Phosphoric acid, 2-ethylhexyl ester

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Testing was conducted between 19 May 2011 and 06 August 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004.

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Version / remarks:

The determination was carried out using a procedure designed to be compatible with Method C7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Preparation of samples:Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 5 x 10-3 g/l in the three buffer solutions. A 0.5% co-solvent of methanol was used to aid solubility.The test solutions were split into individual vessels for each data point.The solutions were shielded from light whilst maintained at the test temperature.Preliminary test/Tier 1Sample solutions at pH 4, 7 and 9 were maintained at 50.0 ± 0.5°C for a period of 144 hours (see Discussion in the Overall Remarks Section of this Summary).Analysis of sample solutionsThe sample solutions were taken from the water bath at various times and the pH of each solution recorded.The concentration of the sample solution was determined by liquid chromatography – mass spectroscopy (LC-MS).SamplesDuplicate aliquots (A and B) of sample solution were analysed neat.StandardsDuplicate standard solutions of test item were prepared in the relevant buffer solution at a nominal concentration of 5 mg/l.Matrix blanksRelevant buffer solution.

Buffers:

Buffer Solution (pH 4)Components: Potassium hydrogen phthalteConcentration (mol dm-3): 0.05Buffer Solution (pH7)Components: Disodium hydrogen orthophosphate (anhydrous), Potassium dihydrogen orthophosphate, Sodium chlorideConcentration (mol dm-3): 0.03, 0.02, 0.02Buffer Solution (pH9)Components: Disodium tetraborate, Sodium chlorideConcentration (mol dm-3): 0.01, 0.02The buffer solutions were diluted to 5% using reverse osmosis water, filtered through a 0.2 µm membrane filter to ensure they were sterile before commencement of the test. Also these solutions were subjected to ultrasonication and degassing with nitrogen to minimise dissolved oxygen content.

Estimation method (if used):

Not used.

Duration:

144 h

pH:

4

Initial conc. measured:

0.005 g/L

Duration:

144

pH:

7

Initial conc. measured:

0.005 g/L

Duration:

144

pH:

9

Initial conc. measured:

0.005 g/L

Number of replicates:

Duplicate aliquots (A and B) of sample solution were diluted by a factor of 100 using glass double-distilled water.

Positive controls:

no

Negative controls:

no

Statistical methods:

Not specified.

Preliminary study:

The component concentrations at the given time points are shown in the Results section including tables.

Test performance:

ValidationThe linearity of the detector response with respect to concentration was assessed over the nominal concentration range of 0 to 8 mg/l in the relevant buffer solution. These were satisfactory with correlation coefficients in the range of 0.994 to 1.000 being obtained.

Transformation products:

not specified

pH:

4

Temp.:

25 °C

DT50:

> 1 yr

Type:

other: pseudo-first order reaction

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

Type:

other: pseudo-first order reaction

pH:

9

Temp.:

25 °C

DT50:

> 1 yr

Type:

other: pseudo-first order reaction

Other kinetic parameters:

None.

Details on results:

At pH4, 7 and 9 there was less than 10% hydrolysis after 6 days at 50°C, equivalent to a half-life greater than 1 year at 25°C.ConclusionThe estimated half-life at 25°C of the test item is shown in the following table:pHEstimated half-life at 25°C4>1 year7>1 year9>1 year

The 2 -ethylhexyl dihydrogen phosphate concentrations at the given time points are shown in the following tables.

Table 11.4    pH 4 at 50.0 ± 0.5ºC

Time (Hours)	Mean Concentration (g/l)		% of mean initial concentration (g/l)
	A	B	A	B
0	5.15 x 10^-3	4.54 x 10^-3	-	-
24	5.26 x 10^-3	4.85 x 10^-3	109	100
96	6.48 x 10^-3	6.95 x 10^-3	134	144
120	4.78 x 10^-3	5.48 x 10^-3	98.7	113
144	5.15 x 10^-3	4.56 x 10^-3	106	94.1

Result:          Less than 10% hydrolysis after 6 days at 50°C, equivalent to a half-life greater than 1 year at 25°C.

