Bis(2,6-diisopropylphenyl)carbodiimide

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

other: Expert statement

Adequacy of study:

key study

Study period:

2011 - 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: An extensive assessment of the toxicological behaviour of bis(2,6-diisopropylphenyl)carbodiimide was performed, taking into account the chemical structure, the available physico-chemical-data and the available (eco-)toxicological data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

no guideline required

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2,6-diisopropylphenyl)carbodiimide

EC Number:

218-487-5

EC Name:

Bis(2,6-diisopropylphenyl)carbodiimide

Cas Number:

2162-74-5

Molecular formula:

C25H34N2

IUPAC Name:

N,N'-bis[2,6-bis(propan-2-yl)phenyl]methanediimine

Details on test material:

not applicable

Radiolabelling:

other: not applicable in this expert statement

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 routes of administration are discussed in the expert statement

Vehicle:

other: not applicable

Details on exposure:

all routes of administration are discussed in the expert statement

Details on study design:

not applicable

Details on dosing and sampling:

not applicable

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Bis(2,6-diisopropylpheny)carbodiimide is favourable for absorption by oral route of exposure due to clear systemic effects observed in repeated dose toxicity studies. Dermal absorption and absorption by inhalation will be limited.

Main ADME results 2

Type:

distribution

Results:

If absorbed, the distribution of the target substance is extensive throughout the body due to the affected organs in the 28-day study.

Main ADME results 3

Type:

excretion

Results:

The hydrolysis products and/or hydroxylated derivatives are expected to be eliminated via the urine (mostly conjugated). Small amounts of the unchanged substance is expected to be excreted via the GI tract or be subject to enterohepatic recycling.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Bis(2,6-diisopropylphenyl)carbodiimide is favourable for absorption, when taking its molecular weight (362.55 g/mol) into account. Its non-existing water solubility (< 0.05 mg/L) and the value for logPow above 4 do not hinder absorption to such an extent, that absorption into the body will not occur (even though logPow between 0 and 4 are favourable for absorption), as hydrolysis occurs and the subacute oral 28-day repeated dose study and the OECD 421 study showed toxic effects.
Regarding oral absorption, bis(2,6-diisopropylphenyl)carbodiimide is expected to be subject to hydrolysis, the final hydrolysis product DIPA is rather favoured for absorption in the small intestine. So available data suggest that orally administered bis(2,6-diisopropylphenyl)carbodiimide will be absorbed.
Regarding absorption in the respiratory tract, as bis(2,6-diisopropylphenyl)carbodiimide has a very low calculated vapour pressure (1.89 E-05 Pa at 25°C), it is clear that there is only a low availability for inhalation. However, the little amount of substance, which might be available for inhalation, is expected to be absorbed directly across the respiratory tract epithelium, due to its lipophilicity.
Regarding dermal absorption, the molecular weight of bis(2,6-diisopropylphenyl)carbodiimide indicates a low potential to penetrate the skin. However, the low vapour pressure can be judged advantageous for dermal uptake. Based on this knowledge, bis(2,6-diisopropylphenyl)carbodiimide is expected to be absorbed following dermal exposure into the stratum corneum and to a limited extent into the epidermis, due to its molecular weight and its logPow.

Details on distribution in tissues:

The distribution of bis(2,6-diisopropylphenyl)carbodiimide is expected to be more extensive in fat tissues than in other tissues, due to its better solubility in octanol than in water (predicted logPow > 6.2). Accordingly, the possibility to reach the central-nerve-system is given. The first target will be the gastrointestinal tract, where the substance and possibly bacterial metabolites will be absorbed in small quantities and transferred via the blood stream to the liver. After first pass metabolism, 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 affected.

