6-amino-1,3-dimethyluracil

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Remarks:

Expert statement

Type of information:

other: Expert statement

Adequacy of study:

key study

Study period:

2018-01-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP-conform guideline study, but scientifically valid expert statement based i.a. on studies assessed with Klimisch 1 or 2.

Data source

Reference

Reference Type:

other: Expert statement

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

An extensive assessment of the toxicokinetic behaviour of 1,3-Dimethyl-4-aminouracil (DIMAU) was performed, taking into account the chemical structure, the available physico-chemical and toxicological data.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-amino-1,3-dimethyluracil

EC Number:

229-662-0

EC Name:

6-amino-1,3-dimethyluracil

Cas Number:

6642-31-5

Molecular formula:

C6H9N3O2

IUPAC Name:

6-amino-1,3-dimethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

Test material form:

solid

Radiolabelling:

other: not applicable

Test animals

Species:

other: not applicable

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

not applicable

Administration / exposure

Route of administration:

other: All relevant routes of administration are discussed in the expert statement.

Vehicle:

other: not applicable

Details on exposure:

not applicable

Duration and frequency of treatment / exposure:

not applicable

Doses / concentrations

Remarks:

not applicable

Control animals:

other: not applicable

Positive control reference chemical:

not applicable

Details on study design:

not applicable

Details on dosing and sampling:

not applicable

Statistics:

not applicable

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

The relevant absorption rates were estimated to: Oral absorption: approx. 100%, Dermal absorption: approx. 100%, Inhalative absorption: approx. 10%.

Main ADME results 2

Type:

distribution

Results:

Systemic bioavailability of the substance is very high. A high peak exposure can be expected, a very relevant AUC is not to be expected.

Main ADME results 3

Type:

metabolism

Results:

Amine hydroxylation, N-dealkylation, and epoxydation were identified as the mode of action during Phase-I-metabolism, and subsequent conjugation is expected.

Main ADME results 4

Type:

excretion

Results:

DIMAU and its estimated metabolites are small, sufficiently hydrophilic and soluble in water. A very fast excretion of the compounds via the kidneys and urine can be expected. DIMAU has a minor potential for bioaccumulation, and will be excreted rapidly.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Absorption
In this chapter, the physico-chemical properties of the substance are used to draw general conclusions for its behaviour and how these properties will influence its oral, inhalatory and dermal absorption. Furthermore, these conclusions will be supported by the available data and studies.
In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 g/mol are favourable for absorption). Generally, the absorption of chemicals which are surfactants or irritants may be enhanced, because of damage to cell membranes.
DIMAU was found to be non-corrosive and non-irritating to both skin and eyes. Further, when dissolved in water as shown in the partition coefficient study, the resulting pH value is 6.2-6.9, which is in between of neutral and the physiological pH value of the skin. Hence, the possibility of an enhanced absorption due to damaged cell membranes can be excluded.
Due to the lack of experimental absorption data, the following physico-chemical parameters of DIMAU will be taken into account when discussing its absorption into the body:
- Molecular weight = 155.1546 g/mol
- Physical state: solid
- Water solubility = 5.46 g/L at 25°C
- Partition Coefficient log Pow = -0.4 at 20°C
- Vapour pressure = 1*10-7 at 25°C
- Melting point = 299°C

