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Effects on fertility

Additional information

The key study for reproductive toxicity, i.e. a combined repeated dose and reproductive/ developmental toxicity screening test, has been performed according to the OECD TG 422 and GLP (Hatano Research Institute, 2000). 4-methoxybenzaldehyde has been 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) for parental toxicity 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. 

The number of pregnant dams and the respective fertility index (nr. animals pregnant /nr. animals copulated x 100) was significantly decreased in the 500 mg/kg bw/d dose group. However, no test substance related effects on the number of animals having copulated, on the time to copulation and on estrous cycling has been observed. No test substance related pathological abnormalities in reproductive organs were found (see chapter “Repeated dose toxicity”). A significant decrease in absolute and relative epididymis weights (91%/88% vs. ctrl.) was reported for the high dose males, however, this finding lacked a histopathological correlate. In epididymis, only a single animal finding (slight unilateral sperm granuloma) was reported in the mid dose group (100 mg/kg bw/d). In testis, only single animal findings were found, such as a very slight granulation tissue in the seminiferous tubule interstitium (1 animal in the 500 mg/kg bw/d dose group) and a very slight atrophy in the seminiferous tubule (1 animal of control and 100 mg/kg bw/d dose groups). None of these abnormalities was deemed to cause a failure of conception. Sperm parameters have not been assessed in this study.

Further, no significant differences were observed in the number of corpora lutea in pregnant animals, number of implantation sites, implantation index, gestation index and gestation length. No abnormalities were observed during parturition and lactation. Under the conditions of this study, the no observed effect level (NOEL) of 4-methoxybenzaldehyde is to be set at 100 mg/kg/day for reproductive toxicity, i.e. fertility and/or developmental toxicity.

No valid data on reproductive toxicity are available for the key metabolite anisic acid. However, information from a multigeneration study performed with another benzyl derivative with similar structural features as the key metabolite anisic acid, i.e. benzoic acid, was taken into account. The respective structure only differs in an additional methoxy group in para-position. In terms of systemic toxicity after single or repeated administration, the toxicological profile of benzoic acid shares similarities with 4-methoxybenzaldehyde (see also OECD SIDS for Benzoates). Acute toxicity is comparably low with LD50 values observed in rats between 2000 and 5000 mg/kg bw. Systemic toxicity after repeated administration of benzoic acid via feed was generally lower as seen for 4-methoxybenzaldehyde, however the liver has been identified as target organ for both substances. Differences in potencies might be explainable by differences in ADME after administration via feed versus gavage. Furthermore, similarities exist in pharmacokinetics of orally administered benzoic acid and 4-methoxybenzaldehyde. Benzoic acid is metabolized and excreted predominantly as glycine conjugate and to a lower extent as benzoylglucuronide. A limited capacity for glycine conjugation of benzoates was described at high dose levels only in the OECD SIDS. However, AUCs for benzoic acid in humans were reported to increase disproportional with dose, while that for hippuric acid increased proportionally (see Kubota et al.; J. Chromatography 425, 67-75, 1988). It is therefore suggested that transformation of orally administered benzoic acid to hippuric acid is a saturable process.

In a 4-generation study 0.5 or 1% benzoic acid in the diet (= approx. 375 or 750 mg/kg/day) had no influence on growth (weight, weight gain and food efficiency (measured by protein efficiency)) and organ weights (Kieckebusch, 1960). In all 4 generations, no effects on fertility ("Fortpflanzung") and lactation ("Aufzucht der Jungen") was found. The animals of the 3rd generation were sacrificed and examined histopathologically after 16 weeks (after lactation of the pups). No remarkable histopathological findings were found. In this paper, no information is given on the organs investigated. From other parameters it can be assumed, that as a minimum the brains, heart, liver, kidney, testis and were examined. Feeding of 0.5% led to prolongation of survival compared to controls. In addition, a so-called "Alterspaarung" after 48 weeks gave no influence on start of menopause. The NOAEL for P-, F1- and F2 generation was >= 750 mg/kg bw/day.