Details on excretion:

It is unlikely that the parent substance will be excreted unchanged. However, if unchanged excretion is assumed, based on chemical structure of bis(2,6-diisopropylphenyl)carbodiimide, its molecular weight and its non-existent water solubility, it is unlikely to be excreted via the urine. The excretion, if any, of the parent compound will occur via the gastrointestinal tract (unabsorbed material) and the bile (small amounts of unchanged compound), and it could be subject to enterohepatic recycling. Due to the high lipophilicity excretion is possible via saliva and/or milk, which could endanger the new-born, when repeated exposure occurs.
Concerning the fate of the metabolites formed of bis(2,6-diisopropylphenyl)carbodiimide, the hydroxylated isopropyl groups, in cases linked to glucuronic acid, activated sulphate or activated methionine, could be eliminated via the urine. However, it is likely that 2,6-diisopropylaniline will be linked to glucuronic acid and subsequently excreted via the urine (polar amino-group, lower molecular weight).

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The diisocyanate group is most likely cleaved at the nitrogen-carbon bond, leading to the formation of 2,6-diisopropylaniline (DIPA) and 2,6-diisopropylphenyl isocyanate. The last molecule, however, is unstable and will hydrolyse through 2,6-diisopropylphenyl carbamic acid to 2,6-diisopropylaniline (DIPA).
The formed hydrolysis products will react in phase 2 with different molecules, leading to the formation of conjugations. They will be conjugated to glucuronic acid, activated sulphate or activated methionine.

Any other information on results incl. tables

Background

There is data available on the physico-chemical properties of bis(2,6-diisopropylphenyl)carbodiimide. With the aid of the EPIWIN software some physical-chemical properties were calculated.

The substance is at room temperature a white to light yellowish solid with a slight odour (Rheinchemie, 2011). The substance is insoluble in water (< 0.05 mg/L at 20°C, Erstling, 2008) and has a logPow of > 6.2 (Mullee and White, 2001). It has a very low vapour pressure (≤ 5.8*10E-3 Pa at 20°C, Kintrup, 2012). The melting point has been determined to be 50.97°C (Blaul, 2012) and the decomposition temperature 294°C (Blaul, 2012), respectively. Determination of hydrolysis as a function of pH revealed the substance to be hydrolytically unstable, especially at a low pH and high temperatures, and 2,6-bis(propan-2-yl)aniline was identified as the main hydrolysis product (Čížek, 2012). The substance is, when administered orally to rats toxic (LD50 > 300 mg/kg bw, Gillisson, 2009) and when administered dermally to rats not toxic (LD50 > 2000 mg/kg bw, Driscoll, 2001a). However, when administered orally in a subacute study, toxic effects were evident at low doses (NOAEL = 4 mg/kg, Popp, 2012). Total implantation loss (no implantation cites and no corpora lutea) was the main effect in the Reproduction/Developmental Screening Test (OECD 421, Popp, 2013, Study No. T6084004). NOAEL of 3 mg/kg bw and 1 mg/kg bw were established for systemic and reproduction/developmental toxicity, respectively. It is not an eye (Gmelin, 2010, and Kaufmann, 1993b) or skin irritant (Kaufmann, 1993a), and no skin sensitising properties have been found when tested in the guinea pig maximisation test (Driscoll, 2001b). Additionally, the substance was shown to be not mutagenic in studies according to OECD471 (Herbold, 2009 and Thompson, 2001), 473 (Ciethier, 2011) and 476 (Wollny, 2011).

Absorption

In general, absorption of a chemical is possible, if the substance crosses biological membranes. 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 are favourable for absorption). Generally, the absorption of chemicals which are surfactants or irritants may be enhanced, because of damage to cell membranes.

Bis(2,6-diisopropylphenyl)carbodiimide is favourable for absorption, when taking its molecular weight (362.55 g/mol) into account. Though, the substance is insoluble in water (< 0.05 mg/L), so it is apparent, that its absorption is hindered. The value of the logPow (> 6.2) shows the substance to be better soluble in octanol than in water (positive logPow for lipophilic substances, negative logPow for hydrophilic substances). Its non-existing water solubility (< 0.05 mg/L) and the value for logPow above 4 do not hinder absorption to such an extent, that absorption into the body will not occur (even though logPow between 0 and 4 are favourable for absorption), as hydrolysis occurs and the subacute oral 28-day repeated dose study and the Screening Test showed toxic effects. Bis(2,6-diisopropylphenyl)carbodiimide is not irritating to the skin or to the eyes. Therefore, the above mentioned enhancement of absorption for irritants, does not apply.