Absorption from the gastrointestinal tract
In the small intestine absorption occurs mainly via passive diffusion or lipophilic compounds may form micelles and be taken into the lymphatic system. Additionally, metabolism can occur by gut microflora or by enzymes in the gastrointestinal mucosa. However, the absorption of highly lipophilic substances (LogPow of 4 or above) may be limited by the inability of such substances to dissolve into gastrointestinal fluids and hence make contact with the mucosal surface. The absorption of such substances will be enhanced if they undergo micellular solubilisation by bile salts. Substances absorbed as micelles enter the circulation via the lymphatic system, bypassing the liver. Consequently, immediate Cytochrome P450 metabolism is less important here as for substances which directly enter the hepatic system via the portal vein.
According to ECHA’s guidance R.7c [ECHA 2008] [ECHA 2014], it is stated that the smaller the molecule the more easily it may be taken up. Molecular weights below 500 g/mol are favourable for absorption. With a molecular weight of 155.1546 g/mol, absorption in general can be considered as possible, and absorption is in general favourable. Also, substances with moderate log P values (between -1 and 4) are favourable for absorption by passive diffusion. With a LogPow of -0.4 at 20°C, a passive diffusion in the gastrointestinal fluids is indicated; as it is directly in between those boundary values, absorption is also highly likely based on the LogPow. Water-soluble substances will readily dissolve into the gastrointestinal fluids [ECHA 2014], and the determined solubility of 5.46 g/L at 25°C is magnitudes above the guidance value of 1 mg/l, so water solubility does also not indicated a hindered absorption.
The substance is not readily biodegradable, and hydrolytically stable. So in general a degradation via gut bacteria can be assumed not to occur. No neither bacterial metabolites nor hydrolysis products need to be regarded when assessing the absorption of DIMAU, and it is sufficient to focus on the substance itself only. Also, a possible metabolism as described in chapter 3.4 Metabolism does only have to be regarded after absorption already has occurred.
Taking into account the acute and subchronic oral toxicity data, at least some information can be taken to prove the conclusions that absorption occurs. Via both oral and dermal exposure routes, LD50 values were determined to be above the limit dose, no systemic effects were noted, which could be either indicative of a low toxicity of the substance or of non-absorption. So it cannot be clearly deducted from these results whether the substance is poorly absorbed or not very toxic as such. However, the NO(A)EL of 110 mg/kg bw/d derived from the oral OECD 408 study on rats, indicates in general that absorption occurs. And, due to the fact that minor effects on systemic toxicity occurred, a wide distribution of the test items throughout the body is strongly indicated.
Hence, a preliminary occurrence of oral absorption of the test item after gavage can reasonably deducted.
So in summary, taking into account the available physico-chemical data of DIMAU, especially its small molecular weight and suitable partition coefficient and water solubility, its absorption via the GI tract can be considered to be very high. This conclusion is supported by effects after oral application in an OECD 408 study indicating a distribution of the compounds throughout the body. Due to the lack of further data, an absorption of 100% should be taken into account when performing the subsequent assessment.

