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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other:
Remarks:
This study was a part of the original dossier for MMTE. It remains in the dossier to provide continuity to reviewers and an historical perspective of the changes which are being made to the dossier. New data on in vitro metabolism, cited in section 7.1.1 of this dossier, caused a change in the interpretation of the data cited. The new in vitro metabolism data support the conclusion that MMTE does not metabolise to MMTC under simulated mammalian gastric conditions [pH ~2 and 37 °C as was formerly believed. It then follows that the toxicology of MMTE via the oral route in mammalian species cannot be accurately predicted based on studies conducted with MMTC via the oral route in mammalian species. The implications are clear: (a) dietary feeding and oral gavage studies conducted with MMTC cannot be read-across to MMTE, and (b) for studies conducted with MMTC, their relevance for hazard classification of MMTE must be reduced or eliminated. Therefore, the Klimisch score of this study has been reduced to Klimisch 3 because the read-across strategy from this study to MMTE is no longer valid (though the study itself is still considered to be reliable).
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance was tested under low pH conditions (0.07 N HCl) at 37 °C in order to simulate the possible hydrolytic action on mammalian gastric contents. The hypothesis is that in the hydrochloric acid solution the tin-EHMA bond breaks leading to formation of the corresponding alkyltin chloride and simultaneous liberation of the ligand, 2-Ethylhexylmercaptoacetate (EHMA). Under these conditions EHMA, that contains an ester group, can (partially) hydrolyse to thioglycolic acid (TGA) and 2-ethylhexanol (EH).

A stock solution of the test substance in acetonitrile (2.87 mg/mL) was freshly prepared by dissolving 340.81 mg of the test substance in 100.0 mL acetonitrile. A correction for the percentage of the test substance (84.14 %) was used; the amounts of free EHTG and EH present in the test substance were taken into account in the calculations. Into a series of 4 Teflon vessels, 175 µL of a stock solution of the test substance (2.87 mg/mL) was added to 50 mL of 0.07 N HCl. In this way, the concentration of the test substance in the final 0.07 N HCl solution was 10.0 mg/L. The solution was stirred for predetermined periods at 37 °C. The temperature was maintained using an oven. A sample was taken from one of the Teflon vessels after 0.5, 1.0, 2.0 and 4.0 hours, respectively. Once a vessel had been sampled, no other sample was collected from that vessel. Fifty mL of the sample (0.07 N HCl solution) was extracted with 25 mL of heptane. The amount of EHMA and EH in the heptane layer was analysed by GC-FID. The experiments were performed in duplicate.
GLP compliance:
no
Radiolabelling:
no
Metabolites identified:
yes
Details on metabolites:
The recovery of EHMA spiked to 0.07N HCl at a level of 10.0 mg/L was 125 ± 3 %.
The recovery of EH at a level of 12.7 mg/L was 92 ± 3 %.

It was observed that the simulated gastric hydrolysis of the test substance to EHMA and EH was rapid, to a level of 93.9 % after 0.5 h. Looking at the later time points, the measured amounts of EHMA and EH decreased resulting in a calculated percentage of hydrolysis of 78.0 % after 4 hours.

Based on the percentage of completion at 0.5 hours, the half-life time was estimated to be 0.27 hours.

Results (as % conversion to MMTC) for MMT(2-EHMA), by sample collection time:

0.5 h: 94 %

1 h: 91 %

2 h: 85 %

4-h: 78 % 

t1/2 (estimated) = 0.27 hours 

 

The data show that within 0.5 hours, most of the available EHMA ligands have been released and there is greater than 90 % hydrolysis of the test substance. Following the reaction for an additional amount of time it is evident that the amount of free EHMA in solution decreases from the initial level. In previous studies, reanalysing the samples in reverse order confirmed that this trend was real and not caused by the GC drifting out of calibration. This effect was thought to be due to a loss of free EHMA by adsorption, oxidation or the formation of other hydrolysis or reaction products.

Conclusions:
These results support the use of monomethyltin chloride as an appropriate surrogate for mammalian toxicology studies of monomethyltin (ethylhexylthioglycolate) via the oral route.
Executive summary:

These results support the use of monomethyltin chloride as an appropriate surrogate for mammalian toxicology studies of monomethyltin (ethylhexylthioglycolate) via the oral route.