However, 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 treatment resulted in adverse effects on the epididymis and sperm formation(BASF 2018;50C0538/11S273).In this study, the test substance 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. In addition to the required examinations, special attention was given to the reproductive organs of male and female animals.

Food consumption and body weights 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 treatment related findings affecting to reproductive organs/parameters were noted in the 500 mg/kg bw/d dose group (for further details on other parameters see Chapter “Repeated dose toxicity”):

·        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.

 

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 the pathological 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%).Under the conditions of this studythe oral administration of 4-methoxybenzaldehyde by gavageto Wistar rats over a period of 3 months revealed adverse effects on male reproductive organs at a dose levelof 500 mg/kg bw/d.The no observed adverse effect level (NOAEL) for was set to 100mg/kg bw/d.

It needs to be pointed out, that the decrease in numbers of pregnant dams / fertility index (OECD 422) and the adverse effects on the epididymis and sperm formation (OECD 408) was observed at a dose level only, which leads to an evident disproportionate increase of anisic acid in plasma and a temporary persistence of high anisic acid plasma levels (for details, see. Chapter “Toxicokinetics, metabolism and distribution”). This is supported by the drop in the urinary pH values in treated Wistar rats, observed in the subchronic repreated dose toxicity study.At the dose level of 500 mg/kg bw/d, a saturation of the formation of the respective glycine conjugate as detoxifying mechanism has been identified. The disproportionate increase of endogenous anisic acid plasma levels based on overwhelmed elimination pathways is assumed to be related to the adverse effects observed. 

 

Overall, the present data provide some evidence from experimental animals for an adverse effect on sexual function and fertility. However, due to the discrepancies between the available study results (i.e. effects on the epididymis in the OECD 422 versus OECD 408) and the effect levels at saturated processes in metabolism and/or excretion, the evidence is currently inconclusive for a final decision on the classification of 4-methoxybenzaldehyde concerning fertility.

 

Therefore, the dossier submitter is planning to execute further mechanistic studies to clarify the mode of action and the human relevance of the effects on epididymides and sperm formation in the rat. Furthermore, additional studies to investigate the toxicokinetic behavior of 4-methoxybenzaldehyde in comparative gavage and feeding studies in relation to the adverse effects observed are intended, because the relevance of gavage/bolus administration for humans is low and the toxicokinetic profile of anisic acid in plasma might be substantially different (see eg. TK information of Benzylacetate: Yuan et al. 1995).

 

 

Based on the final decision on a compliance check (CCH-D-2114359254-48-01/F), the dossier submitter is obliged to test 4-methoxybenzaldehyde in an Extended one-generation reproductive toxicity study in rats according to OECD TG 443. As outlined in the comments to this final decision, the dossier submitter considers the following text of the OECD TG 443 relevant for an adequate dose selection:

 

“If TK data are available which indicate dose-dependent saturation of TK processes, care should be taken to avoid high dose levels which clearly exhibit saturation, provided of course, that human exposures are expected to be well below the point of saturation. In such cases, the highest dose level should be at, or just slightly above the inflection point for transition to nonlinear TK behaviour.“

 

Such a saturation process has been identified for 4-methoxybenzaldehyde upon gavage administration, and the effects on reproductive organs/ parameters seen were observed at doses above this point of saturation.

It is stated in EChAs final decision, that the dossier submitter has not provided any exposure estimation in the chemical safety report and the potential level of exposure of the human population to 4-methoxybenzaldehyde is not known. To demonstrate the absence of a high worker and consumer exposure to 4-methoxybenzaldehyde and to exclude an internal human exposure to toxicologically relevant concentrations, the dossier has been updated with an exposure estimation and risk characterization.