Absorption from the respiratory tract
Concerning absorption in the respiratory tract, any gas, vapour or other substances inhaled as respirable dust (i.e. particle size ≤ 15 µm) has to be sufficiently lipophilic to cross the alveolar and capillary membranes (moderate LogPow values between 0-4 are favourable for absorption). The rate of systemic uptake of very hydrophilic gases or vapours may be limited by the rate at which they partition out of the aqueous fluids (mucus) lining the respiratory tract and into the blood. Such substances may be transported out of the lungs with the mucus and swallowed or pass across the respiratory epithelium via aqueous membrane pores. Lipophilic substances (LogPow >0) have the potential to be absorbed directly across the respiratory tract epithelium. Any lipophilic compound may be taken up by micellular solubilisation but this mechanism may be of particular importance for highly lipophilic compounds (log P >4), particularly those that are poorly soluble in water (1 mg/l or less) that would otherwise be poorly absorbed [ECHA, 2008].
DIMAU is a solid which has a very low vapour pressure, 1. x 10exp(-7) hPa at 25°C, and melts at 299°C. The boiling point could not be determined because no suitable method was found and the vapour pressure is too low, indicating that it would be way above normal handling temperatures, provided that no decomposition occurs. However, these results are clearly showing that the inhalative absorption as a gas does not have to be regarded.
According to its particle size distribution, the main agglomerate size was in the range of 250 µm to 2000 µm. 50 % of the test item agglomerates exceeded 500 µm. The amount of test item with a particle size below 250 µm was only about 2 %, 0.05% < 125 µm, none were < 75 µm. According to its particle size distribution, 0% of the particles belong to the respirable (<4 µm), or thoracic fraction (4-10 µm), and a further negligible amount of the particles (0.05% < 125 µm, none were < 75 µm ) are potentially inhalable (10-100 µm). Those boundary values are taken from the recent version of ECHA’s Guidance document [ECHA, 2015], the former version gave the following information: In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract [ECHA, 2008]. So, based on the particle size distribution, max. 0.05% of the applied dose would reach the body via inhalation, all other particles are too large to be bioavailable. Further, most of the minimal amount of particles are subject to nasal clearance, and none to tracheobronchial clearance, and further none of the particles are able to reach the alveoli. As a consequence, the fraction potentially reaching the alveolar region of the respiratory tract beyond the bronchi, where no mechanical excretion mechanism as the ciliary movements is available, is negligible. So, the potential absorption can only be regarded theoretically.
For absorption of deposited material similar criteria as for GI absorption can be applied. In general, either a prolonged exposure due to deposition and subsequent absorption or immediate absorption by micellular solubilisation has to be assumed. The latter mechanism may be of particular importance for highly lipophilic compounds (LogPow >4), particularly those that are poorly soluble in water (1 mg/l or less) and is hence not relevant here. To be readily soluble in blood, a gas, vapour or dust must be soluble in water and increasing water solubility would increase the amount absorbed per breath. However, the gas, vapour or dust must also be sufficiently lipophilic to cross the alveolar and capillary membranes. Therefore, a moderate log P value (between -1 and 4) would be favourable for absorption. Generally, liquids, solids in solution and water-soluble dusts would readily diffuse/dissolve into the mucus lining the respiratory tract. Hence, with a logPow of -0.4 and a water solubility of 5.46 g/L, the absorption of this fraction which may not be subjected to ciliary clearance can be considered as rather high.
However, as only a very minor portion of the particles may reach the respiratory tract at all due to physical limitations, which will also further apply in case safety equipment and precautionary measures fail, absorption via the respiratory tract can be practically excluded. In theory, if absorption was not limited by the particle size, a similar absorption rate a via the oral route, i.e. 100%, could be assumed. Due to the physical limitations however, an inhalative absorption of approx. 10% can be assumed as a worst case.

Absorption after dermal exposure
In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 g/mol are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore, if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally, logPow values between 1 and 4 (especially 2 and 3 are optimal) favour dermal absorption. Values below -1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low. Above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and will limit absorption across the skin [ECHA, 2008]. Uptake into the stratum corneum itself may be slow. Moreover vapours of substances with vapour pressures below 100 Pa are likely to be well absorbed and the amount absorbed dermally is most likely more than 10% and less than 100% of the amount that would be absorbed by inhalation. If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. During the whole absorption process into the skin, the compound can be subject to biotransformation.
In case of DIMAU, an evaporation after skin contact does not need to be regarded due to the high melting point and low vapour pressure, and hence it can be assumed that the substance will remain on the skin until mechanical removal. Furthermore, since the substance is not a skin irritant, additional absorption-enhancing effects can be disregarded, too.
The molecular weight of DIMAU is with 155.1546 g/mol rather low, which in general indicates a certain potential to penetrate the skin. Further, with a water solubility of 5.46 g/L at 25°C, DIMAU is considered sufficiently soluble to pass the epidermis. Also, it is most likely not too high to penetrate the stratum corneum and to be absorbed via the skin. This is supported by its partition coefficient. It is with -0.4 at 20°C below the indicative guidance value of 1 – 4, however not below -1, which is considered not to affect its potential to be absorbed in a manner to exclude complete absorption, especially when applied e.g. as a thin layer without mechanical removal.
Therefore, a nearly non-hindered dermal absorption can be assumed, most reasonably a resulting maximum dermal penetration rate of DIMAU of 100% as a worst case, which is compliant with i.a. the European ECHAs Guidance documents [ECHA, 2008] and scientifically reasonable when e.g. performing route-to-route extrapolations during risk assessment.