In summary, bis(2,6-diisopropylphenyl)carbodiimide will be absorbed into the body.

Absorption from the gastrointestinal tract

Regarding oral absorption, in the stomach, a substance will most likely be hydrolysed, because this is a favoured reaction in the acidic environment of the stomach. This was shown in a hydrolysis study on bis(2,6-diisopropylphenyl)carbodiimide (Čížek, 2012). It was shown that the substance is hydrolysed especially under acidic conditions and high temperatures, i.e. 93% hydrolysis at pH = 4.0 (60°C) after 3.2 days and 82-90% (23°C) after 30 days, respectively. Under these pH conditions, onlyN,N'-bis(2,6-diisopropylphenyl)urea was identifiedas a hydrolysis product in the hydrolysis study. Due to the even lower pH values found in the human stomach it can not be ruled out that this substance is possibly formed in the stomach maybe in relevant amounts. Therefore, this hydrolysis product has to be considered, too. QSAR modelling (Chemservice GmbH, 2012a, b) revealed that this degradation product bears similar physico-chemical properties as its precursor (logPow = 8.79, water solubility 1.25*10^-4mg/L), therefore, the same considerations regarding ADME based on physico-chemical data apply to this compound. Nevertheless, it is rather unstable and decomposes into 2,6-bis(propan-2-yl)aniline (DIPA) and 2,6-diisopropylphenyl isocyanate (DIPI), whereas the latter undergoes easily decarboxylation and will be transformed to DIPA. DIPA is, compared to bis(2,6-diisopropylphenyl)carbodiimide, rather favoured for absorption in the gastrointestinal tract due to its low molecular weight (177,3 g/mol) and logPow of 3.18 (Chemservice GmbH, 2012c). Even though, bis(2,6-diisopropylphenyl)carbodiimide is known to be hydrolysed in the stomach, its retention time in the stomach is rather short compared to its estimated half-life due to hydrolysis. Therefore, bis(2,6-diisopropylphenyl)carbodiimide is the more relevant molecule in the matter of gastrointestinal absorption, and the hydrolysis product DIPA is only relevant to a minor extent.

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.

Although the available physico-chemical and acute toxicity data suggest that orally administered bis(2,6-diisopropylphenyl)carbodiimide will be poorly absorbed, based on its non-solubility in water and its high logPow, it can be assumed from toxic effects in the 28-day subacute study (oral gavage, Popp, 2012) and in the Screening Test (oral, gavage, Popp, 2013), that absorption (and accumulation) occurs. Taking into account the affected organs after a subacute gavage of 16 mg/kg bis(2,6-diisopropylphenyl)carbodiimide, i.e. heart, white blood cells and lymphoid organs, gastro-intestinal tract, liver, kidney and female genital tract, an absorption via passive diffusion and consequent distribution is very likely.

Furthermore, since lymphoid organs are affected, absorption as micelles also seems to be likely. Here, a substance enters the lymphatic system while bypassing the liver and can therefore reach the target organs without being metabolized (see "Metabolism").

Absorption from the respiratory tract

Concerning absorption in the respiratory tract, any gas or vapour has to be sufficiently lipophilic to cross the alveolar and capillary membranes (moderate logPow values between 0-4 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. Very hydrophilic substances can be absorbed through aqueous pores (for substances with molecular weights below and around 200) or be retained in the mucus.

Bis(2,6-diisopropylphenyl)carbodiimide has a very low calculated vapour pressure (1.89 E-05 Pa at 25°C), which indicates a low availability for inhalation. Additionally the high logPow indicates no absorption though aqueous pores.

Based on this data, it is speculated that bis(2,6-diisopropylphenyl)carbodiimide is not available in the air for inhalation, due to its really low vapour pressure. However, if any amount of substance is available for inhalation, it is expected to be absorbed directly across the respiratory tract epithelium, due to its lipophilicity.

