Anisaldehyde

Endpoint summary

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Experimental data on toxicokinetics and metabolism are available from in vivo (rat and rabbit) and in vitro experiments with 4-methoxybenzaldehyde (Anisaldehyde). Its physico-chemical properties, i. e. water solubility (2 g/l at 20°C), log Pow (1.56 at 25°C) and molecular weight (136.15 g/mol) favour bioavailablility via the dermal and oral route. However, on the basis of the low vapour pressure at room temperature (vapour pressure approx. 3 Pa), it can be assumed that exposure via inhalation of 4-methoxybenzaldehyde as a vapour is low.

In a comparative in vitro metabolism study, 4-methoxybenzaldehyde was incubated with cryopreserved hepatocytes from mouse, rat, rabbit and human at concentrations of 1, 10 and 100 µM and incubation times of 0, 1 and 4 h (RIFM, 2011). Cell suspension samples were analysed afterwards by LC-MS to elucidate species dependent metabolite profiles formed. Seven metabolites were identified:

• 4-methoxybenzoic acid (Anisic acid)


• 2-[(4-methoxyphenyl)formamido]acetic acid (Anisic acid - glycine conjugate)


• Anisic alcohol - glucuronide conjugate


• demethylated Anisic alcohol - glucuronide conjugate


• three hydroxylated Anisaldehyde - glucuronide conjugates

After direct application of the test substance, the metabolite anisic acid was the only detectable metabolite in the respective samples. For later incubation timepoints (1h, 4h), the glycine conjugate of 4-methoxybenzoic acid generally represented the largest fraction. For rat hepatocytes, the second largest fraction was the glucuronide conjugate of anisic alcohol, whilst for other species this was typically 4-methoxybenzoic acid for these timepoints. The remaining metabolites were typically observed at low levels and/or in a limited number of incubations. Unchanged Anisaldehyde was not detectable.

Overall, an immediate formation of 4-methoxybenzoic acid was observed followed by the formation of conjugates of the respective alcohol, acid, hydroxylated and demethylated forms of 4-methoxybenzaldehyde . Besides the predominant formation of anisic alcohol - glucuronide conjugate in rats and to a lesser extent in the mouse, interspecies differences in the metabolite profiles were generally small. 

In a 14-day percutaneous and oral repeated dose study in rats, 4-methoxybenzaldehyde  was applied at doses of 0, 100, 250, 500 and 1000 mg/kg bw/day in corn oil (dermal, daily for 6 hours under occlusive conditions) or at doses of 0, 20, 100 and 500 mg/kg bw/d in corn oil (oral, gavage), and blood plasma samples were obtained on the first day of dosage administration, i.e. prior to dosage administration and at approximately 1 hour, 2 hours, 8 hours, 12 hours and 24 hours postdosage (RIFM, 2011). Samples were also collected from rats in the vehicle control groups at approximately 1 hour postdosage and from all animals prior to scheduled euthanasia on day 15.  Key metabolites, as identified in the comparative in vitro metabolism study, i.e. anisic acid, anisic acid glycine conjugate and the glucuronide of anisic alcohol as well as the parent compound were quantified via GC/LC-MS-MS.

No 4-methoxybenzaldehyde  but 4-methoxybenzoic acid, anisic acid glycine conjugate and the glucuronide of anisic alcohol was detectable in blood plasma after oral or dermal application of 4-methoxybenzaldehyde.

Plasma levels of 4-methoxybenzoic acid at comparable doses were found to be 10-100 fold lower after dermal application compared to oral application of 4-methoxybenzaldehyde, whereas levels of anisic acid glycine conjugate were comparable.

After oral administration high levels of 4-methoxybenzoic acid remained for min. 12 hours at 500 mg/kg bw/d, whereas an evident decline was observed within 2 hours at lower doses. An overproportionate increase of the AUD to the applied dose became evident (approx. 15 fold for the mid dose group and approx. 150 fold for the high dose group). In contrast, AUDs for anisic acid glycine conjugate were found to be increased only approx. 5 fold between the high and the low dose group. 

Dermal administration of 4-methoxybenzaldehyde also resulted in an overproportionate increase of the AUD for 4-methoxybenzoic acid (approx. 40 fold and 90 fold for the 500 and 1000 mg/kg bw/d dose group). In line, respective estimated AUDs for anisic acid glycine conjugate were found to be increased only approx. 5 fold between the high and the low dose group. 