Table 11.5    pH 7 at 50.0 ± 0.5ºC

Time (Hours)	Mean Concentration (g/l)		% of mean initial concentration (g/l)
	A	B	A	B
0	4.72 x 10^-3	5.24 x 10^-3	-	-
24	5.27 x 10^-3	4.92 x 10^-3	106	98.8
96	5.34 x 10^-3	5.12 x 10^-3	107	103
120	5.39 x 10^-3	5.15 x 10^-3	108	103
144	4.63 x 10^-3	5.42 x 10^-3	93.0	109

Result:          Less than 10% hydrolysis after 6 days at 50°C, equivalent to a half-life greater than 1 year at 25°C.

Table 11.6    pH 9 at 50.0 ± 0.5ºC

Time (Hours)	Mean Concentration (g/l)		% of mean initial concentration (g/l)
	A	B	A	B
0	4.95 x 10^-3	4.84 x 10^-3	-	-
24	5.14 x 10^-3	4.76 x 10^-3	105	97.2
96	5.28 x 10^-3	5.44 x 10^-3	108	111
120	4.90 x 10^-3	5.29 x 10^-3	100	108
144	5.25 x 10^-3	4.29 x 10^-3	107	87.5

Result:          Less than 10% hydrolysis after 6 days at 50°C, equivalent to a half-life greater than 1 year at 25°C. 

 

The bis(2-ethylhexyl) hydrogen phosphate concentrations at the given time points are shown in the following tables:

Table 11.7    pH 4 at 50.0 ± 0.5ºC

Time (Hours)	Mean Concentration (g/l)		% of mean initial concentration (g/l)
	A	B	A	B
0	6.06 x 10^-3	5.11 x 10^-3	-	-
24	6.67 x 10^-3	5.96 x 10^-3	120	107
96	6.13 x 10^-3	6.40 x 10^-3	110	115
120	5.74 x 10^-3	6.55 x 10^-3	103	117
144	6.10 x 10^-3	6.08 x 10^-3	109	109

Result:          Less than 10% hydrolysis after 6 days at 50°C, equivalent to a half-life greater than 1 year at 25°C.

Table 11.8    pH 7 at 50.0 ± 0.5ºC

Time (Hours)	Mean Concentration (g/l)		% of mean initial concentration (g/l)
	A	B	A	B
0	5.46 x 10^-3	6.01 x 10^-3	-	-
24	5.90 x 10^-3	5.31 x 10^-3	103	92.5
96	5.63 x 10^-3	5.33 x 10^-3	98.1	92.9
120	5.68 x 10^-3	5.35 x 10^-3	98.9	93.3
144	4.73 x 10^-3	5.55 x 10^-3	82.5	96.7

Result:          Less than 10% hydrolysis after 6 days at 50°C, equivalent to a half-life greater than 1 year at 25°C.

Table 11.9    pH 9 at 50.0 ± 0.5ºC

Time (Hours)	Mean Concentration (g/l)		% of mean initial concentration (g/l)
	A	B	A	B
0	5.22 x 10^-3	4.95 x 10^-3	-	-
24	5.28 x 10^-3	4.90 x 10^-3	104	96.3
96	5.51 x 10^-3	5.76 x 10^-3	108	113
120	5.20 x 10^-3	5.54 x 10^-3	102	109
144	5.55 x 10^-3	5.12 x 10^-3	109	101

Result:          Less than 10% hydrolysis after 6 days at 50°C, equivalent to a half-life greater than 1 year at 25°C. 

Conclusion

The estimated half-life at 25°C of the test item is shown in the following table:

Table 11.10

pH	Estimated half-life at 25°C
4	>1 year
7	>1 year
9	>1 year

Validity criteria fulfilled:

yes

Conclusions:

The estimated half-life at 25°C of the test material at pH 4, 7 and 9 is greater than 1 year.

Executive summary:

Method

The determination was carried out using a procedure designed to be compatible with Method C7 Abiotic Degradation, Hydrolysis as a Function of pH of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 111 of the OECD Guidelines for Testing of Chemicals, 13 April 2004.

Discussion

Due to an error in entering method details, two methods with different mobile phase gradients were used. The samples and the method used for the analysis are as follows; Method A was used for all the linearity analyses and for pH 7 and pH 9 initial, 24-Hour, 96-Hour and 120-Hour samples; Method B was used for all pH 4 samples and for pH 7 and pH 9 144-Hour samples. It was considered that since the results for the three pHs and the five time points were consistent and the test item had demonstrated less than 10% hydrolysis over the test period, the test data was valid. 

Since there was some analytical variation observed, the test was extended to 144 hours (6 days) to provide additional data in support of the stability of the test item.

