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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001 - 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study based on international guidelines (EEC, SETAC, OECD) under GLP.
Qualifier:
according to
Guideline:
other: SETAC. Procedures for assessing the environmental fate and ecotoxicity of pesticides. Ed. M. Lynch, 1995
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
yes
Remarks:
see overall remarks
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Reference material I
Identification: MCX , unlabelled, notox test substance 97299)
IUPAC name 2-ethylhexyl-p-methoxycinnamate
CAS 5466-77-3
Molecular formula C18H26 O3
Mol weight 290.403
Batch 9000360523
Appearance: clear pale yellow oil
Chemical purity: >=98 % (GLC: sum of impurities 1.21 %. 98.5 trans isomer, 0.29 % cis isomer, ester value 99.5 %)
Storage: room temperature, protected from ligth
Stabilty: stable
Expiry date: October 11, 2001
Supplier F. Hoffman-La Roche, Basel, Swizerland

Reference material II
Identification: AS 416
IUPAC name p-methoxycinnamic acid,
Description: white powder
CAS 943-89-5
Storage conditions: at room temperature protected from light
Expiry date: October 19, 2001
Supplier F. Hoffmann-La Roche, Basel Switzerland
Date of receipt: October 19, 2000
Batch nr not indicated
Purity: trans isomer 100 % (confirmed by 1H-NMR, MS and IR by the supplier)
Certified: no

Reference material III
Identification AS417
IUPAC name: 2-ethyl-1-hexanol
Apprearance: colourless liquid
CAS : 104-76-7
Batch nr: not indicated
Purity: not indicated. the identity of the material was confirmed by 1H NMR, MS and IR byt the supplierand according to supplier could be considered as being pure for the purpose of co-chromatography.
Certified: no
Storage: room temperature in the dark
Expiry date: October 19, 2001
Supplier; F. Hoffmann- La Roche
Date of receipt: 19 October 2000

Reference material IV
Identification: AS 418
IUPAC name; E/Z -MCX
Appearance: yellow liquid
Cas 55466-77-3
Batch nr: not indicated
Purity : not indicated: according to the supplier the sample could be considered pure for purposes of co-chromatography. identity was confirmed by 1H NMR, MS, IR and GC analysis by the supplier. GC analysis gave trans cis(E/Z) ratio of 60.7 / 39.3 %. 1H NMR confirmed this (cis ~32 %).
Certified: no
Storage: room temperature, dark
Expiry date: October 19, 2001
Supplier: F. Hoffmann-La Roche
Supply date: 19 October 2000
Radiolabelling:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals for the parent/transformation products: after 0, 4, 7, 14, 21, 31 days after incubation in the second test
- Sampling method: At sampling, the metabolism flask and its traps were removed from the climate room, the metabolism flask was weighed. Duplicate samples of 200 µL each were taken for LSC analysis. For TLC analysis, directly after sampling, also duplicate samples of 200 µL each were taken.
the remainder of the sample was transferred from the container to a glass LSC vial ans stored in the freezer.
- Sampling intervals/times for pH measurements:the pH of the buffer/acetonitrile (80/20 v/v) mixtures was measured directly after preparation. also at t = 14 days(halfway) and the end (t = 31 days)
- Sampling intervals/times for sterility check: At t = 0, 14 and 30 days to 2 mL of test solution was added aprox. 2 mL of sterile Brain heart Infusion medium and this mixture was incubated for at least 2 days at 37 °C. Absence of turbidity was regarded as proof for sterile conditions.
Buffers:
First test, performed in flow through system

As test media were used:

Buffer medium pH 4: 83 mL of a 0.05M sodium acetate solution (0.43 g in 100 mL Milli-Q water) mixed with 417 mL of a 0.05 M acetic acid solution (1.51 g in 500 mL Milli-Q water) and diluting the obtained mixture 2.5 times with Milli-Q water rendered a 0.02 M acetate-buffer with pH 4.04.
Buffer medium pH 7: 3.44 g of sodium dihydrogen phosphate dissolved in 250 mL Milli-Q water in a 500 mL volumetric flask, adjusted to pH 7 with 1 M sodium hydroxide and then diluted to 500 mL, which solution was subsequently diluted 2.5 times with Milli-Q water to render a 0.02 M phosphate-buffer with pH 7.15.
Buffer medium pH 9: 1.55 g of boric acid and 1.88 g of potassium chloride dissolved in 250 mL MilliQ water in a 500 mL volumetric flask, adjusted to pH 9 with 1 M sodium hydroxide and then diluted to 500 mL, which solution was subsequently diluted 2.5 times with Milli-Q water to render a 0.02 M borate-buffer with pH 9.08.