Overall, oral or dermal application of 4-methoxybenzaldehyde starting at 500 mg/ kg bw/d leads to an evident disproportionate increase of anisic acid plasma concentrations. Furthermore, oral application led to an elongation of high 4-methoxybenzoic acid plasma levels. At these dose levels, a saturation of the formation of the respective glycine conjugate as detoxifying mechanism has been observed. 

 

Data from literature further confirm the metabolites identified. Anisoylglucuronide was detected in the 24-hour urine of 4-methoxybenzaldehyde fed rabbits (Sammons 1945). The formation of anisyl alcohol and anisic acid has been detected in rat caecal extracts, incubated with 4-methoxybenzaldehyde under anaerobic conditions for 46 hours (Scheline 1972). Incubation of 4-methoxybenzaldehyde in nasal and liver microsomes in vitro resulted in trace amounts only or no formation of formaldehyde, respectively (Dahl 1983). In the fragrance raw materials monograph on anisic aldehyde, it is reported that anisic aldehyde undergoes a very slight degree of demethylation with oxidation of its aldehyde group to an acid group, the major excreted metabolite being anisic acid (Opdyke 1974).

 

In a comparative toxicokinetics study in rats (BASF 2021; 99C0220/19S022) plasma kinetics of 4-methoxybenzaldehyde after oral application for a period of 4 weeks was determined and administration via the diet versus gavage was compared.

Encapsulated 4-methoxybenzaldehyde was administered via the diet to groups of 5 male Wistar rats at adjusted concentrations to achieve target dose levels of 20, 100 and 500 mg/kg bw/d. Dietary concentrations of 4-methoxybenzaldehyde (encapsulated) for each test group were adjusted according to the food consumption and body weight determination intervals. The concentrations were calculated according to body weight and food consumption values from the recent week to achieve the respective target dose levels. To archive a constant concentration of starch (capsule material) in the diets of test groups, HI-CAP 100 was supplemented to the diets for low and mid dose groups.

Comparative to the administration via the diet, 4-methoxybenzaldehyde was administered orally by gavage to groups of 5 male Wistar rats at dose levels of 20, 100 and 500 mg/kg bw/d. Corn oil served as vehicle; control animals were dosed daily with the vehicle. In addition, all animals of administration by gavage were fed with the control food containing a defined amount of HI-CAP 100 (capsule material).

Food consumption and body weights were determined and animals were examined for signs of toxicity or mortality including a detailed clinical examinations in an open field.

Clinico-chemical and sperm examinations as well as plasma kinetics were performed during the administration period.

Blood sampling for plasma kinetics was performed on treatment day 1 (before, 1, 2, 4, 8, 24 hours after gavage administration), day 7 and 14 (2 hours after gavage administration, respectively. After administration via feed, blood sampling occurred on treatment day 1 (7:00 am, 10:00 am, 01:00 pm, 04:00 pm), day 2 (7:00 am), day 7 and 14 (10:00 am, respectively).

After the administration period and subsequent sacrifice, gross pathology was performed and organ weights were determined followed by histopathological examinations.

Data on plasma kinetics demonstrate that 4-methoxybenzaldehyde (Anisaldehyde) was metabolized intensively after oral administration to the rat. Analytical results for the selected metabolites including the major metabolites 4-methoxybenzoic acid and 2-[(4-methoxyphenyl)formamido]acetic acid (i.e. glycine conjugate of 4-methoxybenzoic acid) were dependent from plasma sampling times, dose and route of administration with generally higher plasma concentrations after administration of 4-methoxybenzaldehyde via gavage than administration of the same dose levels via the feed. There was no accumulation of internal doses between the sampling intervals under the test conditions used. The measured concentrations lead to calculated internal doses of the metabolite 4-methoxybenzoic acid, that is higher by factors of approx. 4, 10 and 16 for doses of 20, 100 and 500 mg/kg bw/d after administration by gavage than by feeding. The internal doses of metabolite 2-[(4-methoxyphenyl) formamido]acetic acid is higher by factors of approx. 6, 2 and 3 for doses of 20, 100 and 500 mg/kg bw/d after administration by gavage than by feeding.

For 4-methoxybenzoic acid, the internal dose factor from dose 20 to 100 mg/kg bw/d is more or less comparable to the external dose factor after administration via the diet. After administration by gavage, the internal dose factor is approx. 3.5-fold higher than the external dose factor, the internal dose factor being 17 and the external dose factor being 5. The internal dose factor from dose 100 to 500 mg/kg bw/d are 19 and 12 fold higher for the high dose versus the next lower dose after gavage and feed administration, respectively, and thus approx. 2 to 4 fold higher than the external dose factor of 5 for both routes of administration.