Human exposure is well below such a point of saturation. Exposure of workers via the oral route is not expected and dermal exposure in an occupational setting is controlled and limited due to the use of closed systems and/or containment and personal protective equipment as industry standard. Inhalative exposure is limited due to the low vapor pressure, the inherent odor and sufficient risk management measures when handled as undiluted substance. Since the calculation of the exposure estimates as provided in the CSR is made with a Tier 1 exposure tool (i.e. ECETOC TRA), which are based including highly conservative assumptions, the real exposure in an occupational setting is considered lower than the estimates given. Consumer exposure is even lower, since use levels of 4-methoxybenzaldehyde as a component in often complex fragrance mixes are mostly well below 1% or in the ppm range when used as flavor.

 

Altogether, with regard to human exposure with 4-methoxybenzaldehyde it is very likely that the expected exposure estimates do not induce a metabolic saturation and temporary persistence of anisic acid plasma levels, however, with regard to hazard characterization and the OECD 443 animal study requested by EChA, the TK data of 4-methoxybenzaldehyde are of crucial importance for the dose selection (MTD) as outlined above.

 

References:

·        Yuan et al. 1995. Effects of Gavage Versus Dosed Feed Administration on the Toxicokinetics of Benzyl Acetate in Rats and Mice. FdChem. Toxic. Vol. 33, No. 2, pp. 151-158.

Effects on developmental toxicity

Additional information

In a combined repeat dose and reproductive/ developmental toxicity screening test according to the OECD TG 422 and GLP (Hatano Research Institute, 2000), 4-methoxybenzaldehyde has been administered orally via gavage 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) for parental toxicity 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. 

 

Concerning the offspring, the number of pups born, liveborns and live pups at day 4 of lactation per litter was affected at 500 mg/kg bw/d when compared the control group. The respective delivery index (nr. pups born / nr. implantation sites x 100) was significantly decreased and the birth index (nr. live pups d0 / nr. implantation sites x 100) showed a non-significant trend for a decrease. However, the live birth index (nr. live pups / nr. pups born x 100) and the viability index (nr. live pups d4 / nr. live pups d0 x 100) were not affected by test substance treatment. Overall, these findings at the high dose of 500 mg/kg bw/d 4-methoxybenzaldehyde are indicative for post-implantation losses. Postnatal viability was shown to be not affected under the chosen testing conditions. Furthermore, there were no effects of the test article on pup weights and sex ratio. No external malformations and no abnormalities in internal organs were found in the pups observed.

Under the conditions of this study, the no observed effect level (NOEL) of 4-methoxybenzaldehyde is to be set at 100 mg/kg/day for reproductive toxicity, i.e. fertility and/or developmental toxicity.

No valid data on developmental toxicity are available for the key metabolite anisic acid. However, information from developmental toxicity studies performed with another benzyl derivative with similar structural features as the key metabolite anisic acid, i.e. benzoic acid, was taken for further assessment of this endpoint via read across. The respective structure only differs in an additional methoxy group in para-position. In terms of systemic toxicity after single or repeated administration, the toxicological profile of benzoic acid shares similarities with 4-methoxybenzaldehyde (see also OECD SIDS for Benzoates). Acute toxicity is comparably low with LD50 values observed in rats between 2000 and 5000 mg/kg bw. Systemic toxicity after repeated administration of benzoic acid via feed was generally lower as seen for 4-methoxybenzaldehyde, however the liver has been identified as target organ for both substances. Differences in potencies might be explainable by differences in ADME after administration via feed versus gavage.

Furthermore, similarities exist in pharmacokinetics of orally administered benzoic acid and 4-methoxybenzaldehyde. Benzoic acid is metabolized and excreted predominantly as glycine conjugate and to a lower extent as benzoylglucuronide. A limited capacity for glycine conjugation of benzoates was described at high dose levels only in the OECD SIDS. However, AUCs for benzoic acid in humans were reported to increase disproportional with dose, while that for hippuric acid increased proportionally (see Kubota et al.; J. Chromatography 425, 67-75, 1988). It is therefore suggested that transformation of orally administered benzoic acid to hippuric acid is a saturable process. If administered as sodium and potassium salts, benzoic acid is expected to immediately dissociate and form benzoic acid in an aqueous environment.

