1,4-dioxacyclohexadecane-5,16-dione

Endpoint summary

Description of key information

Experimental toxico-kinetic data are lacking for assessing adsorption, distribution, and excretion of the substance. Based on the molecular structure, physico-chemical properties, the presence minor and non-adverse effects seen in the human health toxicity studies and physico-chemical parameters Zenolide is expected to be readily absorbed via the oral and inhalation route and somewhat lower via the dermal route. Using the precautionary principle for route to route extrapolation the final absorption percentages derived are: 100% oral absorption, 100% dermal absorption and 100% inhalation absorption.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

In vitro rat plasma stability test with Zenolide

A study on the stability of Zenolide (CRL, 2019) in rat plasma has been performed. Stability of Zenolide was evaluated in rat plasma in two separate experiments in triplicate using LC-MS/MS. In the first preliminary experiment, rat plasma was spiked with 1-50 µg/ml Zenolide and incubated at 37°C and no Zenolide could be detected. Thereafter, Zenolide was also incubated with denaturated proteins (thus without carboxyl esterases breaking down the d-ester bond) and Zenolide could be detected. Further work was carried out to see if the metabolisation of Zenolide could be slowed down. In the next experiment Zenolide was incubated at 500 µg/ml on ice (0°C) and there it could be seen that Zenolide disappeared within 1 minutes (<1% remaining) in plasma. In this test a metabolite appeared on the LC-MS detector. To further follow the pathway of this intermediate metabolite, another test was carried out. In this third experiment 100 µg/ml Zenolide was incubated in rat plasma at 37°C. Here it was shown that this intermediate metabolite disappeared with a half-life of 20 minutes. Though this intermediate has not been characterized, it has almost the same molecular weight as Zenolide. Therefore, it is hypothesized that this intermediate is a cleaved Zenolide on one ester bond as is shown in Figure 1 in the toxico-kinetics statement.

Toxico-kinetics statement

Introduction

Zenolide (Cas no 54982-83-1) is a cyclic aliphatic double ester. It is a clear colourless liquid with a molecular weight of 256 that does not preclude absorption. The test material is likely to hydrolyse in alkaline conditions rather than in acidic conditions because it is an ester. The substance has a low volatility of 0.028 Pa.

Absorption

Oral: The acute oral LD50 of 4500 mg/kg bw and the adverse effects of the oral Reproduction/Developmental Toxicity Screening Test (Repro-screen, OECD TG 421) of the substance shows that it is being absorbed by the gastro-intestinal tract following oral administration. The relatively low molecular weight and the moderate octanol/water partition coefficient (Log Kow 3.65) and water solubility (75 mg/l) would favour absorption through the gut. According to Martinez and Amidon (2002) the optimal log Kow for oral absorption falls within a range of 2-7. Based on this full oral absorption is expected.

Complete absorption (100%) is supported with the kidney effects showing Oxalic acid nephropathy, including Calcium oxalate crystals, found in the Repro-screen study (OECD TG 421), which can be fully contributed to the exposure of Ethylene glycol (EG) as is presented below.

In this Repro-screen study with Wistar rats, EG type of Oxalic acid nephropathy was seen after oral exposure to Zenolide (MW 256) at 1000 mg/kg bw (almost 3.9 mMol), which is equivalent to 242 mg/kg bw EG (3.9 mMol, MW 62). In EG studies with Wistar rats this Oxalic acid nephropathy is seen at 300 mg/kg bw (4.8 mMol), while the NOAEL in EG studies with Wistar rats is 150 mg/kg bw (2.4 mMol) (Cruzan et al., 2004). The NOAEL – LOAEL for Zenolide, 1.2 – 3.9 mMol is in the overlapping range of the NOAEL – LOAEL for EG: 2.4 – 4.8 mMol. Comparing this EG range with Zenolide it can be seen that surely at 1.2 mMol EG no effects are seen but surely at 4.8 mMol.

Based on this reasoning above the oral absorption conclusion is that Zenolide’s systemic effects can be fully attributed to EG and that 100% absorption occurred. This is further supported in the metabolic fate paragraph below. In the Zenolide Repeated dose study (OECD TG 407) with Sprague Dawley rats no EG type of Oxalic acid nephropathy was seen after oral exposure to 1000 mg/kg/bw. This discrepancy in rat strain difference is further discussed in the repeated dose toxicity endpoint summary and read-across justification.

Skin: Based on the physico-chemical characteristics of the substance, being a liquid, its molecular weight (256), log Kow (3.65) and water solubility (75), indicate that (some) dermal absorption is likely to occur. The optimal MW and log Kow for dermal absorption is <100 and in the range of 1-4, respectively (ECHA guidance, 7.12, Table R.7.12-3). Zenolide is just outside optimal range and therefore the skin absorption is not expected to exceed oral exposure. The oral absorption is set to 100% and therefore also the dermal absorption will be set to 100% to be conservative and not to underestimate the exposure via the dermal route.

Lungs: Absorption via the lungs is also indicated based on these physico-chemical properties. Though the inhalation exposure route is thought minor, because of its low volatility (0.028 Pa), the octanol/water partition coefficient (3.65), indicates that inhalation absorption is possible. The plasma/air (BA) partition coefficient is another partition coefficient indicating lung absorption. Buist et al. (2012) have developed BA model for humans using the most important and readily available parameters:

Log PBA = 6.96 – 1.04 Log (VP) – 0.533 (Log) Kow – 0.00495 MW.

