1,4-dioxacyclohexadecane-5,16-dione

Endpoint summary

Description of key information

Key information for the parent compound

Zenolide did not show systemic toxic effects in an oral gavage OECD TG 407 study (GLP) with Sprague Dawley rats in which a NOAEL ≥1000 mg/kg bw/day was derived.

Zenolide did show systemic toxic effects in an oral OECD 421 study (GLP) with Wistar rats in which a NOAEL of 300 and 1000 mg/kg bw/day was derived for male and female rats, respectively.

Zenolide is expected to have a NOAEL of 300 mg/kg bw in a chronic study because its toxicity can be based on its key metabolite Ethylene glycol.

Key information for the metabolites

Ethylene glycol did show systemic toxic effects in an oral 90 -day toxicity study in mice (Similar to OECD 408, GLP) in which NOAELs of 3000 and 12000 mg/kg bw/day were derived for male and female mice, respectively.

Literature data revealed that Ethylene glycol did show systemic toxic effects in an oral 90 -day toxicity study in Wistar rats in which a NOAEL of 150 mg/kg bw/day was derived. In another study with F344 rats, the NOAEL was ca. 3 times higher. The 90-day NOAEL can be extrapolated to a chronic value because NOAELs for EG were the same independent from the repeated dose toxicity duration.

Information from the ECHA website showed that Dodecanedioic acid did not show toxic effect in an OECD TG 422 and in an OECD TG 408 study. In both tests, a NOAEL of ≥1000 mg/kg bw was derived.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available studies were all considered reliable and the overall conclusion can be considered adequate for assessment of the toxicological effects after repeated oral exposure.

System:

urinary

Organ:

kidney

Additional information

For repeated dose toxicity several sources of information are available. Data on Zenolide, Ethylene glycol and Dodecanedioic-acid are presented first, followed by a discussion on DNEL derivation and at the end of the section the read across justification is outlined.

Essential Toxico-kinetics information: As discussed in the toxico-kinetics section and as observed in the in vitro test with rat blood, Zenolide will be fully metabolised into Ethylene glycol and Dodecanedioic acid and in the GI tract, lungs, and skin because of the presence of the carboxyl-esterases in those tissues. The systemic toxicity will therefore be caused by the metabolites of Zenolide. Repeated dose toxicity studies on the metabolites Ethylene Glycol and Dodecanedioic-acid have been taken into account to assess the overall systemic toxicity of Zenolide.

Zenolide: Key study to cover the repeated dose toxicity for 28-days

In a 28-days repeated dose toxicity study (OECD 407, GLP) with a 2-week recovery period, the test substance was administered orally to Sprague-Dawley rats at dosages of 15, 150, 400 or 1000 mg/kg bw/day. Control animals received the vehicle, corn oil, alone.

Results: No adverse effects considered to be associated with treatment were observed for mortality, clinical signs, bodyweight, food consumption, efficiency of food utilisation, haematology, and urinalysis. Lower glutamic oxalacetic transaminase levels were noted in females receiving 1000 or 400 mg/kg bw/day and males receiving 1000 mg/kg bw/day. However, these findings are not considered to be of toxicological importance as these reduced plasma levels are not considered to be associated with cell damage. These findings were also not apparent at day 16 of recovery. Slight increases in plasma sodium and chloride levels were seen among females receiving 1000 or 400 mg/kg bw/day. Also these findings were not apparent at recovery and are not considered to be of toxicological importance.

Organ effects: Relative kidney weights were statistically significantly increased for females receiving 1000 or 400 mg/kg bw/day in comparison with controls. A similar finding was apparent for males at these dosages but not statistically significant different. These increases were not apparent following the recovery period. In addition, there were no macroscopic or histopathological changes associated with treatment, hence these findings are not considered to be of toxicological importance. As there were no adverse treatment-related findings in this study, it is concluded that 1000 mg/kg bw/day, the highest dose tested, represents the NOAEL in the rat for Zenolide when administered orally for at least 28 days.

