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Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
In the study the metabolism of 2-phenylethylamine to phenylacetaldehyde was examined in liver slices and the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity was compared in the oxidation of the test substance with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme.
GLP compliance:
no
Radiolabelling:
no
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 450-950 g
- Diet: FD1 pellets supplemented with ascorbic acid and hay three times weekly, ad libitum
- Water: ad libitum

No further data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18±1
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
other: ex vivo incubation in vials
Vehicle:
not specified
Details on exposure:
Cylindrical cores, measuring 1 cm in diameter and approximately 1 cm in depth, were removed from the isolated livers using a sharpened stainless steel tube attached to a circular handle. The prepared liver slices were stored in oxygenated Krebs-Henseleit buffer pH 7.4 containing 0.024 M bicarbonate at 4 °C until required. 4 fresh liver slices in a total volume of 3 mL Krebs-Henseleit buffer pH 7.4 containing 0.024 M bicarbonate per 20 mL vials were incubated with 0.001 M phenylacetaldehyde at 37 °C in a shaking water bath. The medium was oxygenated with 95% O2 and 5% CO2 initially and every subsequent hour. 0.2 mL aliquots were removed after 0, 5, 10, 15, 30, 45, 60 and 90 min and analysed by HPLC. In addition, incubations without liver slices were performed as control incubations. To determine the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of the test substance, liver slices were co-incubated in the presence of specific inhibitors of each enzyme for 15, 30 and 45 min. The inhibitors used were 0.001 M isovanillin for aldehyde oxidase, 0.0001 M allopurinol for xanthine oxidase and 0.0001 M disulfiram for aldehyde dehydrogenase activity. After analysis, the slices were blotted dry and weighed to determine the total weight of liver used for each incubation. For comparison reasons, the results on the effect of inhibitors in liver slices have been normalized per 100 mg of liver.
Duration and frequency of treatment / exposure:
4 liver slices per vial were incubated with test substance for 0, 5, 10, 15, 30, 45, 60 and 90 min
Remarks:
Doses / Concentrations:
0.001 M
No. of animals per sex per dose:
not data
Control animals:
no

Incubation of the test substance with fresh guinea pig liver slices:

When the test substance (0.001 M) was incubated with freshly prepared guinea pig liver slices, three metabolites appeared on the chromatograms. The major metabolite was phenylacetic acid with small amounts of 2-phenylethanol and a third more polar metabolite, which eluted at 3.2 min and was thought to be phenyl-2-hydroxyacetic acid. However, the identity of the third compound could not be clearly verified. 4-hydroxyphenylacetic acid or N-(phenylacetyl)glycine was also considered as possible metabolites.

 

Incubation of the test substance with fresh guinea pig liver slices in the presence of inhibitors:

Isovanillin (0.001 M) only slightly reduced (about 14%, t= 30 min) the production of phenylacetic acid although breakdown of the test substance was significantly inhibited. A small increase in the formation of the other metabolites was observed.

In the presence of allopurinol (0.0001 M) there was a slight enhancement of phenylacetic acid production. There was also a positive enhancement in both the production of 4-hydroxyphenylacetic and 2-phenylethanol.

The presence of disulfiram (0.0001 M) in test substance incubations caused a 70-80 % inhibition in the production of phenylacetic acid over the incubation time period. It was shown that upon inhibition with disulfiram there was a significant increase in 2-phenylethanol production.

 

Conclusion:

The effects of inhibitors tested in the liver slice incubations showed that disulfiram (specific inhibitor for aldehyde dehydrogenase) almost totally inhibited oxidation of the test substance, whereas isovanillin (specific inhibitor for aldehyde oxidase) inhibited to lesser extent and allopurinol (specific inhibitor for xanthine oxidase) had little or no effect. The results indicate that the test substance is metabolized mainly by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.

Description of key information

The molecular weight, physicochemical properties incl. water solubility and octanol-water partition coefficient of the substance suggest that oral, inhalative and dermal absorption occur. Widely distribution within the water compartment of the body after systemic absorption is because of lipophilicity of the test substance not expected. Based on its log Pow the test substance is not considered to accumulate. The test substance might be metabolized after absorption. Excretion is expected to be predominantly via the urine.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

In accordance with Annex VIII, Column 1, Item 8.8 of Regulation (EC) No 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), assessment of the toxicokinetic behaviour of the test substance was conducted to the extent that can be derived from the relevant available information on physicochemical and toxicological characteristics. There are two publications available evaluating the hepatic metabolism of the test substance in vitro / ex vivo (Martini and Murr, 1996 and Panoutsopoulos et al. 2004).

The test substance is a colourless to pale yellow clear liquid at 20°C with a molecular weight of 120.15 g/mol and a water solubility of 18.0 g/L at 20 °C. The substance has a vapor pressure of 2.09 hPa at 20°C and the log Pow is 1.44 at 25°C.

