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Description of key information

Repeated dose toxicity; oral (gavage) exposure in rats (OECD 408, GLP): NOAEL = 100 mg/kg bw (BASF 2018;50C0538/11S273)

Key value for chemical safety assessment

Additional information

Repeated dose toxicity; oral exposure

In the key study for repeated dose toxicity, i.e. a subchronic repeated dose toxicity study in Wistar rats according to OECD TG 408 and GLP, 4-methoxybenzaldehyde was administeredorally by gavage to groups of 10 males and 10 femalesatdose levels of 0 (vehicle control), 20, 100 and 500 mg/kg bw/day in corn oilover a period of 3 months (BASF 2018;50C0538/11S273).In addition to the required examinations, special attention was given to the reproductive organs of male and female animals.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.Ophthalmological examinations were performed before the beginning and at the end of the administration period. For at least 3 weeks an estrous cycle determination was performed.Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations.Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals for sperm examinations.


The following adverse findings – related tothe treatment with 4-methoxybenzaldehyde- were noted in the 500 mg/ kg bw/d dose group:

Clinical Pathology

·        Decreased absolute and relative eosinophil counts in both sexes.

·        Decreased total protein levels in both sexes.

·        Increased glucose and inorganic phosphate levels in males.

·        Decreased urine pH values in both sexes.

·        Increased specific gravity and incidences of crystals of unknown origin in urine of females.

·        Decreased motility of the sperms and total sperm head counts in the cauda epididymidis in males.

·        Increased incidences of abnormal sperms in the cauda epididymidis in males.


·        Significantly decreased mean absolute epididymides weight by -17%.

·        Significantly decreased mean cauda epididymis weights, i.e. -29% (absolute) and -23% (relative).

·        Ductal atrophy at epididymides’ distal corpus and caudal junction of all male animals (minimal to moderate).

·        Oligospermia in epididymides’ distal corpus and caudal junction of all male animals (minimal to slight).

No treatment-related, adverse effects were observed in the dose groups of 20 and 100 mg/kg bw/d.


Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of all test groups during the entire study period.

Salivation shortly after treatment was observed in all male and female animals of the high dose group (500 mg/kg bw/d) on several days of the application period.From the temporary, short appearance immediately after dosing, it was concluded, that the finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse as no lesions in the upper digestive tract were observed in male and female animals during pathological examinations.No adverse effects on body weight development were obtained in any test group, since mean body weights and body weight change values did not show any significant deviations to the control. Only a slight decrease in male mean body weights (-5.3% deviation vs controls on study day 91) and body weight gains (-8.4% deviation vs controls in the period of day 0 - day 91) has been observed in the high dose group (500 mg/kg bw/d) during the course of the study.

Regarding the clinical pathology parameters, the decreased absolute and relative eosinophil counts as well as lower total protein values in rats of both sexes of the 500 mg/kg bw/d dose group and additionally higher glucose levels in males are due to a chronic stress situation among these individuals. A drop in the mean urine pH values (from pH 6.0 to pH 5.6 in males and from pH 5.9 to pH 5.5 in females) has been observed, which may be the cause for precipitation of crystals of unknown origin due to a high specific gravity of the urine at least in the high dose females.This finding supports the disproportionate increase of endogenous anisic acid due to overwhelmed elimination pathways,as outlined in chapter “Toxicokinetics” and “Reproductive toxicity”.


In high dose males (500 mg/kg bw/d), motility of the sperms and the sperm head counts in the cauda epididymidis were significantly decreased, whereas the incidence of abnormal sperms in the cauda epididymidis was significantly increased. These findings are supported by pathological the examinations performed. Histopathology revealed a ductal atrophy at the distal corpus and proximal cauda of the epididymides, associated with oligospermia in high dose males. In some animals this change was accompanied by a cribiform change. The findings further correlated with an absolute weight decrease of the epididymides (-17%) and the cauda epididymis (absolute -29%, relative -23%).

In the livers of high dose males and females (500 mg/kg bw/d), a minimal centrilobular hypertrophy was observed, which was consistent with significant liver weight increases in males (relative +17%) and females (absolute +14%, relative +18%). These findings were regarded as treatment-related but not adverse, since they were not associated with altered clinical chemical parameters.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Under the conditions of this studythe oral administration of 4-methoxybenzaldehyde by gavageto Wistar rats over a period of 3 months revealed adverse signs of toxicity at a dose levelof 500 mg/kg bw/d in male and female animals taking clinical pathology and pathology findings into account.Thus,the no observed adverse effect level (NOAEL) for was set to 100mg/kg bw/d in male and female Wistar rats.

In support, 4-methoxybenzaldehyde was tested in a combined repeated dose and reproductive/ developmental toxicity screening test according to the OECD TG 422 and GLP (Hatano Research Institute, 2000). The substance was administered orally to male and female (13 each/group) Sprague-Dawley rats at doses of 0 (vehicle), 20, 100 and 500 mg/kg bw/d in corn oil throughout 2-week premating and 2-week mating periods. Male rats were further dosed for 2 weeks after the completion of mating period, while females continued to be dosed throughout pregnancy to day 4 of lactation after parturition.