Second test, performed in closed system

As test media were used:

Buffer medium pH 4: 83 mL of a 0.05M sodium acetate solution (0.41 g in 100 mL Milli-Q water) mixed with 417 mL of a 0.05 M acetic acid solution (1.50 g in 500 mL Milli-Q water) and diluting the obtained mixture 2.5 times with Milli-Q water rendered a 0.02 M acetate-buffer with pH 3.90.
Buffer medium pH 7: 3.41 g of potassium dihydrogen phosphate dissolved in 250 mL Milli-Q water in a 500 mL volumetric flask, adjusted to pH 7 with 1 M sodium hydroxide and then diluted to 500 mL with Milli-Q water, which solution was subsequently diluted 2.5 times with Milli-Q water to render a 0.02 M phosphate-buffer with pH 7.03.
Buffer medium pH 9: 1.55 g of boric acid and 1.88 g of potassium chloride dissolved in 250 mL Milli-Q water in a 500 mL volumetric flask, adjusted to pH 9 with 1 M sodium hydroxide and then diluted to 500 mL with Milli-Q water, which solution was subsequently diluted 2.5 times with Milli-Q water to render a 0.02 M borate-buffer with pH 8.88.
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 1 L amber glass flasks (first test) and 20 mL teflon or glass vials (second test)
- Sterilisation method: autoclave
- Measures taken to avoid photolytic effects: test performed in amber coloured metabolism flasks
- Measures to exclude oxygen: tests peformed under nitrogen
- If no traps were used, is the test system closed/open: first test: flow-through; second test: closed system

TEST MEDIUM (described for first test)
- Volume used/treatment: 15 mL in second test
- Kind and purity of water: Mili-Q water
- Preparation of test medium: A stock solution (E) of 14C-Ethylhexyl Methoxycinnamate in acetonitrile was prepared containing 6.081 MBq/mL. To prepare the stock solution, part part of Homogeneity was checked by LSC of three aliquots.
The stock solution (E) was used to spike an appropriate amount of each buffer solution. For each pH test level the following test solution was prepared: 41 4L of stock solution (E) were diluted to 250 mL of test solution (in a volumetric flask) by first adding 50 mL acetonitrile and then making up to volume with the appropriate buffer solution. Thus, each test solution had a 14C-Ethylhexyl Methoxycinnamate concentration of 1.0 MBq/L, which is equivalent to
0.117 mg/L. Considering a water solubility for Ethylhexyl Methoxycinnamate of 0.22 mg/L, the test concentration was approximately 50% of the water solubility. Molarity of Ethylhexyl Methoxycinnamate in the test solutions was approximately 0.4 µM. The test solutions contained 20% (v/v) organic co-solvent. Although half the water solubility was not exceeded in the test solutions, it was found during the study on the photolytic degradation of Ethylhexyl Methoxycinnamate in water (NOTOX project 285558) [21, that the high amount of organic co-solvent (20 % v/v) was necessary to maintain homogeneous conditions. After homogenising of the test solution, samples were taken for pH measurement, TLC analysis, LSC analysis and sterility test.
- Renewal of test solution: In case of weight loss at sampling time, the appropriate amount of buffer was added to compensate

TEST MEDIUM (described for second test)
A stock solution (F) of 14C-Ethylhexyl Methoxycinnamate in acetonitrile was prepared containing 5.411 MBq/mL. To prepare the stock solution (F), part of the Ethylhexyl Methoxycinnamate stock had been repurified by preparative TLC. Subsequently a spike solution (F50) was prepared by diluting 200 4L of stock solution (F) to 10 mL with acetonitrile. Spike solution (F50) was shown to contain 113.7 kBq/mL, which is equivalent to 13.4 4g 14C-Ethylhexyl Methoxycinnamate/mL. Homogeneity was checked by LSC of triplicate 10 pL aliquots.