Absorption following 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 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 favour dermal absorption (values between 2 and 3 are optimal; TGD, Part I, Appendix VI). 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. 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 bis(2,6-diisopropylphenyl)carbodiimide, the molecular weight is above 100 and below 500, which indicates a low potential to penetrate the skin. Additionally, the low vapour pressure can be judged advantageous for dermal uptake. Based on this knowledge, bis(2,6-diisopropylphenyl)carbodiimide is expected to be absorbed following dermal exposure into the stratum corneum and to a limited extent into the epidermis, due to its molecular weight and its logPow. However, the systemic toxicity of bis(2,6-diisopropylphenyl)carbodiimide via the skin is assumed to be low and this has been shown with the results of the acute dermal toxicity study, which showed no mortality after dermal application of 2000 mg/kg bw in rats. Nevertheless, toxic effects (possibly due to accumulation) after chronic dermal exposure cannot be completely excluded. The contribution of metabolites to the substance’ toxicity cannot be absolutely quantified and therefore, contribution of the parent substance to the toxicity remains unknown.

Distribution

In general, the following principle applies: the smaller the molecule, the wider the 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’s not possible to foresee protein binding, which can limit the amount of a substance available for distribution. 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 bis(2,6-diisopropylphenyl)carbodiimide, no quantitative data is available for distribution patterns. In general, the distribution of bis(2,6-diisopropylphenyl)carbodiimide is expected to be more extensive in fat tissues than in other tissues, due to its better solubility in octanol than in water (predicted logPow > 6.2). Accordingly, the possibility to reach the central-nerve-system is given.Taking into account the affected organs in a subacute study, i.e. heart, white blood cells and lymphoid organs, gastro-intestinal tract, liver, kidney and female genital tract, and the assumed absorption via passive diffusion, the following systemic distribution is likely: The first target will be the gastrointestinal tract, where the substance and possibly bacterial metabolites will be absorbed in small quantities and transferred via the blood stream to the liver. After first pass metabolism (see "Metabolism"), 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 affected. Additionally, accumulation (see "Accumulation") in all directly and indirectly (general supply via bloodstream) involved organs has to be considered. Furthermore, the affection of the lymphatic system via micellar uptake must also be taken into consideration.

Accumulation

It is also important to consider the potential for a substance to accumulate or to be retained within the body. Lipophilic substances have the potential to accumulate within the body (mainly in the adipose tissue), if the dosing interval is shorter than 4 times the whole body half-life. Although there is no direct correlation between the lipophilicity of a substance and its biological half-life, substances with high logPow values tend to have longer half-lives. On this basis, there is the potential for highly lipophilic substances (logPow >4) to accumulate in biota which are frequently exposed. Highly lipophilic substances (logPow between 4 and 6) that come into contact with the skin can readily penetrate the lipid rich stratum corneum but are not well absorbed systemically. Although they may persist in the stratum corneum, they will eventually be cleared as the stratum corneum is sloughed off. A turnover time of 12 days has been quoted for skin epithelial cells.

Accordingly, the predicted logPow of > 6.2 and the experimentally determined water solubility (< 0.05 mg/L) of bis(2,6-diisopropylphenyl)carbodiimide indicate a potential for accumulation in the body. Additionally, the effects observed in the subacute study in rats (Popp, 2012) and in the Screening Test (Popp, 2013) indicate a slow metabolism (> 24 h), because already low doses (16 mg/kg bw in the 28-day study and 8 mg/kg bw (later reduced to 5 mg/kg bw) in the OECD 421 study) showed significant signs of toxicity after repeated exposures. NOAEL of 4 and 3 mg/kg bw were established in the 28-day and in the OECD 421 study, respectively. The half-life was determined in the hydrolysis study (11.75 and 1.76 days at pH 4 and 23°C and 50°C, respectively), therefore, the accumulation of bis(2,6-diisopropylphenyl)carbodiimide and/or its metabolites, is rather likely. Although absorption of only small quantities is likely, its accumulative potential seems to increase its risk for harmful health effects significantly.