Zenolide OECD TG 421 study: Key study for the NOAEL derivation for repeated dose toxicity

Introduction: The potential adverse effects of the test material on reproduction including offspring development were studied in an OECD 421 Reproduction/Developmental Toxicity Screening Test following GLP. The test material was administered by gavage to three groups, each of ten male and ten female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of 10 rats/sex received the same dose volume (5 mL/kg/day) of the vehicle (Corn oil). Males were treated for 28 days, i.e. 2 weeks prior to mating, during mating and up to termination. Females were treated during 2 weeks prior to mating, during mating, during gestation and up to the day before necropsy (i.e. on day 13 of lactation for females that delivered.

Method: Mortality, clinical signs, body weight, food consumption, estrous cycle determination, measure thyroid hormone T4, macroscopy at termination, organ weights and histopathology on a selection of tissues were determined. In addition, the following reproduction/developmental parameters were determined: mating and fertility index, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy).

Mortality: There was no compound-related mortality.

Clinical signs: Compound-related clinical signs were restricted to higher incidences (animals and/or occasions) of hypersalivation, abnormal foraging and pedalling for both males and females in all treated groups generally in a dose-related manner during all phases of the study, including gestation and lactation, compared with sporadic cases in the control group.

Body weight: At 1000 mg/kg bw/day, overall mean body weight gain was slightly lower for the males during the dosing period (Days 1 to 29) and for the females during the pre-mating period (Days 1 to 15) compared with the control. However, in the absence of any effect on absolute mean body weight through to termination for either sex, the differences were considered to be of no toxicological significance. There was no compound-related effect on mean food consumption for either sex in any group.

T4: No biologically relevant differences in total T4 levels were noted among the different groups of F0-males.

Pathology findings at 1000 mg/kg bw/day included irregular surface of the kidneys (bilateral), on a few occasions accompanied by bilateral enlarged appearance, and increased absolute and relative mean kidney weights that correlated histologically with minimal to moderate interstitial inflammation, cortical tubular degeneration/regeneration with tubular dilatation accompanied by multifocal intratubular crystals for males. Microscopic examination with polarised light showed presence of intratubular translucent crystals of Calcium oxalate. These kidney effects and the crystals are expected to be the result of one Phase 1 metabolite of Zenolide: Ethylene glycol, which further metabolised into Oxalic acid (third metabolite). The overall term for this toxicity will be Oxalic acid nephropathy. The formation of these crystals is rat species dependent and Wistar rats (used in the present study and mainly males) are more sensitive compared with other rat strains. This crystal formation considered for the determination of the NOAEL. Only minor increases in mean kidney weights were noted at 300 or 100 mg/kg bw/day but without features of Oxalic acid nephropathy.

Results on fertility: There were no compound-related effects on estrous cycles, mating performance or fertility, including mean sperm counts (testis and cauda epididymis), sperm motility and sperm morphology. There were 9/9, 9/9, 8/9 and 8/10 pregnant females in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively, that completed delivery. The mean duration of gestation was comparable. Two pregnant females in the 1000 mg/kg bw/day group were unable to complete delivery and were sacrificed for ethical reasons. There were no test item-related effects on the number of implantation, pre-birth loss and litter size.

Results on developmental toxicity: All females that delivered had liveborn pups. There were no test item-related effects on pup viability or pup body weight. There was no compound-related effect on male areola/nipple retention in any group. No nipples were observed for any male in any group on PND13. There was no compound-related effect on anogenital distance (normalized for body weight) for the male or female pups. No biologically relevant differences in total T4 levels were noted among the different groups of PND 13 pups.

Conclusion: The NOAEL for parental toxicity was 300 and 1000 mg/kg bw/day for males and females, respectively, due to the presence and absence of compound-related Oxalic acid nephropathy, respectively. The high dose of 1000 mg/kg bw/day was the reproduction and developmental NOAEL for both sexes.

Ethylene Glycol (EG) repeated dose toxicity information covering sub-chronic and chronic exposure for Zenolide:

For one of the two key metabolite: Ethylene glycol, two studies are used for assessing repeated dose toxicity for Zenolide. These two studies are used because

1) The NTP study covers the OECD TG 408 endpoints, and

2) From the sub-chronic study by Cruzan et al. (2004) study it can be concluded that the Wistar strain is more sensitive compared to the F344 rat strain (and other rat strains). Furthermore, from all available studies on EG this study from Cruzan has the lowest reliable NOAEL and LOAEL for EG, and this study has no confounding factors according to the ATSDR and can be used as such. A complete overview of all available data is presented by the ATSDR (2010) and Fowles et al. (2017).