Based on these similarities the following data on sodium benzoate were taken into consideration via read across in a weight of evidence:

·        FDRL (1972; cited in the OECD SIDS for Benzoates) reported about the teratologic evaluation of sodium benzoate. Wistar rats were administered (gavage) concentrations of 1.75, 8, 38 or 175 mg/kg/day from gestation day 6 - 15. No effects on nidation or on maternal or fetal survival were found. The number of abnormalities of soft and skeletal tissues did not differ from the controls; maternal toxicity was not reported at any dose level. The NOAELs for maternal toxicity and teratogenicity were >= 175 mg/kg bw/day.

·        In another FDRL study (1972; cited in the OECD SIDS for Benzoates), CD-1 mice were administered (gavage) concentrations of 1.75, 8; 38 or 175 mg/kg from gestation day 6 - 15. No effects on nidation or on maternal or fetal survival were observed. The number of abnormalities and skeletal tissues did not differ from controls; maternal toxicity was not reported at any dose applied. The NOAELs for maternal toxicity and teratogenicity were >= 175 mg/kg bw/day.

·        Golden hamsters were administered (gavage) concentrations of 3, 14, 65 or 300 mg/kg/day from gestation day 6 - 10. No effects on nidation or on maternal or fetal survival were observed. The number of abnormalities of soft and skeletal tissues did not differ from controls; maternal toxicity was not reported at any dose applied (FDRL, 1972; cited in the OECD SIDS for Benzoates).

·        Onodera et al. (1978; cited in the OECD SIDS for Benzoates) reported about effects of sodium benzoate on fetuses and offspring of Wistar rats. The animals were offered concentrations of 1, 2, 4 or 8% via the diet (about 700, 1400, 2800 or 5600 mg/kg/day) over the whole gestation period (20 days). The authors concluded, that the effects on the dams and fetuses at the 2800 and 5600 dose levels were due to reduced maternal food intake in these groups leading to malnutrition. The intake of sodium benzoate in the highest dose group (8%) was lower than that at the 2% dose level, whereas no adverse effects were seen. The NOAELs for maternal toxicity and teratogenicity were 1400 mg/kg bw/day.

 

It needs to be pointed out, that the indication for post-implantation losses was observed at a dose level, which leads to an evident disproportionate increase and an elongation of high anisic acid plasma levels (for details, see. Chapter “Toxicokinetics, metabolism and distribution”). At this dose level, a saturation of the formation of the respective glycine conjugate as detoxifying mechanism has been observed. In the present reproductive toxicity study, dose levels have not been selected taking into account such toxicokinetic data. The disproportionate increase of endogenous anisic acid plasma levels based on overwhelmed elimination pathways may to lead to a potential acidosis and may be causative for the adverse developmental toxicity effects observed. 

However, occupational and consumer exposure to 4-methoxybenzaldehyde is low, and endogenous levels of 4-methoxybenzaldehyde leading to toxicologically relevant anisic acid concentrations is highly unlikely. Exposure of workers via the oral route is not expected and dermal exposure in an occupational setting is controlled and limited due to the use of closed systems and/or containment and personal protective equipment as industry standard. Inhalative exposure is limited due to the low vapour pressure (approx. 3 Pa) and the inherent odor. Use levels of 4-methoxybenzaldehyde as component in fragrance mixes (mostly below 1%) or as flavor (in the ppm range) for consumer products is low.

Since saturation processes in metabolism and / or excretion are evident at the tested dose level, the relevance of the observed post-implantation losses is highly questionable and do not suffice to form a robust rationale for a classification of 4-methoxybenzaldehyde as developmental toxicant.

Based on the final decision on a compliance check (CCH-D-2114359254-48-01/F), the dossier submitter is obliged to test 4-methoxybenzaldehyde in a prenatal developmental toxicity study. The results of this study will be taken into account for a final evaluation of the endpoint developmental toxicity.

Justification for classification or non-classification

The present data on reproductive toxicity are inconclusive.