% of the F2A female rats were allowed to produce the F3B litter the remaining 75% were allocated to the developmental study.

In this rather unusual study, for four of five generations of rats fed extraordinary high concentrations of butane-1,3-diol, 24% of diet (240,000 ppm), reproduction and lactation parameters were similar to those of controls and no adverse effects were indicated. For the F1A generation mated to produce 5 successive litters, there was an apparent dose-related decrease in fertility for the production of the last 2 litters only (F2D & F2E). This decrease was considered by the authors to be related to a physiological stress induced by the highly ketogenic, semi-synthetic diet rather than a direct effect of butane-1,3-diol on the fertility of the males. However it can be concluded that there were no adverse effects on the fertility of at least three successive matings of rats fed extraordinary high levels (240,000 ppm) of butane-1,3-diol. Furthermore, butane-1,3-diol is one potential metabolite of 3 -methoxybutyl acetate and therefore the corresponding dose level of 3 -methoxybutyl acetate required to deliver such high quantities of this metabolite would be so high that it would not be relevant to human health and probably unachievable in animal studies.

For 3 -methoxybutyl acetate and the read-across candidates discussed above a range of fertility/developmental test protocols have been reported. No significant reproductive potential has been highlighted in these studies. The evidence supports the conclusions that there is sufficient evidence to assess the reproductive potential of 3 -methoxybutyl acetate and that there are no fertility effects associated with 3 -methoxybutyl acetate exposure.

Short description of key information:
The weight of evidence indicates that there are no fertility effects associated with 3-methoxybutyl acetate exposure.

Effects on developmental toxicity

Description of key information

For 3-methoxybutyl acetate and the read-across candidates discussed above a range of developmental test protocols have been reported.  Without exception, no teratogenic potential has been highlighted in these studies.  The evidence clearly supports the conclusions that there is sufficient evidence to assess the developmental potential of 3-methoxybutyl acetate and that there are no developmental effects associated with 3-methoxybutyl acetate exposure.

Additional information

Introduction

In the absence of significant direct information on 3-methoxybutyl acetate, for this endpoint a weight of evidence approach is proposed for the developmental toxicity of 3 -methoxybutyl acetate. After careful consideration of structural analogues of 3 -methoxybutyl acetate, putative metabolism and kinetics, the case for which is presented in more detail in CSR Section 5.1, this summary discusses the candidate data individually followed by an overall weight of evidence discussion. For 3 -methoxybutyl acetate, the candidate read-across substances are 3-methoxybutan-1 -ol, acetate, butane-1,3 -diol, 3 -methyl-3 -methoxy-1 -butanol, n-butyl acetate and n-butanol. These candidate substances are related to 3 -methoxybutyl acetate in that they are either structural analogues, putative metabolites, or structural analogues of putative metabolites.

3 -Methoxybutyl acetate – the substance of direct interest

An oral gavage OECD 414 prenatal developmental toxicity study in which 20 female Wistar rats received daily doses of 1000 mg/kg/day from day 7 to day 16 of pregnancy has been reported (Hoechst, 1997). There was no evidence of either maternal or developmental toxicity at this limit dose.

3-methoxybutan-1 -ol (putative proximate metabolite of 3 -methoxybutyl acetate)

No data are available on the developmental toxicity of 3-methoxybutan-1-ol, a proximate metabolite of 3 -methoxybutyl acetate.

Acetate (putative proximate metabolite)

The recent review of acetic acid for inclusion in Annex I of 91/414/EEC concluded that “Based on human exposure to orally ingested acetic acid from various foods and the lack of evidence that such exposure is related to fertility problems and developmental deficiencies in humans, neither a new multigeneration study nor any other postnatal evaluation or developmental toxicity study are required” (EU DAR, 2008)

3-methoxy-3-methyl-1-butanol (structural analogue of the proximate metabolite, 3-methoxybutan--1-ol)

Relevant data are available on 3-methoxy-3 -methyl-1-butanol. This substance is a close structural analogue, with only an additional methyl group, of putative metabolite 3-methoxybutan-1-ol.

In an OECD Guideline 421 reproduction/developmental toxicity test, rats (CrjCD(SD)IGS) were exposed to 3-methoxy-3-methy-1-butanol by oral gavage at 0, 8, 40, 200, or 1000 mg/kg bw/day. The test included a 14 day pre-mating exposure period and a total test duration of 47 days for males and 42-52 days for females. The NOAEL was 40 mg/kg bw/day for males and 200 mg/kg bw/day for females based on liver and kidney weight increases (in the absence of histopathological change). No changes in estrous cycle, copulation index, fertility index, gestation period, number of corpora lutea, number of implantations, gestation index, delivery index, or delivery and nursing conditions were seen in the parent animals. Moreover, no changes in total number of pups born, number of live newborn pups, sex ratio, live birth index, body weight, morphology or viability index of newborns on day 4 of nursing were seen in the pups.