For Zenolide the B/A partition coefficient would result in:

Log P (BA) = 6.96 – 1.04 x (-1.55) – 0.533 x 3.65 – 0.00495 x 256 = 5.4

This means that Zenolide has a tendency to go from air into the plasma. It should, however, be noted that this regression line is only valid for substances which have a vapour pressure >100 Pa. Despite the substance being somewhat out of the applicability domain and the exact BA may not be fully correct, it can be seen that the substance will be readily absorbed via the inhalation route and therefore inhalation absorption will be set similarly to oral absorption, which is 100%.

Metabolism

Zenolide is a di-ester connected with a long alkyl chain. The cleavage of the ester bonds will occur by carboxyl esterases present in gut, liver and plasma as is presented in Toxicological handbooks, Belsito et al., 2011, which use JECFA reports on ester metabolism and also Saghir et al (1997), which have tested fatty acid esters in the gut and in plasma show half-lives < 1 minute. This means that Zenolide is fully metabolised into Ethylene glycol (EG,(CAS #107-21-1)) and Dodecanedioc-acid (DDDA,Cas #693-23-2) as is depicted in Fig. 1. This ester cleavage is supported with an experimental in vitro study in rat plasma at 37°C in which Zenolide could not be detected in the presence of carboxyl esterases but could be detected in absence of these esterases (see in vitro rat plasma study). In this experimental study an intermediate is seen also depicted in Figure 1.

 

Figure 1: Zenolide and its Phase 1 metabolism in which Zenolide is metabolised into the short living hypothesized intermediate, Ethylene glycol, and Dodecanedioc acid.

 

Beside this metabolic information it can be seen from the repeated dose toxicity studies with Zenolide that Oxalic acid nephropathy can be fully contributed to EG and its metabolite Oxalic acid. This is because the same effects were observed in the Repro-screen study (OECD TG 421) with Zenolide at doses which can be converted back to EG (see oral absorption paragraph). According to ATSDR (2010) and Fowles et al (2017) the EG metabolism into Calcium oxalate crystals can be depicted in Figure 2. The other metabolite, Dodecanedioic acid is a saturated fatty acid and will be further metabolised by beta-oxidation and consumed in the Krebs cycle (Dodecanedioic-acid, ECHA site, 2019).

Figure 2: Metabolic pathway of Ethylene glycol as presented in ATSDR (2010).

 

Distribution

Zenolide will not be distributed in the systemic circulation as such because Zenolide is not stable in plasma, based on theoretical consideration and experimental information in a rat in vitro plasma study and the Oxalic acid nephropathy, which can be contributed to EG.

Excretion

Zenolide will not be excreted as such because it is completely absorbed and metabolised.

Conclusion

Zenolide is expected to be fully absorbed, orally, dermally and via inhalation, based on the human toxicological information and physico-chemical parameters.

Oral to dermal extrapolation: Based on the systemic kidney toxicity in the Repro-screen (OECD TG 421) it can be assumed that Zenolide is completely absorbed in the gastro-intestinal tract. In view of Zenolide’s instability in plasma (< 1 minute) the metabolites causing the toxicity via the oral route will also cause the toxicity via the dermal route.

The overriding principle will be to avoid situations where the extrapolation of data would underestimate toxicity resulting from human exposure to a chemical by the route-to-route extrapolation and therefore it will be assumed that the oral absorption will be equal to dermal absorption, which is 100% dermal absorption in the present case.

Oral to inhalation extrapolation: Though Zenolide is not a volatile substance, inhalation exposure is conservatively calculated. In view of Zenolide’s instability in plasma (< 1 minute) the metabolites causing the toxicity via the oral route will also cause the toxicity via the inhalation route.

The overriding principle will be used to avoid situations where the extrapolation of data would underestimate toxicity resulting from human exposure to a chemical by the route-to-route extrapolation. Therefore, it will be assumed that the oral absorption will be equal to inhalation absorption, which is 100%.

References

- Agency for Toxic Substances and Disease Registry (ATSDR), Toxicological Profile For Ethylene Glycol, 2010,https://www.atsdr.cdc.gov/toxprofiles/tp96.pdf.

- Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M.L., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H, Sipes, I.G, THE RIFM EXPERT PANEL, 2011, Food and Chemical Toxicology, 49, S219–S241

- Buist, H.E., Wit-Bos­ de, L., Bouwman, T., Vaes, W.H.J., 2012, Predicting plasma:air partition coefficient using basis physico-chemical properties, Regul. Toxicol. Pharmacol., 62, 23-28. 

- Charles River Laboratory (CRL), 2019, Feasibility assessment of Zenolide in EDTA Rat Plasma, Study report, 20180030.

- Fowles, J., Banton, M., Klapacz, J., Shen, H., 2017, A toxicological review of the ethylene glycol series: Commonalities and differences in toxicity and modes of action, Toxicology Letters, 278, 66–83,https://www.sciencedirect.com/science/article/pii/S0378427417302345

- Martinez, M.N., And Amidon, G.L., 2002, Mechanistic approach to understanding the factors affecting drug absorption: a review of fundament, J. Clinical Pharmacol., 42, 620-643.

- REACH dossier Dodecanedioic acid: https://echa.europa.eu/nl/registration-dossier/-/registereddossier/14886

- Saghir. M., Werner, J., Laposta, M., 1997, Rapid in vivo hydrolysis of fatty acid ethyl esters, toxic nonoxidative ethanol metabolites, Am. J. Physiol., 273, G184-G190.