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Toxicological information

Basic toxicokinetics

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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
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
hydrolysis
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
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
no
Analytical monitoring:
yes
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
Details on test conditions:
High pH testing (pH 4.0, 7.0 and 9.0)
The test material was used without a co-solvent or a detergent.
1 g (1.3 mMol) of the test material was added to 100 mL of the respective buffer solution in a 250 mL Erlenmeyer flask. The flask was closed with a stopper and heated in a heating cabinet for 5 days (120 h) at 50 °C. The mixture was stirred by a magnetic stirrer using a 40*7, stir bar at approx. 100 rpm. The test was carried out at pH 1.2 and 37 °C.
After the pre-determined reaction time, the reaction mixture was allowed to cool down to room temperature and extracted with hexane. The phases were separated using a separatory funnel. The organic phase was transferred to a pre-weighed flask and the solvent was removed in a rotary evaporator (< 40 °C, 10 mbar). The weight difference was recorded for the mass balance and the samples were analysed by 119Sn-NMR.
The water phase was analysed by AAS for a total tin content.

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:
120 h
pH:
4
Temp.:
50 °C
Initial conc. measured:
1.3 mmol/L
Duration:
120 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
1.3 mmol/L
Duration:
120 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
1.3 mmol/L
Duration:
4 h
pH:
1.2
Temp.:
37 °C
Initial conc. measured:
1.3 mmol/L
Remarks:
Gastric pH testing
Number of replicates:
Not specified
Positive controls:
no
Negative controls:
no
Transformation products:
yes
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
Hydrolysis at pH 4.0, 7.0 and 9.0: samples of the test material were added to the respective buffer solutions at 50 °C for 5 days (120 h). The reaction products were extracted with hexane. The 119Sn-NMR spectra of the extracted reaction products did not show any sign of hydrolysis.

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.


pH:
7
Temp.:
50 °C
DT50:
> 1 yr
Type:
not specified
Remarks on result:
other: St. dev. not reported.
Details on results:
Mass balance: For each tested pH value, a 1 g (1.3 mmol) sample of the test material was added to the respective buffer solution. After the required hydrolysis period of 5 days amounts of the hydrolysate were recovered via hexane extraction from the aqueous phase.
The mass balance showed a high recovery of the initial material of the test material after completing the hydrolysis test over the required period (5 days) and the extraction with hexane. It demonstrates high reliability of the chosen experimental design of the study.

The aqueous phases of the hydrolyses at different pH values have been analysed for tin content by AAS.
Tin content in remaining aqueous phases:
pH 4: 280 mg tin/L.
pH 7: 70 mg tin/L.
pH 9: 115 mg tin/L.
This shows that water soluble tin compounds were only present in trace amounts in the reaction mixture after the hydrolysis test followed by the extraction with hexane.

Composition of test material lysate in the pH 1.2 buffer based on 119Sn-NMR analysis

pH value

MMTE

[Mol%]

MMTEC

[Mol%]

MMTE:MMTCE ratio

Start

96

4

96:4

pH 1.2

69

31

69:31

NMR ppm

6807

-12.7

 

 

Recovery of the tested material

pH

Initial mass

[g]

Recovered

[g]

Recovery rate

[%]

4

1.0

0.89

89

7

1.0

0.97

97

9

1.0

0.89

89

 

Validity criteria fulfilled:
not specified
Conclusions:
Under the conditions of the study the test material at pH 4, 7 and 9 can be considered hydrolytically stable. After 5 days at 50 °C less than 10 % of the test material was hydrolysed (t0.5 25°C > 1 year).
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 the test material at pH 4, 7 and 9 can be considered hydrolytically stable. After 5 days at 50 °C less than 10 % of the test material was hydrolysed (t0.5 25°C > 1 year).

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
according to
Guideline:
other: EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH
Deviations:
not applicable
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Pale yellow liquid
Details on test material:
- Storage: In original container, at 15 °C to 25 °C

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Results and discussion

Metabolite characterisation studies

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.

Applicant's summary and conclusion

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.