EG in NTP study (1993) sufficiently similar to OECD TG 408:

In the 90-day repeated dose toxicity study (NTP, 1993), performed similar to OECD TG 408 and following GLP, the test substance was administrated in feed to male and female B6C3F1 mice for 13 weeks at dose levels of 0, 3200, 6300, 12500, 25000 and 50000 ppm (0, 768, 1512, 3000, 6000 and 12000 mg/kg bw/day, respectively). Mortality, clinical signs, body weight, haematology, biochemistry, urinalysis, gross pathology, organ weights determination and histopathology were determined.

Results: All mice survived to the end of the study. Final mean body weights of dosed male and female mice were similar to those of the controls. No toxicological relevant effects were observed on clinical signs, haematology, biochemistry, and urinalysis. Absolute and relative organ weights of treated mice were generally similar to those of controls throughout the study. Chemical-related kidney and liver lesions, seen only in 25000 and 50000 ppm (6000 and 12000 mg/kg bw/day, respectively) male mice, consisted of nephropathy and centrilobular hepatocellular hyaline degeneration (cytoplasmic accumulation of non-birefringent, eosinophilic, globular, or crystalline material resembling erythrocyte fragments).

Conclusion: The NOAEL is considered to be 3000 and 12000 mg/kg bw for male and female mice, respectively.

The EG studies of Cruzan et al. (2004):

In the study by Cruzan et al. (2004), Ethylene glycol was administrated in two rat strains (Wistar and F-344) via the diet at targeted dosage levels of 0 (control), 50, 150, 500, or 1000 mg/kg bw/day for 16 weeks. Kidneys were examined histologically including the effect on Calcium oxalate crystals and pathology. Samples of blood, urine, and kidneys from were analyzed for Ethylene glycol, Glycolic acid, and Oxalic acid.

Results: Treatment of Wistar rats at 1000 mg/kg bw/day resulted in the death of two rats. At 500 and 1000 mg/kg bw/day, group mean body weights were decreased compared to control throughout the 16 weeks. In F-344 rats exposed to 1000 mg/kg bw/day and in Wistar rats receiving 500 and 1000 mg/kg bw/day, there were lower urine specific gravities, higher urine volumes, and increased absolute and relative kidney weights. Both strains, treated at 500 and 1000 mg/kg bw/day, some or all treated animals had increased calcium oxalate crystals in the kidney tubules and crystal nephropathy. The effect was more severe in Wistar rats than in F-344 rats. Accumulation of Oxalic acid in the kidneys of both strains was consistent with the dose-dependent and strain-dependent toxicity. As the nephrotoxicity progressed over the 16 weeks, the clearance of Ethylene glycol and its metabolites decreased, exacerbating the toxicity.

Conclusion: The NOAEL for F344 rats is almost 500 mg/kg bw/day. Calcium oxalate crystal deposition is seen but does not cause Oxalic acid nephropathy and is therefore doubtful adverse at this stage, while that of Wistar rats is 150 mg/kg bw/day. At 500 mg/kg bw/day the severity of the Oxalic acid nephropathy is much higher for the Wistar rats compared to the F344 rats. This difference is also reflected when applying a Benchmark dose analysis, the BMDL05 for kidney toxicity in Wistar rats is 71.5 mg/kg bw/day; nearly fourfold lower than in F-344 rats (285 mg/kg bw/day). This study confirms that the Wistar rat is more sensitive to EG-induced Oxalic acid nephropathy than the F-344 rat. Further information from Corley et al. (2008) revealed that clearance plays a vital role in the strain differences.

Besides EG also for

Dodecanedioic-acid repeated dose toxicity information is available on the ECHA site

In the ECHA disseminated dossier on Dodecanedioic acid, an OECD TG 422 and an OECD TG 408 study are reported. In both tests, a NOAEL of ≥1000 mg/kg bw has been derived.

