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Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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.  

Description of key information

Short description of key information on bioaccumulation potential result: A simulated gastric hydrolysis study was conducted with MMT(EHTG) to demonstrate that MMTC (monomethyltin trichloride) can be used as a surrogate for oral toxicity studies if compound-specific data are not available. Please refer to the discussion below.

Key value for chemical safety assessment

Additional information

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. 

 

The hydrolysis data support the conclusion that for MMT(EHTG), the rapid hydrolysis to generate MMTC means that MMTC is the only methyltin toxophore from oral exposure. Furthermore, MMTC is soluble in aqueous media, whereas MMT(EHTG) is very insoluble. This means that MMTC is also going to be more readily available in the GI tract than MMT(EHTG), providing further verification of MMTC as being the toxophore. 

 

As noted in ECHA document “Practical Guide 6: How to Report Read-Across and Categories”: Article 13 of REACH and ANNEX XI indicate that read-across may be used to fulfil REACH data requirements. The guide further notes that toxicokinetics can be used as a basis for read-across for endpoints where it is applicable. For MMT(EHTG), the applicability of read across to MMTC is based on the toxicokinetic hydrolysis study. This read-across will be applicable to specific toxicology studies which are conducted by an oral exposure route. As a result, since MMT(EHTG) is rapidly converted in the gastric system to MMTC, this test provides a toxicokinetic justification that the data on MMTC can be read-across to fill appropriate endpoints for MMT(EHTG). In the case for MMT(EHTG) the specific mammalian toxicology endpoints are repeated dose, reproduction, developmental and in vivo toxicity. 

 

It is also important in making a justification for read-across to note the chemical purity as it relates to the substances under investigation. The MMTC which is used as the source substance for read-across data was a mixture comprised of 82.65% monomethyltin trichloride and 9.29 % dimethyltin dichloride. This compares very well to the MMT(EHTG) target substance which is comprised of 84 % monomethyltin and 14 % dimethyltin. Therefore it is concluded that it is appropriate to use the data on this MMTC substance to fill data requirements for the designated MMT(EHTG) substance. 

 

Given the rapid conversion of the target MMT(EHTG) substance to the source MMTC, it is appropriate to conclude that the mammalian toxicology tests on MMTC provide results that apply qualitatively and quantitatively to the endpoints for MMT(EHTG) noted above. In particular, the dosing and effect levels of MMTC can be converted into equivalents of monomethyl tin (MMT), and using the basis of MMT equivalents, these values can be converted to dose and effect levels of MMT(EHTG). As the hydrolysis is very rapid and complete, the dose levels and effect levels observed for MMTC can be quantitatively converted to corresponding dose and effect levels that would apply to MMT(EHTG).   

 

All of this information on MMT(EHTG) and MMTC and the read-across justification of mammalian toxicity test results from MMTC to MMT(EHTG) was presented and accepted internationally by the OECD Chemicals Secretariat at SIAM 23 under the ICCA HPV Program. At this meeting dossiers covering the monomethyl and dimethyltin families, which included multiple monomethyl and dimethyltin substances, were presented as substance categories, and in those dossiers toxicity endpoints related to oral exposure were all covered by reading across test results from the corresponding methyltin chlorides to the other substances. In their review the OECD members accepted the conclusion that the gastric hydrolysis of the methyltins supported use of the methyltin chloride data by read across to fill data gaps for all corresponding methyltin substances. 

 

In summary, the simulated gastric hydrolysis tests fulfil the toxicokinetic endpoint related to metabolism of MMT(EHTG) for exposure via the oral route. The rapid rate of hydrolysis, coupled with the fact that a single methyltin metabolite, MMTC, is formed, provide adequate justification to use MMTC as the source substance for read-across to MMT(EHTG), the target substance. Given that this toxicokinetic result is specific to exposure via the oral route, this read-across is supported only for mammalian toxicology endpoints where oral dosing is applied, i.e. all repeated dose, reproduction, developmental, and in vivo toxicology endpoints. Based on the gastric hydrolysis, this read-across from MMTC to MMT(EHTG) is justified on both a qualitative and quantitative basis.