Peaks were observed in the matrix blank solutions at the approximate retention time of the analytes were most likely due to instrument carry-over. This was not considered significant and the sample concentrations were appropriately corrected for the matrix blanks. 

The kinetics of the study have been determined to be consistent with that of a pseudo-first order reaction as the graphs of log₁₀concentration versus time are straight lines.

Conclusion.

The estimated half-life at 25°C of the test material are shown in the following table:

pH	Estimated half-life at 25°C
4	>1 year
7	>1 year
9	>1 year

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-14 to 2012-01-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conductedin accordance with international guidelines and in accordance with the principles of GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: For pH 4, 7 and 9, samples were taken at test start (0 h) and at test end (120 h).For pH 1.2, samples were taken at test start and at a minimum of 8 spaced points. All test item containing samples were analysed immediately (max. 1 % of total incubation time until start of analyses) via LC-MS.

Buffers:

- pH: 1.2, 4, 7, 9- Type and final molarity of buffer:Buffer solution pH 1.2250 mL of 0.2 mol/L KCl and 425 mL of 0.2 mol/L HClBuffer solution pH 445 mL of 0.1 mol/L NaOH was mixed with 250 mL 0.1 mol/L mono potassium citrate and diluted to 500 mL with double distilled water.Buffer solution pH 7148.15 mL of 0.1 mol/L NaOH was mixed with 250 mL 0.1 mol/L KH2PO4 and diluted to 500 mL with double distilled water.Buffer solution pH 9106.5 mL of 0.1 mol/L NaOH was mixed with 250 mL 0.1 mol/L H3BO3 in 0.1 mol/L KCl and diluted to 500 mL with double distilled water.- Composition of buffer: Buffers were prepared and sterilised before experimental starting from chemicals with analytical grade or better quality. Buffers were purged with nitrogen for 5 min and then the pH was checked to a precision of at least 0.1 at the test temperature and adjusted. Buffers were sterilised by filtration through 0.22 µm sterile filters.

Details on test conditions:

TEST SYSTEM- Type, material and volume of test flasks, other equipment used: Sterile amber HPLC vials, volume: 4 mL- Measures taken to avoid photolytic effects: Photolytic effects were avoided by exclusion of direct light- Measures to exclude oxygen: Buffers were purged with nitrogen for 5 min, pemanent nitogen flow during incubation- Is there any indication of the test material adsorbing to the walls of the test apparatus? NoTEST MEDIUM- Volume used/treatment: 4 mL - Preparation of test medium: 10 mg test item/L in respective buffer solution- Renewal of test solution: None

Duration:

29 d

pH:

1.2

Temp.:

37 °C

Duration:

120 h

pH:

4

Temp.:

50 °C

Duration:

120 h

pH:

7

Temp.:

50 °C

Duration:

120 h

pH:

9

Temp.:

50 °C

Number of replicates:

test item: duplicatescontrol: single

Positive controls:

no

Negative controls:

yes

Remarks:

buffer solutions (pH 1.2, 4, 7 and 9)

Test performance:

Chronological Test Description:-Method validation-Preparation of the sterile test solutions (experimental starting)-Thermostatisation of the test solutions-Analysis of samples-Evaluation of reaction rate constants and half lives for the testitem

Transformation products:

no

Key result

pH:

4

Temp.:

12 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

7

Temp.:

12 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

9

Temp.:

12 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Remarks on result:

hydrolytically stable based on preliminary test

Details on results:

TEST CONDITIONS- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

Hydrolysis Results for the test item at pH 1.2 and 37 °C

                  

Hydrolysis Time [h]	Addition of Ammonia (mobilisation of diester component)		Without Ammonia
	Phosphoric acid monoethylhexyl ester [mg/L]	Phosphoric acid diethylhexyl ester [mg/L]	Phosphoric acid monoethylhexyl ester [mg/L]	Phosphoric acid diethylhexyl ester [mg/L]
0.00	3.50 ^1)	4.04 ^1)	3.42	4.00
21.9	3.62 ^1)	3.58 ^1)	3.52	1.82
40.1	3.88 ^1)	3.42 ^1)	3.12	1.22
141	3.70 ^1)	3.88 ^1)	3.54	0.936
283	3.50 ^1)	3.42 ^1)	3.24	1.42
332	3.84 ^1)	3.90 ^1)	3.58	0.844
377	3.94 ^1)	3.60 ^1)	3.40	0.596
449	3.76 ^1)	3.82 ^1)	3.42	0.806
527	3.76	3.30	n.d.
624	4.20	4.24
691	3.74	3.58