The authors concluded that the no-effect doses for reproductive capability of male and female parent animals and for pup development were both 1000 mg/kg/day.

n-butanol (structural analogue of 3 -methoxybutyl acetate metabolite, 3-methoxybutan-1-ol)

Groups of approximately 15 female rats (Sprague-Dawley) were exposed by inhalation (whole body), to n-butanol (0, 3500, 6000 or 8000 ppm; 0, 11000, 18000, or 25000 mg/m3) on gestation days 1–19 for 7 hours per day . Foetuses were examined on day 20. At 6000ppm (18 000 mg/m3) and above, maternal toxicity and slightly reduced foetal weights were reported. There were no significant treatment-related increases in the incidence of foetal malformations / variations. The NOAEC for both maternal and developmental toxicity was reported as 3500ppm (11000 mg/m3), (Nelson et al.,1989).

In a developmental neurotoxicity investigation, groups of 18 male rats (Sprague-Dawley) were exposed by inhalation, 7 hours per day for 6 weeks, to n-butanol (0, 9200, or 18 000 mg/m3) before mating with untreated females. These females were allowed to deliver pups. Separately, groups of 15 pregnant female rats were exposed from days 1 to 20 of gestation to n-butanol at 0, 9200 or 1800 mg/m3 before being allowed to deliver pups. Offspring from both groups of dams were then observed during postnatal days 10–90 for indication of developmental neurotoxicity. At 6000ppm a small number of behavioural and neurochemical measures differed from those of the control group but there was no discernable pattern of effect (Nelson et al., 1989).

Butane-1,3-diol

Potential developmental and reproductive effects of this substance were evaluated in a complex 2-year, 5-generation study, with dietary incorporation of butane-1,3 -diol at up to 24% (Hess et al, 1981). A brief introduction to the experimental protocols are discussed in the fertility endpoint summary. 

Developmental study

On day 19 of pregnancy, 75% of the F2A dams in each group were given a caesarean section. The numbers of implantations, resorptions, viable and nonviable foetuses, gross abnormalities, weight and gender of foetuses were recorded. Of these F3B foetuses a third were used for soft tissue examination following Bouin’s fixation and the rest were used for skeletal examination following staining with alizarin red.

No treatment-related effects were observed in the soft tissues of the foetuses from the group exposed to 25% butane-1,3-diol. An increased incidence, of incomplete ossification of sternebrae was observed in the foetuses from the groups fed 20 or 25% butane-1,3-diol and an increased incidence of missing sternebrae was observed in foetuses from the group fed 25%. The authors suggest these findings indicate slightly delayed growth of skeletal tissue, rather than a specific developmental effect, per se, particularly as the sternebrae of one quarter of the control group were only partially ossified. In addition, clinical chemistry results revealed a significant increase in the concentration of ketone bodies in blood and urine in animals fed diets containing 20 or 25% butane-1,3-diol. Futhermore, 1,3 –butandiol is one potential metabolite of butoxyl and therefore the corresponding dose level of butoxyl required to deliver such high quantities of metabolite would be so high that it would not be relevant to human health and probably unachievable in animal studies.Therefore, although growth rate changes were unaffected even for four consecutive generations of female animals, there is clearly significant ketosis, and hence physiological stress, associated with ingestion of synthetic diet containing very large concentrations, 20 or 25%, of test substance.

The developmental toxicity of butane-1,3-diol has been investigated to extraordinary high levels of dietary incorporation without highlighting any significant teratogenic potential for this substance.

Discussion

Although developmental toxicity information on 3 -methoxybutyl acetate is limited to a guideline (OECD 414) prenatal developmental toxicity screen using the oral route and a limit dose, a clear NOAEL was established in this study for both maternal and developmental toxicity of 1000 mg/kg/day. In addition there are several studies, of varying design, available for structural analogues of 3 -methoxybutyl acetate and its putative metabolites. The evidence indicates that metabolism of 3 -methoxybutyl acetate will be significant (CSR Section 5.1 for further detail).