Metabolism

Route specific toxicity results from several phenomena, such as hydrolysis within the gastrointestinal or respiratory tracts, also metabolism by gastrointestinal flora or within the gastrointestinal tract epithelia (mainly in the small intestine), respiratory tract epithelia (sites include the nasal cavity, tracheo-bronchial mucosa [Clara cells] and alveoli [type 2 cells]) and skin.

First of all, partial hydrolysis is the first occurring modification of bis(2,6-diisopropylphenyl)carbodiimide in the stomach, because of the acidic environment and elevated temperature (approx. 37°C) in the stomach. The substance was determined to be hydrolytically unstable at low pH and elevated temperatures (Čížek, 2012). However, hydrolysis rate was slow at acidic conditions: a rate constant of 0.0685/day and DT50 of 10.13 days at pH 4 was determined at 25°C. The percentage of metabolised parent substance was 6-9% during 6 days at 50°C. It can be expected that a faster hydrolysis rate will occur at lower pH (pH in the stomach is between 1.5 and 3.5). The major degradation rate products (at least >= 10 % occurrence) were identified in the hydrolysis study: 2,6-diisopropylaniline (CAS 24544-04-5) with molecular weight of 177, and Urea, N,N´-bis[2,6-bis(1-methylethyl)phenyl] (CAS 76460-94-1) with molecular weight of 380.

 

Additionally, the target substance’ and its hydrolysis products are very likely to be metabolized via the Cytochrome P450 group of metabolising enzymes, as it has been predicted with the TOXTREE modelling tool and the OECD QSAR Toolbox (v3.1, 2013) (Chemservice, 2011 & 2012). There, the chemical and its main hydrolysis product 2,6-diisopropylaniline have been identified to bear primary, secondary, tertiary and quaternary sites and 4 or more sites for metabolism by the Cytochrome P450 group of metabolising enzymes. For bis(2,6-diisopropylphenyl)carbodiimide, the primary sites of metabolism are the isopropyl-groups, which are predicted to be subject to aliphatic hydroxylation. The secondary and tertiary sites of metabolism are the carbon-atoms of the heterocyclic cycles, which are predicted to be subject to aliphatic hydroxylation. The quaternary sites of metabolism were also identified to be the isopropyl-groups, which are predicted to be subject to aliphatic hydroxylation. So the 4 isopropyl-groups and the carbon-atoms of the ring are likely to be oxidised by cytochrome P450 enzymes, as already identified via TOXTREE modelling, yielding hydroxyl-groups.

For 2,6-diisopropylaniline, the primary site of metabolism is the amine, which is predicted to be subject to amine hydroxylation. The secondary site of metabolism is the carbon atom inp-position of the aromatic ring, which is predicted to be subject to aromatic hydroxylation. The tertiary sites of metabolism are the carbon-atoms of the isopropyl-groups, which are predicted to be subject to aliphatic hydroxylation. The sites of metabolism with Rank>=4 were also identified to be the carbon-atoms in m-position of the aromatic ring, which are predicted to be also subject to aliphatic hydroxylation.

As determined in the hydrolysis study (Čížek, 2012), the carbodiimide group of bis(2,6-diisopropylphenyl) carbodiimide is likely cleaved at the nitrogen-carbon-bond, leading to the formation of 2,6-diisopropylaniline (DIPA, substance 1) and 2,6-diisopropylphenyl isocyanate (substance 2). The last molecule, however, is unstable and will hydrolyse (releasing carbon dioxide) to 2,6-diisopropylphenyl carbamic acid (substance 3). Carbamic acid derivatives are unstable compounds and are known to decompose to a corresponding amine releasing carbon dioxide (Morrison and Boyd, 1987). Therefore, it is likely that 2,6-diisopropylphenyl carbamic acid (substance 3) will decarboxylate to DIPA (substance 1). This was confirmed by the profiling results of the OECD QSAR Toolbox (v3.1). Four hydrolysis products are predicted for the target substance:

Hydrolysis products^1
Substance 1: 2,6-di(propan-2-yl)aniline	Substance 2: 2-isocyanato-1,3-di(propan-2-yl)benzene	Substance 3: [2,6-di(propan-2-yl)phenyl]carbamic acid	Substance 4: Carbon dioxide

¹(obtained by Hydrolysis simulator under acidic conditions, Toolbox, v3.1, 2013)

 

Amines reacting with isocyanates form urea linkage (Screening assessment for TDIs, 2008). In this case, it is very likely that DIPA (substance 1) reacts with 2,6-diisopropylphenyl isocyanate (substance 2) yielding Urea, N,N´-bis[2,6-bis(1-methylethyl)phenyl] (CAS 76460-94-1), identified in the hydrolysis study.