The percentage of hydrolysis of monomethyltin (ethylhexylthioglycolate) under simulated gastric hydrolysis conditions (0.07 N HCl at 37 °C) reached a level of 93.9 % after 0.5 hours. The corresponding half-life time of the test substance was estimated to be 0.27 hours.

 

The chemistry of the alkyl organotins has been well studied. For organotins, like MMT(EHTG), the alky groups are strongly bound to tin and remain bound to tin under most reaction conditions. However, other ligands, such as carboxylates or sulfur based ligands (EHTG), are more labile and are readily replaced under mild reaction conditions. To assess the reactivity of MMT(EHTG) under physiological conditions simulating the mammalian stomach, an in-vitro hydrolysis test was performed. This in vitro test provides chemical information that strongly suggests both the probable in vivo metabolic pathway and the toxicokinetics of the MMT(EHTG) substance. This result verifies that under physiological conditions MMT(EHTG) is rapidly and essentially completely converted to the corresponding monomethyltin chloride, MMTC.

 

Specifically, in the simulated gastric hydrolysis studies at low pH (0.07 N HCl) the EHTG ligands are rapidly displaced from tin and replaced by chloride ligands; the methyl group remains attached to tin. For MMT(EHTG), > 90 % hydrolysis of the test compound occurred within 0.5 hours, and the estimated half-life was 0.27 hours. The replacement of all three EHTG ligands under these conditions is therefore a rapid and complete reaction with respect to the overall metabolic timescale.   

 

This same type of simulated gastric hydrolysis study was also performed on other monomethyltin and dimethyltin compounds having sulfur based ligands with the same result, rapid hydrolysis to release the sulfur based ligands and generate the corresponding methyltin chloride compounds. These studies were all conducted as part of the OECD HPV program covering the monomethyltin and dimethyltin chemicals.

 

Since this hydrolysis was done under simulated gastric conditions, the result is entered into this dossier as fulfilling a toxicokinetic endpoint and as a justification for read-across. As there is rapid conversion of MMT(EHTG) to MMTC, this dossier uses studies on MMTC as the source substance to fill certain specific endpoints for MMT(EHTG), the target substance, by read-across. This read-across is justified because oral exposure to MMT(EHTG) places it in the gastro-intestinal tract where, based on this study, it is hydrolysed to MMTC as the initial metabolic action. Therefore, MMTC studies can be used to fulfil the REACH requirements for MMT(EHTG) related to exposure via the oral route, in particular the mammalian toxicology endpoints of repeated dose, reproduction, developmental and in vivo toxicity. Use of studies on MMTC in a “read-across manner” to cover these specific MMT(EHTG) endpoints is fully supported both by the ECHA guidelines on when and how to apply read-across (see below). In addition, the read-across of MMTC data to MMT(EHGT) was also accepted by the OECD under the HPV program for all methyltin substances on the basis of the simulated gastric hydrolysis.

 

The ECHA document “Guidance on Information Requirements and Chemical Safety Assessment, Part B: Hazard Assessment”, Version 2.1, 2011, discusses the role of toxicokinetics in part B.6.2.1 “Guidance on Toxicokinetics”. This section notes the role of toxicokinetics as an important component in providing information regarding metabolism and absorption of chemicals. It comments further that data on the toxicokinetic behaviour of a substance should be considered in conducting the human health hazard assessment. In this regard, the toxicokinetics directly identify MMTC as the principal and sole organotin metabolite of MMT(EHTG) via oral exposure. 

 

The ECHA document “Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7.c: Endpoint Specific Guidance” section R.7.12 notes that toxicokinetics can be used as a means to assist testing strategies, study design, and the application of read-across for building substance categories. It further notes that the appropriateness or applicability of toxicokinetic studies needs to be made on a case-by-case basis, because the toxicokinetic parameters will affect the hazard profile in determining the concentration of the ultimate toxicant at the target site.  