Information used for DNEL derivation for Zenolide

The dose description starting point and assessment factors (AF) used are presented here to prevent repetition of text in all AF sections of Chapter 7 of IUCLID and 5.7 in the CSR.

Dose descriptor starting point: The lowest NOAEL derived from Ethylene Glycol is 150 mg/kg bw/day (ATSDR, 2010 and Cruzan, 2004). For each gram of Zenolide administered, 0.24 gram of Ethylene Glycol may be formed. As such, the NOAEL of 150 mg/kg bw/day of Ethylene Glycol is similar to 625 mg/kg bw/day of Zenolide. As the NOAEL derived from the OECD TG 421 study with Zenolide is 300 mg/kg bw/day, this value will be taken forward to the DNEL derivation.

Duration of exposure: The chronic oral toxicity of Ethylene Glycol has been evaluated in three diet studies in rats and two studies in mice and in it can be concluded that rats were more sensitive compared to mice. The key findings in these rat studies were similar: kidney lesions (oxalate nephrosis), which is the same effect as found in the OECD TG 421 study with Zenolide and in the 90-day study from Cruzan et al. (2004) with Ethylene glycol. Also in studies with longer exposure duration in the same rat species similar kidney effects were seen. In a follow-up study of Cruzan et al (2004), a study by Corley et al. (2008) was performed with Wistar rats. These rats were exposed for 12 months and a NOAEL of 150 mg/kg bw was derived (LOAEL: 300 mg/kg bw). According to these authors, 150 mg/kg bw seemed to be a threshold dose below which no renal toxicity occurs, regardless of exposure duration. Other Ethylene glycol literature data further support this absence of the need of applying AFs: In a study by Blood (1965), Sprague-Dawley rats were exposed for two years and a NOAEL of 150 mg/kg bw was derived (LOAEL: 375 mg/kg bw). In a study by DePass et al. (1986), F344 rats were exposed via diet for 2 years and a NOAEL of 200 mg/kg bw was derived (LOAEL: 1000 mg/kg bw). Although the duration of exposure was substantially longer (12-24 months) compared to the study by Cruzan et al. (2004), which lasted only 3 months, extending the exposure period does not result in lower NOAEL levels. Therefore the 150 mg/kg bw can be used for chronic toxicity of EG. This EG toxicity can be converted to Zenolide as is shown in the toxico-kinetic section and therefore for Zenolide a chronic NOAEL of 300 mg/kg bw can be used. Subsequently, no assessment factor for “differences in duration of exposure” is needed.

Gender, strain and species sensitivity (intra- and interspecies differences): The (sub) chronic oral toxicity of Ethylene Glycol has been evaluated in several studies in rats and mice. From this it can be concluded that males are more sensitive than females, Wistar rats were more sensitive than other rats and rats are more sensitive than mice (ATSDR, 2010 and Fowles, 2017). These differences are related to differences in the rate of metabolism of Ethylene Glycol to Glycolic acid to Glyoxylic acid and to Oxalic acid (Fowles et al, 2017). In addition, clearance of Oxalic acid plays an important role. Clearance of Oxalic acid is a factor 1.7 higher in Sprague Dawley rats compared to Wistar rats and even 3.2 times higher in humans compared to Wistar rats (Corley et al, 2008).

These factors are considered for the DNEL for Zenolide. For Zenolide this means that the Repro-screen (OECD 421) from which the NOAEL of 300 mg/kg bw will be used for the derivation of the DNEL, because in this study the most sensitive species is used. Because humans are at least 3 times less sensitive compared to the Wistar rats, the standard assessment factor of 4 for "interspecies differences" can be reduced to 2.

References

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

Corley, R., Wilson, D., Hard, G., Stebbins, K., Bartels, M., Soelberg, J., Dryzga, M., Gingell, R., McMartin, K., Snellings, W., 2008, Dosimetry considerations in the enhanced sensitivity of male Wistar rats to chronic ethylene glycol-induced nephrotoxicity, Toxicol Appl Pharmacol. Apr 15;228(2).

Cruzan, G., Corley, R., Hard, C., Mertens, J., McMartin, K., Snellings, W., Gingell R., Deyo, J., 2004, Subchronic toxicity of ethylene glycol in Wistar and F-344 rats related to metabolism and clearance of metabolites, Toxicol Sci. Oct;81(2):502-11.