Metabolic considerations and read-across candidates

The first metabolic step is a simple esterase mediated hydrolysis, the products of which are acetic acid (acetate at physiological pH) and 3-methoxybutan-1-ol.  Acetic acid has been discussed above. There is no additional developmental information for 3-methoxybutan-1 -ol, however, it has been reported that a structural analogue, n-butyl acetate, is also hydrolysed by esterases to acetate and n-butanol. Therefore developmental studies on n-butanol (inhalation exposure) have been considered as read-across candidates for the developmental toxicity of 3 -methoxybutyl acetate. At 18000 mg/m3 and above maternal toxicity and slightly reduced foetal weights were reported for n-butanol but there was no effect on the incidence of foetal malformations or variations. Therefore the NOAEC for both maternal and developmental toxicity of n-butanol was considered to be 11 000 mg/m3 (Nelson et al.,1989).

Another substance of interest is a close structural analogue of 3-methoxybutan-1 -ol. 3 -methoxy-3 -methyl-1-butanol differs only in that is has an additional methyl group. A reproduction/developmental toxicity study (OECD 421) is available for this substance using the oral route of exposure. The NOAEL was 1000 mg/kg bw/day for parents and offspring with NOAEL of 40 mg/kg bw/day males and 200 mg/kg bw/day for females based on liver and kidney weight increases (in the absence of histopathological change). 

The second metabolic step (CSR section 5.1 for further detail) is oxidation to 3-methoxybutanoic acid (for which there are no data have been identified) or by O-demethylation to butane-1,3-diol; both pathways may be active.

For butane-1,3-diol there is a significant database available. This substance is incorporated at low levels into human foodstuffs and cosmetic products and a considerable reproductive and developmental database has been published. As part of a 2-year investigation of several endpoints including developmental and reproductive toxicity, a well-conducted teratology study was incorporated. As the substance was then being considered for use as a high-level dietary component, the amounts of butane-1,3-diol incorporated into rat feed were extremely large. Indeed at the highest levels of incorporation (20 and 25% in diet), when presumably the normal elimination routes for the proximal metabolite of butane-1,3-diol (3-hydroxy-butanoic acid, a natural product) were exhausted, ketosis resulted. Despite this maternal ketosis (blood, urine and liver) induced by exposure to extraordinary high levels of dietary incorporation of butane-1,3-diol, no teratogenic potential was indicated for this substance.

For the potential metabolite 3-methoxybutanoic acid no data have been identified. Read-across to alkoxy derivatives of C2 carboxylic acids has been considered but it is known that for these substances small changes in chemical structure produce significant change in both the intrinsic biological activity and metabolic fate. Therefore direct read-across from these C2 acid substances to the C4 acid, 3-methoxybutan-1-ol / 3 -methoxybutyl acetate, has been considered unreliable. However experimental research has indicated that the potency of developmental effects attributed to alkoxy acids falls off sharply with increasing number of carbon atoms within the acid moiety. For example, Rawlings et al. (1985) compared the in vitro embryotoxicity of several alkoxy acids; they found that whereas 2-methoxyacetic acid (C2) was highly potent, 3-methoxypropionic acid (C3) and 4-methoxybutanoic acid (C4) were significantly less active. Additionally in in vivo studies, Carney et al.(2003) concluded that there were no developmental effects in the absence of maternal toxicity with the C3 acid, 2-methoxypropionic acid. Therefore it seems likely that as the carbon backbone of the acid is further increased from C3 to C4, (3-methoxybutanoic acid) the likelihood of developmental effects will diminish even further.

Citations

Rawlings, SJ et al. (1985) The teratogenic potential of alkoxy acids in post-implantation rat embryo culture: structure-activity relationships. Toxicol. Lett., 28, 49-58.

In the Final Decision number: CCH-D-2114453510-59-01/F (2018) issued for 3 methoxybutyl acetate, ECHA identified the need to fulfill information requirements for a Pre-natal developmental toxicity study in a second species (rabbit), oral route. Based on the results in this prenatal developmental toxicity study (i.e. adverse effects on body weight and food consumption and unscheduled deaths in dams treated at 1000 mg/kg/day) the maternal No Observed Adverse Effect Level (NOAEL) for 3-methoxybutyl acetate was established as being 300 mg/kg/day. Due to the absence of any test item-related effects on development and fetal morphology in rabbits, the developmental No Observed Adverse Effect Level (NOAEL) for 3-methoxybutyl acetate was established as being at least 300 mg/kg/day.

Justification for classification or non-classification

According to criteria in Regulation (EC) No.1272/2008, the substance is not classified for reproductive or developmental effects. The weight of evidence assessment indicates there are no developmental or reproductive effects associated with 3 -methoxybutyl acetate exposure.