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 2015 to 03 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Objective of study:
metabolism
Qualifier:
according to
Guideline:
other: EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH
Deviations:
not applicable
GLP compliance:
no
Species:
other: Not applicable to in vitro simulated gastric pH testing
Strain:
other: Not applicable to in vitro simulated gastric pH testing
Sex:
not specified
Details on test animals and environmental conditions:
Not applicable to in vitro simulated gastric pH testing
Route of administration:
other: Not applicable to in vitro simulated gastric pH testing
Vehicle:
unchanged (no vehicle)
Details on exposure:
Analytical Method:
119Sn-NMR spectroscopy:
The 119Sn-NMR has been chosen to analyse the test material as well as the breakdown products of the test material since it combines several unique aspects of analysing tin substances.
- 119Sn-NMR detects all tin-containing substances in a sample qualitatively and quantitatively at the same time.
- 119Sn-NMR is a direct and non-destructive method. It does not require any sample digestion or derivatisation. Thus it avoids errors associated with a) the sample derivatisation and b) misinterpretation of the results associated with analysing and quantifying derivatives.
- The 119Sn spectra signals are highly selective. They directly represent the corresponding tin compounds. Chemical shifts of differently substituted tin atoms are highly characteristic of the specific atom coordination.
- The 119Sn-NMR spectroscopy is very sensitive and reliable. Its detection limit was established to be 0.5 %
- The 119Sn-NMR method has been used for decades by the industry as a standard analytical method on tin compounds for the purpose of quality control, process development and research.

Apparatus: Bruker Advance 200
Temperature: Ambient temperature
Sample preparation: 370 µL / 330 µL toluene-d8 (10 mg/mL CrAcAc)
AAS: Analytik Jena ContraAA 300

Temperature of the measurements: Ambient temperature (outside the apparatus); test temperature 50 °C / 37 °C.
The test material was dissolved in buffer solutions for specific pH values and kept at 50 °C for 5 days assessing the environmental date and to 0.1 M HCl pH 1.2 at 37 °C for 4 h simulating the metabolism under gastric conditions.

Buffers:
The buffer systems were selected according to the guidelines. The chosen buffers provided the required pH values. Commercially available solutions were used:
pH 1.2: HCl 0.1 M
pH 4.0: HCl / NaCl / Citric acid
pH 7.0: Na2HPO4 / NaH2PO4
pH 9: H3BO3 / KCl / NaOH

Gastric pH testing (pH 1.2 / 37 °C)
The test material was used without a co-solvent or a detergent.
1 g (1.3 mMol) test material was added to 100 mL of 0.1 < aqueous solution of hydrochloric acid that was pre-heated to 37 °C in a 250 mL Erlenmeyer flask with ground.
The flask was closed with a stopper and heated on a heating cabinet for 4 h at 37 °C. The mixture was stirred by a magnetic stirrer using a 40 x 7 mm stir bar at approximately 100 rpm.
After the pre-determined exposure time, the solution was allowed to cool down to room temperature; extracted 2 times with 25 mL hexane; the phases were separated using a separatory funnel. The organic phase was transferred into a flask, and the solvent was removed in a rotary evaporator (< 40 °C, 10 mbar). The sample was analysed by 119Sn-NMR spectroscopy.
Duration and frequency of treatment / exposure:
4 h
Dose / conc.:
1.3 other: mmol/L
Control animals:
no
Details on study design:
Not applicable to in vitro simulated gastric pH testing
Details on dosing and sampling:
Not applicable to in vitro simulated gastric pH testing
Metabolites identified:
yes
Details on metabolites:
Hydrolysis at pH 1.2: A sample of the test material was added to an excess of a 0.1 M hydrochloric acid at 37 °C for 4 h. The 119Sn-NMR spectrum of the recovered reaction product showed that the test material is partially hydrolysed to MMTEC. Both substances were present in equilibrium in a ca. 70/30 MMTE / MMTEC mol % ratio.
MMTEC a product of hydrolysis, has been identified based on the 119Sn-NMR signal at -12.7 ppm. The substance was already present in the non-treated test material as an impurity of ca. 4 % (NMR).
No signal corresponding to MMTC (typically present at 133 ppm) was detected.
Conclusions:
Under simulated gastric conditions (0.1 M HCl / pH 1.2 / 37 °C) the test material was partially hydrolysed to its monochloro ester.
It can be concluded that the monochloro ester is the only metabolite of the test material that was formed in the simulated mammalian gastric environment. 
Executive summary:

The hydrolysis of the test material was assessed according to OECD Test Guideline 111 and EU Method C.7. Quantitative ^119Sn-NMR spectroscopy has been used as a valuable analytical tool to directly identify and quantify all organotin components, which are formed as a result of hydrolysis of the tested substance.

The study shows that under the simulated gastric conditions (0.1 M HCl / pH 1.2 / 37 °C) the test material was partially hydrolysed to its monochloro ester.