Dodecanedioic acid, REACH dossier: https://echa.europa.eu/nl/registration-dossier/-/registereddossier/14886, site visited April 2019

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.

The read across justification is presented below.

Repeated dose toxicity of Zenolide (CAS #54982-83-1) using data from Zenolide and its metabolites Ethylene Glycol (EG, CAS #107-21-1), and Dodecanedioic-acid (DDDA, CAS # 693-23-2)

Introduction and hypothesis for the analogue approach

Zenolide is a cyclic aliphatic double ester. For this substance sub-acute repeated dose toxicity information is available but information covering the 90-day repeated dose toxicity is missing, which is needed in view of its tonnage level (REACH Annex IX). In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the repeated dose toxicity of Zenolide, data on Zenolide are used in combination with data of its metabolites, which information can be used for read across.

Hypothesis: Zenolide has the same systemic toxicity as Ethylene glycol (EG) and Dodecanedioc-acid (DDDA) when the toxicity is compared on a molar base.

Available information: For Zenolide, a 28-day repeated dose study (OECD TG 407) was performed using Sprague-Dawley rats resulting in a NOAEL of ≥1000 mg/kg bw. In addition, a reproductive toxicity screening test (Repro-screen, OECD TG 421) in Wistar rats is available resulting in a systemic NOAEL of 300 mg/kg bw. The Oxalic acid nephropathy seen in the latter study are the result of EG, a key metabolite of Zenolide, further metabolising into Oxalic acid.

For EG a 90-day repeated dose toxicity study in B6C3F1 mice is available which resulted in a NOAEL of 3000 mg/kg bw (NTP, 1993, Kl. 2, because similar to guideline). In addition, for EG a 90-day repeated dose toxicity study in Wistar is presented in which a NOAEL of 150 mg/kg bw was derived. In the same study F344 rats were tested and a NOAEL close to 500 mg/kg bw was derived (Cruzan et al., 2004, Kl. 2, because it is a publication). The differences in NOAELs between strains and species show sensitivity differences, which are further investigated by Corley et al. (2008) and reviewed by Fowles et al. (2017). In addition, information on sub-acute and sub-chronic effects on EG are included to show that effects are not exposure duration depending and can therefore be used for chronic toxicity (Corley et al., 2008, Kl. 2 because it is a publication).

For DDDA, the other key metabolite of Zenolide, the supporting information includes a Repeated Dose-Repro-screen study (OECD TG 422) and a 90-day (OECD TG 408) study. In both tests NOAELs of ≥1000 mg/kg bw have been derived.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemicals are shown in the data matrix, including relevant physico-chemical properties.

Purity / Impurities

Zenolide is a mono-constituent with a high purity> 95% and therefore the impurities are not expected to influence the results.

Justification for using the information from Zenolide’s key metabolite EG and supported with information from DDDA to assess the toxicity of Zenolide

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a metabolite when the rate of metabolisation is fast. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation, which is presented in the current document.

Metabolite selection: Zenolide is absent in the systemic circulation. It is fully converted to its key metabolites EG and DDDA. Therefore, repeated dose toxicity information on Zenolide, EG and DDDA will be used to complete the repeated dose toxicity of Zenolide.

DDDA has NOAELs of≥1000 mg/kg bw and is considered not to influence the toxicity of Zenolide and/or EG and will therefore only be presented in the Data matrix. In the earlier dossier version of Zenolide, Habanolide (an aliphatic mono-ester) was presented as a supporting analogue for Zenolide. This substance also has a 90-day repeated dose NOAEL of≥1000 mg/kg bw and is therefore only used as supporting information. In view of the new Repro-screen data of Zenolide, in which it was shown that EG is the key toxicant, the focus in the present read across is on EG.

Structural similarities and differences between Zenolide and its metabolites: These structural differences between Zenolide and its metabolites are not relevant for internal exposure because the backbone and functional groups of Zenolide are separated during metabolisation.