The cleavage of the benzene ring is rather unlikely in mammalians, leading to the formation of either methyl groups or methyl groups and 2R-CHOH group.

The above mentioned functional groups will react in phase 2 of the biotransformation with different molecules, leading to the formation of conjugations. For the hydroxyl-groups of the ring it is most likely that they will be conjugated to glucuronic acid, activated sulphate or activated methionine.

Additionally, since a certain potential for absorption (see "Absorption") ofbis(2,6-diisopropylphenyl)carbodiimide is indicative, the substance will not only accumulate in the affected organs directly after absorption, but also, and possibly to a larger extent, in fatty tissues (see "Accumulation"). Here, the distribution of metabolizing enzymes is rather low, CYP2 and 3 expression is nearly undetectable (Ellero, 2010). In the cytoplasm of the adipocytes degradation due to hydrolysis might also take place. Taking into account the experimental results under physiologically relevant pH-conditions, the compounds half-life at 37°C can be estimated to be 10-12 days. Since the final hydrolysis product, 2,6-diisopropylaniline, has a logPow of 3.18 and therefore does not tend to accumulate as extensively as bis(2,6-diisopropylphenyl)carbodiimide, secondary effects of poisoning due to DIPA can occur. This was confirmed by the results of the subacute study, as the affected organs are those which are affected after exposure via the blood stream. Toxic effects resulting from the hydrolysis product 2,6-diisopropylphenyl isocyanate (substance 2)., e.g. via addition on biomolecules, could also be possible. In this context, the formed 2,6-diisopropylphenyl isocyanate can bind to proteins by the mechanism of acyl transfer (via nucleophilic addition reaction, Toolbox, v3.1, 2013). Certainly, the depletion of the target substance to the amine under CO₂release is the most likely reaction, and an addition to amines would result in urea derivatives, which were already shown to be unstable (Morrison and Boyd, 1987). Addition to an alcohol, on the other hand, would result in a carbamate, which is rather stable.

In conclusion, it is most likely that the substance of interest will be subject to metabolism by cytochrome P450 enzymes and subsequent glucuronidation. In addition, the diisocyanate-group will be cleaved and the resulting 2,6-diisopropylphenyl isocyanate hydrolysed through 2,6-diisopropylphenyl carbamic acid to 2,6-diisopropylaniline.

 

Excretion

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 gastrointestinal 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 gastrointestinal 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 bis(2,6-diisopropylphenyl)carbodiimide no data is available concerning its elimination. Concerning the above mentioned behaviour predicted for its metabolic fate, it is unlikely that the parent substance will be excreted unchanged. However, if unchanged excretion is assumed, based on the chemical structure of bis(2,6-diisopropylphenyl)carbodiimide, its molecular weight and its non-existent water solubility, it is unlikely to be excreted via the urine. The excretion, if any, of the parent compound will occur via the gastrointestinal tract (unabsorbed material) and the bile (small amounts of unchanged compound), and it could be subject to enterohepatic recycling. Due to the high lipophilicity excretion is possible via saliva and/or milk, which could endanger the new-born, when repeated exposure occurs.

Concerning the fate of the metabolites formed of bis(2,6-diisopropylphenyl)carbodiimide, the hydroxylated isopropyl-groups, in cases linked to glucuronic acid, activated sulphate or activated methionine, will be eliminated via the urine. However, it is likely that 2,6-diisopropylaniline will be linked to glucuronic acid and subsequently excreted via the urine (polar amino-group, lower molecular weight).