1) Reanalysed from retained samples after 527 hours of hydrolysis.

n.d. = no determination (general addition of ammonia until sampling event 8)

Degradation [%] of the test item at pH 4, 7 and 9 and 50 °C after 120 Hours

Hydrolysis Time [hours]	Degradation [%]
	pH 4	pH 7	pH 9
120	0.00	0.00	0.00

Validity criteria fulfilled:

yes

Conclusions:

During these testing no hydrolysis for both, the mono and di- ester component of the test item was observed at all tested conditions. With respect to the guideline a half life of > 1 year could be assumed for the test item at pH 4, 7 and 9 and environmental typical temperatures.At the pH 1.2 test condition, an adsorption effect of the diester component of the test item to the test container walls was observed. This adsorption was reversed by addition of ammonia prior to test item analysis. The fully mobilised fraction of the diester component in the test solution might be reduced due to this effect, also reducing the amount of diester component available for hydrolysis. Nevertheless, as the monoester component was not affected and with respect to the long hydrolysis time, the test item might be considered as hydrolytically stable at the pH 1.2 test condition.

Executive summary:

Hydrolysis as a function of pH was determined according to OECD Guideline No. 111 and Council Regulation (EC) No. 440/2008, Method C.7 for the test item (batch number: DEH2024638) from 2011-12-14 to 2012-01-27 at Dr.U.Noack-Laboratorien, Sarstedt, Germany.

For pH 4, 7 and 9 a 5 day testing at 50 °C was performed. Samples were taken at test start and test end. For pH 1.2 the definitive testing (overall 691 h) at 37 °C was performed. The testing was conducted with a test item concentration of 10 mg/L in buffer solution. Samples for the 37 °C testing were taken at test start (0 h) and at least 8 spaced points until test end. Buffer solutions (free of test item) were analysed at test start and test end and there was no analytical interference with the test item. During these testing no hydrolysis for both, the mono and di- ester component of the test item was observed at all tested conditions (Table 1). With respect to the guideline a half life of > 1 year could be assumed for the test item at pH 4, 7 and 9 and environmental typical temperatures.

At the pH 1.2 test condition, an adsorption effect of the diester component of the test item to the test container walls was observed. This adsorption was reversed by addition of ammonia prior to test item analysis. The fully mobilised fraction of the diester component in the test solution might be reduced due to this effect, also reducing the amount of diester component available for hydrolysis. Nevertheless, as the monoester component was not affected and with respect to the long hydrolysis time, the test item might be considered as hydrolytically stable at the pH 1.2 test condition.

Analyses of the test item were performed via LC-MS on a reversed phase column. The method was validated with satisfactory results in regard to linearity, accuracy and specificity.

Reaction Rate Constants and Half Lives of the test item

	pH 1.2	pH 4	pH 7	pH 9
	29 days, 37 °C	120 h, 50 °C
Reaction rate constant kobs [1/s]	Stable, no change in test item concentration over study duration	Stable, half life of > 1 year at environmental typical temperatures
Half life T½ [h]
Half life T½ [d]

Description of key information

Half-life = >1 year (25°C, pH = 4, 7, 9); Butler & White (2011); OECD 111
Half-life = >1 year (25°C, pH = 4, 7, 9); Lange (2012); OECD 111

Key value for chemical safety assessment

Half-life for hydrolysis:

1 yr

at the temperature of:

25 °C

Additional information

Two key studies are presented assessing the hydrolysis of the substance, conducted in accordance with OECD Guideline 111 - "Hydrolysis as a Function of pH".

Both studies indicate that the test item is stable in aqueous solution at pH 4, 7 and 9, each with a half-life >1 year.

An additional supporting study is presented (Plietzsch, 2011) on a form of this substance that contains pyroesters, designed to mimic the physiological environment of the stomach and intestines. In this study, rapid hydrolysis of the pyroester component of the tested batch of this substance was observed. This indicates that the toxicological tests conducted on forms of this substance not containing the pyroester are applicable to the forms containing ≤20 % pyroesters. This applies particularly to the toxicity studies conducted via the oral route of exposure.