Absorption

The major routes by which the test substance can enter the body are via the lung, the gastrointestinal tract, and the skin. To be absorbed, the test substances must transverse across biological membranes either by active transport mechanisms or - as being the case for most compounds - by passive diffusion. The latter is dependent on compound properties such as molecular weight, lipophilicity, or water solubility (ECHA, 2017).

Oral

In general, low molecular weight (MW ≤ 500) and moderate lipophilicity (log Pow values of -1 to +4) are favourable for membrane penetration and thus absorption. The molecular weight of the test substance is low with 120.15 g/mol, favouring oral absorption of the compound. This is supported by the determined log Pow value of 1.44, being advantageous for oral absorption. In addition the considerable water solubility of 18.0 g/L leading to a ready dissolving of the compound in the gastrointestinal fluids favours oral absorption.

Moreover, the observation of systemic toxicity following exposure by any route is an indication for substance absorption; however, this will not provide any quantitative information.

In an acute oral toxicity study conducted in 10 rats per dose group marked signs of toxicity were observed at test substance doses between 0.84 and 6.25 g/kg bw. In the 0.84 g/kg bw dose group one animal died within 24 h, two additional died on Day 5 and 6. In the 2.05 and 4.0 g/kg bw dose groups five animals died within 24 h. In the 4.0 g/kg bw dose group seven animals died within 24 h, two additional animals died on Day 2 and one on Day 6. In the dose groups of 5 and 6.25 g/kg bw five and seven animals were found dead within 24 h and one additional animal on Day 2, respectively. Animals were found lethargic, flaccid and showed ptosis, piloerection and convulsion. At gross pathology reddening or yellowish colour of the stomach, abdominal wall and intestines, dark liver, kidneys and lungs, exudate of mouth and nose, blood in lumen of intestine and bladder and signs of cannibalism were found. The LD50 for rats was determined to be 1.55 g/kg bw.

A combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study (2017) with dose levels of 25, 100 and 400 mg/kg bw/day administred by oral gavage was conducted in rats. One female was found moribund at 400 mg/kg bw/day. Two males and one female were found dead at 400 mg/kg bw/day. These females showed irregular respiration before their moribund state or death. Thickening of the forestomach with diffuse hyperkeratosis and squamous cell hyperplasia was observed in males at 100 mg/kg bw/day and in females at 400 mg/kg bw/day. Submucosal hemorrhage/edema and multifocal necrosis/focal erosion were observed in one dead female at 400 mg/kg bw/day. These effects can presumably be attributed to the corrosive properties of the test substance (Skin corr. 1B). Additionally, erythrophagocytosis and diffuse lymphoid hyperplasia were observed in the mesenteric lymph nodes at some males and females at 400 mg/kg bw/day. Furthermore, post-implantation loss was prominently increased at 400 mg/kg bw/day and the live birth index and viability index of pups at postnatal day (PND) 4 were markedly decreased at 400 mg/kg bw/day. These effects can presumably be attributed to secondary effects of parental local and systemic toxicity at the same dose level and thus no hazard on reproduction or development was identified.

In conclusion, the NOAEL for local toxicity was considered to be 25 mg/kg bw/day for males and 100 mg/kg bw/day for females and the NOAEL and LOAEL for systemic repeated dose toxicity were found to be 100 and 400 mg/kg bw/day respectively in both sexes. The NOAEL for parental fertility was considered to be 400 mg/kg bw/day for males and 100 mg/kg bw/day for females and the NOAEL for development of pups was considered to be 100 mg/kg bw/day.

Based on available data from the acute and repeated dose/reproduction and developmental toxicity studies and apart from local effects on the forestomach due to corrosivity of the test item, clinical signs of systemic toxicity were observed after oral exposure. Thus, absorption of the test substance via the gastrointestinal tract can be assumed.

Dermal

The dermal uptake of liquids and substances in solution is generally expected to be higher than that of dry particles. Molecular weights below 100 g/mol favour dermal uptake, while for those above 500 g/mol the molecule may be too large. Thus, for the respective molecular weight level of the test substance dermal uptake can be estimated to be moderate. Log P values between 1 and 4 of the test substance are favourable for dermal absorption. Also for dermal uptake sufficient water solubility is needed for the partitioning from the stratum corneum into the epidermis. Therefore, if the water solubility is above 10000 mg/L, dermal uptake is considered to be high.

The dermal permeability constant Kp of the substance was estimated to be 0.003 cm/h using DermwinTM (v.2.01) taking into account an estimated log Pow of 1.44 and the molecular weight of 120.5 g/mol. Thus the dermal absorption of the test substance is anticipated to be moderate to high.