For each pH test level 200 mL of the appropriate buffer was mixed with 50 mL of acetonitrile under sterile conditions. Then in each (preweighed) Teflon container 14 mL of the appropriate buffer/acetonitrile (80/20 v/v) solution were brought in. To each Teflon container were then added 123 µL of spike solution (F50). After spiking, the samples were purged with nitrogen for 5 - 10 seconds, closed immediately with a cap and parafilm and mixed well by shaking by hand. Finally, the samples were placed in the climate room at 20 °C ± 2 °C in the dark until sampling. Samples were neither shaken nor stirred during incubation. LSC-analysis of three 123 .tL aliquots (one taken previous to the spiking procedure, one during, one directly afterwards) indicated that this was equivalent to a mean dose of 93.2 mg spike solution (F50). Thus, each sample had a 14C-Ethylhexyl Methoxycinnamate concentration of 0.94 MBq/L, which is equivalent to 0.111 mg/L. Considering a water solubility for Ethylhexyl Methoxycinnamate of 0.22 mg/L (the lowest value of the solubilty range in water, as determined under NOTOX project 284243)[1], the test substance concentration was approximately 50 % of the water solubility. Molarity of Ethylhexyl Methoxycinnamate in each Teflon container was approximately 0.4 µM. The test solutions contained 20 % (v/v) organic co-solvent. Although half the water solubility was not exceeded in the samples, it was found during the study on the photolytic degradation of Ethylhexyl Methoxycinnamate in water (NOTOX project 285558)[2], that the high amount of organic co-solvent (20% v/v) was necessary to maintain homogeneous conditions.

Samples of t = 0 were prepared as described above after preparation of the other samples. However, immediately after mixing, the samples were weighed and subsamples were taken for pH measurement, TLC analysis, LSC analysis and sterility test.

The samples in the glass containers were prepared in the same way as described for the samples in the Teflon containers, except that 15 mL of buffer/acetonitrile (80/20 v/v) solution were spiked with 132 4L of spike solution (F50). LSC-analysis of three 132 µL aliquots (one taken previous to the spiking procedure, one during, one directly afterwards) indicated that this was equivalent to a mean dose of 101 mg spike solution (F50). Thus, each sample had a 14C-Ethylhexyl Methoxycinnamate concentration of 0.95 MBq/L, which is equivalent to 0.112 mg/L. Molarity of Ethylhexyl Methoxycinnamate in each glass container was approximately 0.4 µM.

Spiking took place on April 13, 2001 and incubation of samples lasted until May 14, 2001 (31 days).

OTHER TEST CONDITIONS
- Adjustment of pH: not during test
- Dissolved oxygen: none
- in the second test two kinds of vessel materials where used: teflon and glass, a total of six samples were prepared for each container material at each pH level.
Duration:
30 d
Temp.:
20 °C
Initial conc. measured:
>= 0.11 - <= 0.12 mg/L
Number of replicates:
3 in the first test, 18 per container type in the second test.
Positive controls:
no
Negative controls:
no
Statistical methods:
The DT50 and DT90 values of Ethylhexyl Methoxycinnamate were calculated using the total amounts of Ethylhexyl Methoxycinnamate as determined by TLC. First order kinetics were fitted to the data. Calculations were performed with the prohram Model Manager version 1.1 (1999, Cherwell Scientific Limited, Oxford, United Kingdom). Simple first order (SFO) degradation kinetics were assumed. The DT50 value of Ethylhexyl Methoxycinnamate was calculated as ln2/k, the DT90 value as ln10/k. M0 and k were not initially fixed.

The DT50 values for Ethylhexyl Methoxycinnamate at the three different pH values were calculated using the total amounts of trans-Ethylhexyl Methoxycinnamate as determined by straight phase TLC results. Not surprisingly in view of the TLC results obtained, the program was unable to perform the calculation in a correct way, as a result of a negative correlation coefficient, resulting in DT50 values of 1.4*10^7 days for all three pH-values. It is known that cis-trans isomerisation of Ethylhexyl Methoxycinnamate may occur easily under certain conditions. Hence, the calculation was performed using the total amount of MCX (trans-isomer plus cis-isomer as presented in APPENDIX I) as well. Unfortunately, the calculation program was again unable to perform the calculation in a correct way, as a result of a negative correlation coefficient, resulting in DT50 values of 1.4*10^7 days for all three pH-values as before.