For 2-[(4-methoxyphenyl)formamido]acetic acid, the internal dose factor from dose 20 to 100 mg/kg bw/d is comparable to the external dose factor after administration via the diet. After administration by gavage, the internal dose factor is approx. 2.5-fold lower than the external dose factor, the internal dose factor being 2.0 and the external dose factor being 5. The internal dose factors from dose 100 to 500 mg/kg bw/d are approx. 1.2 to 2 fold higher than the external dose factor of 5 for both administration routes.

Thus, the toxicokinetic assessment in this study confirmed a disproportional increase of internal doses of metabolite 4-methoxybenzoic acid in relation to the external dose factor (5 fold), whereas no comparable and consistent increase was observed for the respective glycine conjugate (2-[(4-methoxyphenyl)formamido]acetic acid).

The concentrations of 4-methoxybenzaldehyde (Anisaldehyde) and its metabolites measured at different sampling dates demonstrate that no bioaccumulation occurs under the test conditions used when taking into account the comparable metabolite levels between study days 1, 7 or 14 or the complete clearance of the main metabolite 4-methoxybenzoic acid within 24h.

Overall, the data on plasma kinetics demonstrate that 4-methoxybenzaldehyde is metabolized intensively after oral administration to the rat yielding to no or very low internal doses of 4-methoxybenzaldehyde. Analytical results for the formed metabolites 4-methoxybenzoic acid and 2-[(4-methoxyphenyl)formamido]acetic acid were dependent from plasma sampling times, dose and route of administration. Generally, dosing by gavage yielded higher plasma concentrations and corresponding higher internal doses (calculated as AUD_24h) than administration of the same dose levels via the feed.

The concentrations of 4-methoxybenzaldehyde and its metabolites measured demonstrated, that no bioaccumulation occurs under the test conditions used.

This difference in internal doses at comparable dose levels was unexpected and the design of the comparative toxicokinetics study discussed (BASF 2021; 99C0220/19S022) contains several shortcomings, which complicate the estimation of the overall bioavailability after administration of 4-methoxybenzaldehyde via gavage versus diet. No precise determination of the test substance uptake was possible due to estimations from 3 day intervals. Furthermore, the plasma sampling in the feed groups did not fully cover the uptake for 24 hours including the uptake at night, which leads to an underestimation of the internal doses due to the sampling regimen applied. The bioavailability can only be estimated by plasma AUDs (representing the internal dose) and no determination of an integral sum parameter (e.g. urine levels) was made for a better comparison of bioavailabilities.

These shortcomings triggered follow-up investigations with an expanded and optimized study design to exclude potential technical reasons for the differences observed. The design of the currently running follow-up study (BASF, 02B0220/19B008) contains additional urine collections as an integral parameter for a better comparability of test substance uptake between the different application strategies and allows a more precise estimation of the actual food (test substance) uptake in the feed dosing groups.

Based on the preliminary assessment of this study, plasma kinetics indicate a confirmation of higher internal doses after gavage of the neat test substance versus diet administration, however, the difference was less pronounced. In urine, the glycine conjugate (2-[(4-methoxyphenyl)formamido]acetic acid) was found to be the prominent metabolite of parent/metabolites investigated, and dose corrected excreted amounts were comparable between feed (test substance encapsulated) versus gavage (test substance neat) dose groups. The content of parent/metabolites investigated in feces was found to be very low.

 

Taken together the kinetic data of the test substance / its metabolites demonstrate that the internal dose (as indicated by calculated AUDs) is overproportional with the administered dose, especially at high dose levels. Consequently, saturation processes in metabolism and / or excretion are evident and should be taken carefully into account for the assessment of the toxicity profile of 4-methoxybenzaldehyde. Such saturation processes have been described for other organic acids such as 2,4-dichlorophenoxyacetic acid, where differences in active renal transport mechanisms between species or body burden overwhelming renal clearance mechanisms are consistent with increased sensitivity to 2,4-D toxicity (Ravenzwaay et al., 2003).

 

Based on the available data, a bioaccumulative potential of 4-methoxybenzaldehyde is not to be expected.

 

• B. VAN RAVENZWAAY, T. D. HARDWICK, D. NEEDHAM, S. PETHEN and G. J. LAPPIN. Comparative metabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) in rat and dog. Xenobiotica, 2003, vol. 33, no. 8, 805–821.