Data from an in vivo skin corrosion test in human keratinocytes revealed corrosive properties of the test substance (2016). After exposure to the test substance the relative absorbance value decreased to 39.3% (3 minutes exposure) and 10.9% (1 h exposure). Both values are below the threshold for corrosivity which is defined to be 50% (3 min exposure) and 15% (1 h exposure), respectively. These data suggest that the test substance evidently damages the skin surface and therefore may enhance skin penetration. Additionally, an in vivo skin sensitisation test in mice (LLNA) was conducted with the test substance (2001). The test substance produced stimulation indices of 0.73, 1.76, 7.84, 8.76 and 18.96 at concentrations of 1, 2.5, 5, 10 and 25% (w/v) in 4:1 v/v acetone:olive oil, respectively and an EC3 value of 3.0% was calculated. Therefore the test substance was considered to be a moderate sensitiser. Based on available data from skin sensitisation, dermal uptake during topical exposure has evidently occurred.

Inhalation

The test substance has a vapour pressure of 2.09 hPa at 20 °C. Therefore, under normal use and handling conditions, inhalation exposure and thus availability for respiratory absorption of the substance in the form of vapour can be considered low. The log P value of 1.44 at 25°C is indicating a favourable absorption across the respiratory tract epithelium by passive diffusion. Nevertheless, since the substance is skin corrosive destruction of the mucosa can presumably occur when inhaled.

Distribution

Distribution of a compound within the body depends on the physico-chemical properties of the substance especially the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider is the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than the extracellular concentration, particularly in fatty tissues (ECHA, 2017).

Since the test substance is lipophilic (log Pow 1.44), distribution into cells is likely to occur and the intracellular concentration may be higher than the extracellular concentration particularly in fatty tissues, if the substance is absorbed systemically. Substances with log P values of 3 or less would be unlikely to accumulate in adipose tissues with the repeated intermittent exposure patterns normally encountered in the workplace but may accumulate if exposures are continuous. Once exposure to the substance stops the substance will be gradually eliminated at a rate dependent on the half-life of the substance.

 

Metabolism

A publication is available investigating the metabolism of the test substance in the presence/absence of specific inhibitors of hepatic enzymes in fresh liver slices of guinea pigs (Sup, Panoutsopoulos et al., 2004). In liver slices, the test substance is rapidly oxidized by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase. The major metabolite of the parent compound was found to be phenylacetic acid with small amounts of 2-phenylethanol. In addition, a third more polar metabolite was discovered, which eluted at 3.2 min and was thought to be phenyl-2-hydroxyacetic acid. However, the identity of the third compound could not be clearly verified. 4-hydroxyphenylacetic acid or N-(phenylacetyl)glycine were also considered as possible metabolites.

In another publication metabolisation of the test substance and other aromatic or aliphatic aldehydes by rat hepatic microsomal aldehyde dehydrogenase (mALDH) was studied. Phenylacetaldehyde was shown to be effectively dehydrogenated by mALDH to the corresponding acid (Martini and Murray, 1996).

Prediction of compound metabolism based on physico-chemical data is very difficult. Structure information gives some but no certain clue on reactions occurring in vivo. The potential metabolites following enzymatic metabolism were predicted using the QSAR OECD toolbox (v3.4, OECD, 2016). This QSAR tool predicts which metabolites may result from enzymatic activity in the liver and in the skin, and by intestinal bacteria in the gastrointestinal tract. Only one hydroxylated metabolite occurring in skin and liver was predicted for the test substance. In general, introduction of hydroxyl groups into the molecule make the substances more water-soluble and susceptible to metabolism by phase II-enzymes. Up to 37 metabolites were predicted to result from all kinds of microbiological metabolism for the test substance. Most of the metabolites were found to be a consequence of the degradation of the molecule.

There was no evidence for differences in genotoxic potencies due to metabolic changes in in vitro genotoxicity tests. The studies performed on genotoxicity (Ames test and HPRT test and micronucleus test in mammalian cells in vitro) were all negative, with and without metabolic activation (2016; 2016; 2016).

Excretion

Only limited conclusions on excretion of a compound can be drawn based on physicochemical data. Due to metabolic changes, the compound finally excreted may have few or none of the physico-chemical properties of the parent compound. In addition, conjugation of the substance may lead to very different molecular weights of the final product as compared to the parent. The molecular weight (< 300 g/mol) and the water solubility of the molecule are properties favouring excretion via urine. Thus the test substance is expected to be excreted predominantly via the urine.

 

References

ECHA (2017): Guidance on information requirements and chemical safety assessment – Chapter 7c: Endpoint specific guidance. European Chemicals Agency, Helsinki

Martini and Murray (1996): Rat Hepatic Microsomal Aldehyde Dehydrogenase. Identification of 3- and 4-Substituted Aromatic Aldehydes as Substrates of the Enzyme. Chemical Research in Toxicology, Vol. 9, pp. 268 - 276