Toxico-kinetics:

Absorption: The absorption of Zenolide versus its metabolites is not relevant because Zenolide is fully converted to its metabolites during the uptake phase and the systemic circulation. Metabolisation: Zenolide will be fully metabolised into EG and DDDA by carboxyl esterases, which are abundantly available in all tissues, including gut, liver and plasma (Toxicological handbooks, Belsito et al., 2011 and Saghir et al. 2017). This is supported with experimental in vitro rat plasma, where no Zenolide could be detected within seconds at 37°C (CRL, 2019, See toxico-kinetic section, and Figure 1).

Figure 1: Zenolide and its Phase 1 metabolism in which Zenolide is metabolised into the hypothesized intermediate, Ethylene glycol and Dodecanedioic-acid

 

Zenolide will not be present in the systemic circulation. In view of the Oxalic acid nephropathy seen in the Zenolide study also the metabolic fate of EG is relevant and is depicted in Figure 2.

 

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

 

Distribution and Excretion of Zenolide: Based on the full metabolisation of Zenolide there will be no distribution or excretion of Zenolide as such.

Mode of Action (MoA) comparison between Zenolide and EG is discussed using three subjects:

1) MoA repeated dose toxicity the overlap between effects seen in Zenolide and EG: In the Repro-screen study of Zenolide with Wistar rats, EG type of Oxalic acid nephropathy effects were 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). The NOAEL for Zenolide was 300 mg/kg bw (1.2 mMol).

In EG studies with Wistar rats this Oxalic acid nephropathy is seen at 300 mg/kg bw (4.8 mMol). The NOAEL in EG studies with Wistar rats is 150 mg/kg bw (2.4 mMol). 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.

This means that the EG effects in Wistar rat in the Repro screen study with Zenolide can only occur when full metabolisation of Zenolide into EG (and DDDA) occurs. EG exposure results in the same Oxalic acid nephropathy including these Calcium oxalate crystals at almost equivalent doses.

2) MoA considering exposure duration: Though for Zenolide only sub-acute repeated dose toxicity information is available, a chronic value for Zenolide can be derived because the Oxalic acid nephropathy can be fully contributed to EG.

The (sub) chronic oral toxicity of EG focusses on the Wistar rat studies because these are the most sensitive. In 90-days and 12 month studies the same Oxalic acid nephropathy is seen at the same 150 mg/kg bw dose (Cruzan et al., 2004 and Corley et al., 2008). According to these authors, 150 mg/kg bw is a threshold dose below which no Oxalic acid nephropathy occurs, regardless of exposure duration. Therefore, the 150 mg/kg bw can be used for chronic toxicity of EG.

This EG toxicity can be converted to Zenolide as is shown in the toxico-kinetics section and would result in a calculated Zenolide NOAEL of 625 mg/kg bw (2.4 mMol Zenolide, MW is 256), which aligns with the NOAEL of Zenolide of 300 mg/kg bw and the LOAEL of 1000 mg/kg bw (1.2 and 3.9 mMol Zenolide).

3) MoA considering gender, strain and species sensitivity: The sensitivity to Oxalic acid nephropathy effects differs between strains of rats as was observed in the two Zenolide rat studies. The Oxalic acid nephropathy observed in the Repro-screen study (OECD TG 421) with Wistar rats are absent in the repeated dose toxicity study (OECD TG 407) with Sprague-Dawley rats.

For EG it has been shown that toxicity differs substantially between gender, strain and species. Among rats, males are more sensitive than female, Wistar rats are more sensitive than other rat species, and rats are more sensitive than mice and humans. These differences are related to at least differences in the rate of metabolism of EG to Glycolic acid and Oxalic acid (Figure 2, ATSDR, 2010 and Fowles et al., 2017) and the clearance of Oxalic acid. Corley et al. (2008) showed that the clearance is 1.7 times higher in F344 rats compared to Wistar rats, and even 3.2 times higher in humans.

Also, in the Repeated dose toxicity study with Zenolide (OECD TG 407) this factor of at least 3 difference between Wistar rat and Sprague-Dawley rats is observed. In Wistar rats, effects were seen at 1000 mg/kg bw while in Sprague –Dawley rats no such effects were seen at this dose.