Conclusion

In order to assess the toxicological behaviour of bis(2,6-diisopropylphenyl)carbodiimide, the available and predicted physico-chemical and toxicological data have been evaluated. The substance is expected to be poorly absorbed after oral exposure, based on its low molecular weight, its low water solubility and its logPow of >6.2. In the stomach however, as the substance is known to hydrolyse under acidic conditions, hydrolysis occurs and therefore the smaller hydrolysis product DIPA can be absorbed in the small intestine (where pH is higher and DIPA is no longer protonated). Concerning the absorption after exposure via inhalation, as the chemical has really low calculated vapour pressure and a high boiling point (294°C), it is clear, that the substance has a low availability for inhalation. Given its lipophilicity (logPow of >6.2), if any of the substance is available for inhalation, it is expected to be absorbed directly across the respiratory tract epithelium. Bis(2,6-diisopropylphenyl)carbodiimide is expected to be absorbed following dermal exposure into the stratum corneum to a certain extent and into the epidermis, due to its molecular weight and its logPow. However, the systemic toxicity via the skin is assumed to be low, which can be concluded from the results of the acute dermal toxicity study, which showed no mortality after dermal application of 2000 mg/kg bw in rats. The substance might bear accumulative potential after repeated exposures (i.e. via entero-hepatic recycling) and possibly subsequent poisoning from this depot by DIPA formation, which might be the reason for the toxic effects seen in the subacute study. Bis(2,6-diisopropylphenyl)carbodiimide is expected to be extensively metabolised via the Cytochrome P450 group of metabolizing enzymes. A likely metabolite is 2,6-diisopropylaniline, which is formed after cleavage of the diisocyanate group leading to the formation of the unstable 2,6-diisopropylphenyl isocyanate, which hydrolyses through 2,6 -diisopropylphenyl carbamic acid to 2,6-diisopropylaniline. 2,6-diisopropylaniline will be eliminated mainly via the urine, either without or after conjugation to glucuronic acid, activated sulphate or activated methionine.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: expert statement indicates a potential for bioaccumulation
An extensive assessment of the toxicological behaviour of bis(2,6-diisopropylphenyl)carbodiimide was performed (expert statement), taking into account the chemical structure, the available physico-chemical-data and the available (eco-)toxicological data.

Executive summary:

In order to assess the toxicological behaviour of bis(2,6-diisopropylphenyl)carbodiimide, the available and predicted physico-chemical and toxicological data have been evaluated. The substance is expected to be poorly absorbed after oral exposure, based on its low molecular weight, its low water solubility and its logPow of >6.2. In the stomach however, as the substance is known to hydrolyse under acidic conditions, hydrolysis occurs and therefore the smaller hydrolysis product DIPA can be absorbed in the small intestine (where pH is higher and DIPA is no longer protonated). Concerning the absorption after exposure via inhalation, as the chemical has really low calculated vapour pressure and a high boiling point (294°C), it is clear, that the substance has a low availability for inhalation. Given its lipophilicity (logPow of >6.2), if any of the substance is available for inhalation, it is expected to be absorbed directly across the respiratory tract epithelium. Bis(2,6-diisopropylphenyl)carbodiimide is expected to be absorbed following dermal exposure into the stratum corneum to a certain extent and into the epidermis, due to its molecular weight and its logPow. However, the systemic toxicity via the skin is assumed to be low, which can be concluded from the results of the acute dermal toxicity study, which showed no mortality after dermal application of 2000 mg/kg bw in rats. The substance might bear accumulative potential after repeated exposures (i.e. via entero-hepatic recycling) and possibly subsequent poisoning from this depot by DIPA formation, which might be the reason for the toxic effects seen in the subacute study. Bis(2,6-diisopropylphenyl)carbodiimide is expected to be extensively metabolised via the Cytochrome P450 group of metabolizing enzymes. A likely metabolite is 2,6-diisopropylaniline, which is formed after cleavage of the diisocyanate group leading to the formation of the unstable 2,6-diisopropylphenyl isocyanate, which hydrolyses to 2,6-diisopropylaniline. 2,6-diisopropylaniline will be eliminated mainly via the urine, either without or after conjugation to glucuronic acid, activated sulphate or activated methionine.