It can be concluded that the monochloro ester is the only metabolite of the test material that was formed in the simulated mammalian gastric environment. No MMTC was formed under the conditions of the study.

Description of key information

The study shows that under the simulated gastric conditions (0.1 M HCl / pH 1.2 / 37 °C) the test material was partially hydrolysed to its monochloro ester.

It can be concluded that the monochloro ester is the only metabolite of the test material that was formed in the simulated mammalian gastric environment. No MMTC was formed under the conditions of the study.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

As part of the original dossier for MMTE a study was included which utilized read-across from MMTC. This study remains in the dossier to provide continuity to reviewers and an historical perspective of the changes which are being made to the dossier. New data on in vitro metabolism, cited in section 7.1.1 of this dossier, caused a change in the interpretation of the data cited. The new in vitro metabolism data support the conclusion that MMTE does not metabolise to MMTC under simulated mammalian gastric conditions [pH ~2 and 37 °C] as was formerly believed. It then follows that the toxicology of MMTE via the oral route in mammalian species cannot be accurately predicted based on studies conducted with MMTC via the oral route in mammalian species. The implications are clear: (a) dietary feeding and oral gavage studies conducted with MMTC cannot be read-across to MMTE, and (b) for studies conducted with MMTC, their relevance for hazard classification of MMTE must be reduced or eliminated. Therefore, the Klimisch score of the studies have been reduced to Klimisch 3 because the read-across strategy from this study to MMTE is no longer valid (though the study itself is still considered to be reliable). The data now provided were conducted on MMTE.

No experimental studies of the absorption, distribution, metabolism or elimination of2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-methyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate (synonym: MMTE; EC Number 260-828-5; CAS Number 57583-34-3) in mammals are available. However, the physical chemical properties and the existing toxicity studies on the substance have been used to infer as far as possible, its potential toxicokinetics.

 

The substance MMTE is a clear liquid, with amolecular weight (MW) of 743.71g/mol. Water solubility was not determined because the substance was believed to be hydrolytically unstable (although this is being further considered in light of some of the recent new data). The partition coefficient (Log Kow/Log Pow) was also not determined but generated by a property-estimation software (e.g., KOWWIN version 1.67), by a calculation method based on the theoretical fragmentation of the molecule into substructures for which reliable log Pow increments are known. The log Pow obtained by summing the fragment values and the correction terms for intramolecular interactions was estimated to be 11.0. However, because this substance was believed not to exist in contact with water, the log Pow of the expected/stable daughter product (2-ethylhexylthioglycolate) was also calculated and was determined to be 3.68. The vapour pressure of MMTE was determined to be 0.38 Pa at 25°C.

In an in vitro study simulating gastric conditions (0.1 M HCl / pH 1.2 / 37 °C) the test material was partially hydrolysed to its monochloro ester. The monochloro ester is the only metabolite of the test material that was formed in the simulated mammalian gastric environment and no MMTC, as was previously postulated, was formed under the conditions of the study.

 

Absorption

Toxicity studies with MMTE showed that oral absorption does occur, because in rats the LD50 was 880 mg/kg bw and the NOAEL in a dietary 90-d study was 2000 ppm, approximately 163 mg/kg bw/day. For human health risk assessment purposes, oral absorption of MMTE is assumed to be complete (100%).

 

The high molecular weight (MW743.71g/mol) and high estimated Log Pow values indicate that micellar solubilisation would play a major role for absorption by inhalation. In the absence of any quantitative data, for human health risk assessment purposes absorption by inhalation of MMTE is assumed to be 100%.

 

As the molecular weight of MMTE is > 500 g/mol and the estimated Log Pow values are in general > 4, a default value of 10 % dermal absorption is considered appropriate for human health risk assessment.

 

Distribution

The fact that following repeated oral (dietary) exposure lesions were observed in the central nervous system indicates that notwithstanding the high molecular weight, wide distribution occurs. Micellar solubilisation and preferential partition to tissues with high lipid content are expected to occur.

 

Metabolism and Excretion

In an in vitro study simulating gastric conditions the only identifiable breakdown product was the monochloro ester; the stable daughter product 2-ethylhexylthioglycolate is also expected to occur.

 

Excretion is expected to occur mainly via the faces.

 

The potential for bioaccumulation is considered low.