Details on distribution in tissues:

Distribution
In general, it can be stated that the smaller the molecule, the wider is its distribution. A lipophilic molecule (LogPow >0) is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues. It is not possible to absolutely foresee protein binding, which can limit the amount of a substance available for distribution. In the case of DIMAU, ToxTree modelling [Ideaconsult Ltd, 2004-2013] gave no protein binding alert. The epoxy group of a possible metabolite however was identified to potentially undergo a SN2-nucleophilic aliphatic substitution. Hence, the possible metabolite may be partially retained in the tissue of first impact due to covalent binding, and hence the distribution of DIMAU through the body may be reduced by that amount. However, this metabolite is only formed in theory and at higher rank sites of metabolism, so this possibility should be neglected. Furthermore, if a substance undergoes extensive first-pass metabolism, predictions made on the basis of the physico-chemical characteristics of the parent substance may not be applicable.
In case of DIMAU, no quantitative data is available for distribution patterns. Taking into account its rather low molecular weight of 155.1546 g/mol, its hydrophilicity and high water solubility, the absolute systemic bioavailability is very high, both being able to distribute via the in the aqueous compartment and to pass biological membranes.
After oral exposure, the first target will be the gastrointestinal tract, where the substance and possibly bacterial metabolites will be absorbed in high quantities, most likely close to 100% of the ingested amount, and transferred via the blood stream to the liver.
After reaching the liver via the portal vein, the substance will be further distributed via the bloodstream. Here, especially the kidneys due to their filter function and the heart due to its enormous need for nutrients and consequently large blood flow through coronary arteries will be exposed.
Possible metabolites and degradation products are expected to be of the same or smaller size and at least same or higher hydrophilicity as the parent compound. Hence, similar distribution patterns can be expected and no differentiation between the parent compound and metabolites has to be made regarding distribution. Due to the hydrophilicity, sufficient water solubility and small size, a possible accumulation can be neglected. Since the solubility and hence absorption via the GI tract of DIMAU and its metabolites is rather complete, a high peak exposure to the compound(s) and hence high systemic bioavailability can be expected. However, due to their tendency to be excreted rather fast, a very relevant AUC is not to be expected. This applies to all three possible exposure routes, although the formation of metabolites will be most relevant for the oral route. The affection of the lymphatic system via micellar uptake however is only of minor importance.
These conclusions are based on the physico-chemical properties of DIMAU, and which are also applicable for its metabolites, are furthermore supported by the results of the repeated dose toxicity OECD 408 study. Several minor effects occurred body-wide at 700 mg/kg bw/d, clearly shows that the substance was distributed throughout the body. As a consequence, a wide distribution of the test items throughout the body can be reasonably assumed.

Details on excretion:

Excretion
In general, the major routes of excretion for substances from the systemic circulation are the urine and/or the faeces (via bile and directly from the gastrointestinal mucosa). For non-polar volatile substances and metabolites exhaled air is an important route of excretion. Substances that are excreted favourable in the urine tend to be water-soluble and of low molecular weight (below 300 in the rat) and be ionized at the pH of urine. Most will have been filtered out of the blood by the kidneys though a small amount may enter the urine directly by passive diffusion and there is the potential for reabsorption into the systemic circulation across the tubular epithelium. Substances that are excreted in the bile tend to be amphipathic (containing both polar and nonpolar regions), hydrophobic/strongly polar and have higher molecular weights and pass through the intestines before they are excreted in the faeces and as a result may undergo enterohepatic recycling which will prolong their biological half-life. This is particularly a problem for conjugated molecules that are hydrolysed by gastrointestinal bacteria to form smaller more lipid soluble molecules that can then be reabsorbed from the GI tract. Those substances less likely to recirculate are substances having strong polarity and high molecular weight of their own accord. Other substances excreted in the faeces are those that have diffused out of the systemic circulation into the GI tract directly, substances which have been removed from the gastrointestinal mucosa by efflux mechanisms and non-absorbed substances that have been ingested or inhaled and subsequently swallowed. Non-ionized and lipid soluble molecules may be excreted in the saliva (where they may be swallowed again) or in the sweat. Highly lipophilic substances that have penetrated the stratum corneum but not penetrated the viable epidermis may be sloughed off with or without metabolism with skin cells.
For DIMAU no test data is available regarding its elimination. Concerning the above mentioned behaviour predicted for its metabolic fate, it is unlikely that the parent substance will be excreted unchanged. Nevertheless a similar behaviour regarding excretion can be assumed for the parent compound and most of its estimated metabolites. DIMAU and its estimated degradation products are small, rather hydrophilic and soluble in water. So a very fast excretion of the compounds via the kidneys and so urine can be expected. Excretion via the GI tract (unabsorbed material) and via the bile and consequent subjection to enterohepatic recycling can be neglected.