No animal died in any dose group. Transient salivation was found at 500 mg/kg bw/d. Continuous increase in food consumption has been found in males from day 7 onward and in females during the premating period (day 7 and 13) at 500 mg/kg bw/d. Statistical significant increases in body weights have been observed for females in the 100 mg/kg bw/d dose group during lactation and in the 500 mg/kg bw/d dose group during gestation and lactation. Male animals showed slight increases in body weights at 100 and 500 mg/kg bw/d without reaching statistical significance. Furthermore, hyperplasia of squamous epithelium in the forestomach was detected in the 100 and 500 mg/kg bw/d dose groups. In line with the findings described above, these findings are likely to stem from local irritating effects of 4-methoxybenzaldehyde. 

Platelet counts were significantly decreased in females (100, 500 mg/kg bw/d) and in males (500 mg/kg bw/d). However, no changes in the bone marrow have been observed, no significant bleeding became evident and no significant changes in the prothrombin time and activated partial thromboplastin time were observed. Therefore, the relevance of this isolated finding is questionable.

A/G ratio, GOT activity and inorganic phosphorus concentration was increased in males, whereas increased glucose levels and decreased calcium levels have been observed in females at 500 mg/kg bw/d. Increased absolute/relative liver weights and centrilobular hypertrophy of hepatocytes has been observed for both sexes at 500 mg/kg bw/d. Overall, these findings indicate a test substance related effect on the liver.

From these test results, under the conditions of this study, the no observed effect level (NOEL) is set at 20 mg/kg/day and the stomach (local irritative effects at 100 mg/kg bw/d) and the liver (systemic effects at 500 mg/kg bw/d) represent target organs after oral (gavage) administration of 4-methoxybenzaldehyde. 


In further supportive studies, the chronic toxicity potential of test substance in rats was evaluated in a study from Hagan et al. (1967) after continuous administration of the test substance in feed a single dose level of 1000 ppm (67 mg/kg bw/ day) in diet for 27-28 weeks or 10000 ppm (670 mg/kg bw/ day) for 15 weeks. The animals were then observed for clinical signs, body weights, food consumption and changes in hematological parameters. The surviving animals were sacrificed at the end of the observation period for necropsy and gross pathology and histopathology. At the respective unique dose evaluated, there was no effect on body weights or haematology, and no macroscopic / microscopic organ changes at necropsy.


Furthermore, 4-Methoxybenzaldehyde was applied by gavage for 14-days to male and female Sprague-Dawley rats (RIFM, 2011). The test substance was ingested at concentrations of 0, 20, 100 and 500 mg/kg bw/d. No mortality or test substance related clinical signs were observed. Body weight gains in male animals were generally comparable to or increased over the vehicle control group value, with the exception of sporadic reductions in weight gain at 500 mg/kg bw/d. Body weight gains on DS 1 to 14 were reduced in the 500 mg/kg/day dosage group by 12% (compared to vehicle control group) although the average body weight on DS 14 was comparable to control animals. This reduction in body weight gains in male rats occurred without corresponding reductions in feed consumption values. Following the initiation of dosage administration, transient losses in body weight, with corresponding reductions in feed consumption values, occurred in female rats given 500 mg/kg bw/d of the test substance. However, the initial losses in body weight and the reductions in feed consumption did not affect the overall weight gain or feed consumption values for the cumulative dosage period. Oral administration of 4-Methoxybenzaldehyde did not result in any gross lesions, changes in organ weights or microscopic findings at any dose level investigated.

The no observed adverse effect level (NOAEL) has been set to 100 mg/kg bw/d for male animals, whereas in females, no persistent adverse effect was observed up to 500 mg/kg bw/d.

Further data from literature were insufficient for assessment due to limited documentation or invalid due to administration of 4 -methoxybenzaldehyde in mixtures.



Repeated dose toxicity, dermal exposure

In a 14-day percutaneous study in rats, 4-methoxybenzaldehyde has been administered dermal to male and female Sprague-Dawley rat at doses of 0, 100, 250, 500 and 1000 mg/kg bw/day daily for 6 hours over a period of 2 weeks under occlusive conditions (RIFM 2011).

No animal died in any dose group. Skin observations attributed to percutaneous administration of 4-Methoxybenzaldehyde included flaking and erythema grade 1 at 500 and 1000 mg/kg bw/d and erythema grade 2 at 1000 mg/kg/day in males. In females, flaking grade 1 was observed in each dosage and the control group, however, the incidence and number of rats affected was greatest at 500 and 1000 mg/kg bw/d. Erythema grade 1 occurred in each test substance treated group. Skin changes at the application site were confirmed in gross pathology/histopathology, i.e. minimal to mild, focal cellular infiltration of the dermis and focal epithelial hyperplasia at 500 and 1000 mg/kg bw/d. No other test substance related clinical findings were observed.

In male rats, net losses in body weight occurred in each 4-methoxybenzaldehyde-treated group the day after the first application of the test substance. In the 1000 mg/kg bw/day dose group, body weight gains in male rats on DS 1 to 14 were reduced by 32% compared to the controls, although the average body weight on DS 14 was comparable to control animals.

There were no apparent test substance related effects on body weights, body weight gains or feed consumption in female rats at any dosage level.

No test substance related effects in absolute and relative organ weights, gross pathology and histopathology (besides skin effects described above) were observed.

From these test results, under the conditions of this study the NOAEL addressing systemic effects is set at 500 mg/kg bw/day for male animals whereas in females, no persistent adverse test substance related effect was observed up to 1000 mg/kg bw/d. Based on the confirmed adverse effects observed at the skin application site, a NOAEL addressing local effects is indicated at 100 mg/kg bw/day.

Justification for classification or non-classification

Based on available repeated dose toxicity data and on the criteria laid down in the 67/548/EEC / (EU) 1272/2008 regulations, a non-classification is warranted.