Conversion to Zenolide from EG considering NOAEL: For 1000 mg/kg bw (3.9 mMol) Zenolide administered, 3.9 mMol EG is formed, which is 240 mg/kg bw. The NOAEL of 150 mg/kg bw of EG (1.2 mMol) can be converted to 2.4 mMol (625 mg/kg bw) Zenolide. As the NOAEL derived from the Repro-screen (OECD TG 421) study with Zenolide is 300 mg/kg bw (based on EG Oxalic acid nephropathy), which is lower than the calculated 625 mg/kg bw, the value derived from the Zenolide study will conservatively be taken forward to the risk characterisation.

Uncertainty of the prediction: There are no remaining uncertainties other than those addressed above.

Data matrix

The relevant information on physico-chemical properties and other toxicological properties are presented in the Data Matrix below.

Conclusions on repeated dose toxicity

For Zenolide, repeated dose toxicity information is available from a 28-day repeated dose toxicity study (OECD 407) and a Reproduction Developmental Toxicity Screening Test (OECD TG 421). For the current Annex IX registration, data on at least a 90-day exposure period is needed. Chronic type of information is available from Ethylene glycol, which is the key toxic metabolite of Zenolide. For the other non-toxic metabolite Dodecanedioic-acid information is available at the ECHA dissemination site.

When using read across the result derived should be applicable for C&L and/or risk assessment and accompanied with adequate documentation. This documentation is presented here.

The effects of Zenolide are caused by the metabolite Ethylene Glycol. The chronic NOAEL of Ethylene glycol is Ethylene Glycol is 150 mg/kg bw (2.4 mMol, MW 62), which is equivalent to 625 mg/kg bw Zenolide (2.4 mMol, MW 256). Because the lowest NOAEL derived with Zenolide is 300 mg/kg bw from the Repro-screen (OECD TG 421) this 300 mg/kg bw NOAEL will be used as a chronic value for the risk characterisation.

Final conclusion: The chronic NOAEL for Zenolide is 300 mg/kg bw.

Data matrix to support the read across to Zenolide from Ethylene Glycol and Dodecanedioic-acid for repeated dose toxicity

Common names	Zenolide	Ethylene glycol	Dodecanedioic-acid
Chemical structures
	Target	Key source	Supporting source
CAS no	54982-83-1	107-21-1	693-23-2
EC no	259-423-6	203-473-3	211-746-3
Registration information	Yes	Yes	Yes
Empirical formula	C14H24O4	C2H6O2	C12H22O4
Molecular weight	256	62	230
Phys-chem data not relevant for comparison
Human health
Repeated dose toxicity (mg/kg bw) - 28 days	≥1000 (OECD TG 407) 300 (OECD TG 421)	-	≥1000 (OECD TG 422)
Repeated dose toxicity (mg/kg bw) - Chronic toxicity	300, including read across Ethylene glycol	150 (Chronic value, see text)	≥1000 (OECD TG 408)

References

- ATSDR, 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

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

- Corley, R., Wilson, D., Hard, G., Stebbins, K., Bartels, M., Soelberg, J., Dryzga, M., Gingell, R., McMartin, K., Snellings, W., 2008, Dosimetry considerations in the enhanced sensitivity of male Wistar rats to chronic ethylene glycol-induced nephrotoxicity, Toxicol Appl Pharmacol. Apr 15;228(2)

- Cruzan, G., Corley, R., Hard, C., Mertens, J., McMartin, K., Snellings, W., Gingell R., Deyo, J., 2004, Subchronic toxicity of ethylene glycol in Wistar and F-344 rats related to metabolism and clearance of metabolites, Toxicol Sci. Oct;81(2):502-11.

-Dodecanedioic-acid, REACH dossier:https://echa.europa.eu/nl/registration-dossier/-/registered-dossier/14886, site visited April 2019. 

-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.

- NTP, 1993, Toxicology and carcinogenesis studies of ethylene glycol in B6C3F1 mice, NIH Publication No. 93-3144.https://ntp.niehs.nih.gov/ntp/htdocs/lt_rpts/tr413.pdf

- 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.

Justification for classification or non-classification

Based on the results of the sub-acute oral toxicity study and reproscreening study of Zenolide and the repeated dose toxicity studies of its metabolites, Zenolide does not have to be classified for repeated dose toxicity according to EU CLP (EC 1272/2008 and its amendments).