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

For details, see attached file. Metabolites of the substance are estimated to be formed via amine hydroxylation, N-dealkylation, and epoxydation.

Any other information on results incl. tables

See attached expert statement.

Applicant's summary and conclusion

Conclusions:

The present expert statement covers all relevant toxicokinetic parameters to assess the behaviour of 1,3-Dimethyl-4-aminouracil (DIMAU) in the body, the available information is sufficient to enable one to perform a proper further assessment. Hence, no further information needs to be gathered and further studies can be omitted due to animal welfare. In conclusion, the substance has a minor potential for bioaccumulation in its non-metabolized or metabolized form.

Executive summary:

In order to assess the toxicokinetic behaviour of 1,3-Dimethyl-4-aminouracil (DIMAU), the available toxicological, ecotoxicological and physico-chemical data were evaluated, as toxicokinetic test data is lacking.

The molecular weight of 155.1546 g/mol, a LogPow of -0.4 at 20°C, and a water solubility of 5.46 g/L at 25°C, a high potential for oral absorption is given. The substance is not ready biodegradable and hydrolytically stable, so degradation products do not need to be considered. Taking into account the oral OECD 408 study, a certain absorption of the test compounds is evident, reproduction was impaired. Hence, a preliminary occurrence of oral absorption of the test item after gavage can reasonably deducted. DIMAU is a high melting solid with very low vapour pressure, and max. 0.05% of the particles would reach the body via inhalation as they are otherwise too large to be respirable, which is a negligible amount. Hence, inhalative absorption is not relevant. In case of DIMAU, an evaporation after skin contact does not need to be regarded, and it will remain on the skin until mechanical removal. Since the substance is not a skin irritant, additional absorption-enhancing effects can be disregarded. With the above-mentioned molecular weight, logPow and water solubility, a nearly non-hindered dermal absorption can be assumed.

So in summary, the absorption rates may be estimated to:

-      Absorption via oral route: 100%

-      Absorption via inhalative route: 10%

-      Absorption via dermal route: 100%

Taking into account DIMAU’s rather low molecular weight and its sufficient water solubility, the absolute systemic bioavailability is very high. Similar distribution patterns can be expected for its metabolites and a possible accumulation can be neglected. A high peak exposure to the compound(s) and hence high systemic bioavailability can be expected, but not a very relevant AUC. This is supported by the results of the OECD 408 study, in which minor systemic effects strongly indicate a wide distribution of the test item throughout the body.

The low logPow and sufficient water solubility are clearly indicating that for DIMAU a certain potential for accumulation in the body can be excluded and a rather fast excretion can be expected. So, the potential of DIMAU for bioaccumulation in its classic sense is virtually not existing.

Regarding its metabolic fate, amine hydroxylation, N-dealkylation, and epoxydation were identified as the mode of action during Phase-I-metabolism. The metabolites formed are not expected to modify essentially the molecular weights, physico-chemical properties and hence ADME behaviour of DIMAU. These metabolites are expected to be either excreted directly or to react in Phase II of the biotransformation with different molecules, leading to the formation of conjugations. Hence, it is unlikely that DIMAU will be excreted unchanged. DIMAU and its estimated metabolites are small, charged and hence hydrophilic and very soluble in water. So a very fast excretion of the compounds via the kidneys and so urine can be expected.

In conclusion, DIMAU has a minor potential for bioaccumulation, and will be excreted rapidly after metabolism.