It can be concluded that Ethylhexyl Methoxycinnamate is slightly hydrolysing at pH 7 (DT50 > 30 days) at 20°C, based on the classification scheme by Mensink.
Preliminary study:
The radioactivity in the CO2 traps never exceeded 0.65 % of the initially applied radioactivity. No radioactivity was recovered in the organic volatile compounds traps. The amount of radioactivity detected in the test solutions decreased with time. This was mainly due to adsorption of the test substance to the container walls. Due to low recovery of the applied radioactivity obtained with this test set-up, a revised test set-up was developed, and the test was repeated in a closed test system.
Test performance:
Because of unsatisfactory results obtained with the first test set-up, a revised test set-up was developed, and the test was repeated in a closed test system.
Transformation products:
yes
Remarks:
but not fully identified
Details on hydrolysis and appearance of transformation product(s):
Based on co-chromatography with reference samples, the major metabolites were thought most likely to be p-methoxycinnamic acid (straight phase TLC) and Ethylhexyl Methoxycinnamate cis-isomer (reversed phase TLC). However, in both cases this identification could not be confirmed.
% Recovery:
98.13
pH:
4
Duration:
0 d
% Recovery:
99.54
pH:
4
Duration:
4 d
pH:
4
Temp.:
20 °C
DT50:
> 1 yr
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
20 °C
DT50:
> 1 yr
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
20 °C
DT50:
> 1 yr
Type:
(pseudo-)first order (= half-life)
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

See attached tables 13, 14 and 15 for further details on results (Ethylhexyl Methoxycinnamate and metabolites in test solution of pH 4, 7 and 9).

Quantification of the amount of (trans)Ethylhexyl Methoxycinnamate, as determined by normal phase TLC, was not confirmed by reversed phase TLC. In some cases, the difference of the quantified amount of trans Ethylhexyl Methoxycinnamate amounted up to 25 % in terms of applied radioactivity. between the two TLC methods.

Although the sum of other metabolites exceeded the 10 % level in several cases, the amount of each individual metabolite did not exceed the significancy level of 10 % in terms of radioactivity applied. Only at pH 4 on t = 0, one radioactive fraction (Rf 0.27) was found at an amount of 10.9 % in terms of applied radioactivity.

 

The cis isomer of Ethylhexyl Methoxycinnamate was detected at amounts of 1.1 to 9.7 % of initially applied radioactivity in all samples except one. It was identified by co-chromatography using its reference substance on normal phase and reversed phase TLC.  However, trans to cis isomerisation was also observed in the spike solution (F50) immediately after spiking of samples.

The presence of p-methoxycinnamic acid could not be confirmed by reversed phase TLC in any of the samples. Thus its identification by normal phase TLC was considered as not reliable.

Tables 13, 14 and 15 present the detected amount of radioactive fractions in terms of applied radioactivity at pH 4, 7 and 9 on different sampling intervals (see tables 13, 14 and 15 in the attached file tables.doc).

Validity criteria fulfilled:
yes
Conclusions:
The half-life (hydrolysis) of Ethylhexyl Methoxycinnamate is > 1 year at 20 °C.
Executive summary:

The hydrolysis as a function of pH was determined for Ethylhexyl Methoxycinnamate using a SETAC protocol under GLP. Since results with a flow-through test were unsatisfactory due to low recovery of radioactivity, a second closed test system was performed. The half-life (hydrolysis) of Ethylhexyl Methoxycinnamate was found to be > 1 year at pH 4, 7 and 9 at 20 °C. The degradation products that were formed could not be identified.

Description of key information

Hydrolysis as a function of pH: T1/2 > 1 year at pH 4, 7 and 9 and 20 °C (OECD TG 111)

Key value for chemical safety assessment

Additional information

The study consisted of two separate tests, the first test was under flow-through conditions, the second one in a closed system. This second closed system test was performed because the results with the flow-through test were unsatisfactory. The half-life (hydrolysis) of Ethylhexyl Methoxycinnamate was found to be > 1 year at pH 4, 7 and 9 at 20 °C. The degradation products that were formed could not be successfully identified, as both TLC methods (straight phase and reversed phase) did not confirm each other.