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Hydrolysis

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Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was performed on the primary hydrolysis product of BPA-DA, 4,4-Bisphenol A Tetra-Acid (BPA-TA; CAS 38103-05-8). Testing on BPA-TA was in addition to testing on BPA-DA as BPA-DA was concluded to be hydrolytically unstable at pH 2, 5-6 and 9.
Reason / purpose:
reference to other study
Reason / purpose:
other: read-across target
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes
Remarks:
OECD GLP
Analytical monitoring:
yes
Buffers:
Buffer solutions used during this study were prepared in the following manner: pH 4 - 180 mL of 0.05 M sodium acetate (3.40 g of sodium acetate trihydrate dissolved and diluted to a final volume of 500 mL with purified reagent water) was added to 820 mL of 0.05 M acetic acid (2.88 mL of acetic acid diluted to 1000 mL with purified reagent water). The pH of the resultant solution was 3.44 and was adjusted to 4.00 with 1 N sodium hydroxide; pH 7 - 1000 mL of 0.1 M potassium dihydrogen phosphate solution (13.65 g potassium phosphate dissolved in 1000 mL purified reagent water) was added to 580 mL of 0.1 M sodium hydroxide (100 mL of 1.0 M sodium hydroxide dissolved in 1000 mL purified reagent water) and diluted to 2000 mL with purified reagent water. The pH of the resultant solution was 7.01 and required no adjustment; and pH 9 - 100 mL of 0.5 M boric acid solution (30.9 g of boric acid dissolved in 1000 mL purified reagent water) was diluted to 1000 mL with purified reagent water. The pH of the resultant solution was 9.00 and required no adjustment.

Buffers were autoclaved at approximately 120°C at 15 psi for 30 minutes. Prior to dosing, the buffer solutions were purged with sterile nitrogen for approximately 5 minutes to exclude oxygen. The sterility of the prepared buffers was confirmed on day 0 and day 5. Sterility was evaluated using aerobic count plates. A 0.5-mL aliquot of each dosed sample was pipetted onto the center of the bottom film and spread using a spreader. Plates were incubated at room temperature for at least 48 hours and then observed for microbial growth on days 0 and 5. Plates were interpreted as positive, indicating the presence of microbial growth, or as negative, indicating the absence of microbial colony formation.
Details on test conditions:
Hydrolysis in Buffers Experiment:
All standard reagent solutions were prepared using sterilised reagent water. The filter-sterilised water typically shows greater than 16.7 Mohm-cm resistivity and less than 1 mg/L total organic carbon, which is the established detectable limit at this laboratory. All chemicals were obtained from commercial sources and are at least reagent grade.

A 2.5 mg/mL BPA-TA primary stock solution was prepared by placing 0.2521 g of the test substance in a 100-mL volumetric flask and bringing it to volume with methanol. This stock solution was used to fortify calibration standards, test and QC samples during the preliminary test.

Prior to testing, the vials and pipettes used to transfer solutions were autoclaved at approximately 121 °C at 15 psi for 30 minutes, to minimize the potential for microbial degradation of the test substance. Individual 10-mL amber vials (eight vials per buffer solution) were filled with 4.96 mL of the appropriate buffer solution (i.e., pH 4, 7 or 9). To each 10-mL vial was added 40 microL of the 2.5 mg/mL BPA-TA stock solution to provide a nominal concentration of 20.0 mg/L. The vials were capped with aluminum crimp caps fitted with Teflon-lined rubber septa and vortexed for 20 seconds. The vials were placed in an incubator designed to maintain a temperature of 50°C for a period of 5 days. Analysis of the test solutions for BPA-TA concentration was performed on day 0 and day 5. In addition, two QC samples were prepared at a concentration of 20.0 mg/L in buffers of varying pH (i.e., 4, 7 or 9). The QC samples were prepared at each sampling interval and were analyzed along with the test samples. The results of the QC sample analyses were used to judge the precision and quality control maintained during the analytical process. All samples were mixed with 1.25 mL of 0.5% phosphoric acid in acetonitrile prior to HPLC analysis. Acceptance criteria for the recovery of QC samples were set at 80.0 to 120% of the prepared concentration. All hydrolysis solutions and QC samples were analyzed for BPA-TA using high performance liquid chromatography with ultraviolet detection (HPLC/UV) based on methodology validated at the testing laboratory. The method validation study was conducted prior to the initiation of the test and established an average recovery of 99.8% ± 5.93% from pH 5, 7, and 9 buffer solutions. Conditions and procedures used throughout the analysis of the test and quality control (QC) samples during this study were the same as those described in the method validation study with the following exceptions: 1) the pH buffers used during the hydrolysis study were pH 4, 7 and 9, while the pH buffers used during the method validation were pH 5, 7, and 9. The method validation was conducted to support Kow testing, requiring pH 5, and hydrolysis testing required pH 4, and 2) the nominal concentration used during the hydrolysis test was 20.0 mg/L, while the method validation range was up to 10.0 mg/L. Since both studies used acetate buffer solutions and QC samples (nominal concentration of 20.0 mg/L) were all within the required range, these exceptions did not negatively impact the study results.

The pH of the test solutions was measured on test day 0 (before and after dosing) and day 5. The pH was determined to the nearest 0.01 pH unit using a Jenco Model 60 pH meter. The temperature was monitored continually.

The percent of nominal at each time point is calculated by:
% nominal = (Ct / C0)* 100
where:
C0 = initial concentration, mg/L
Ct = measured concentration at time t in days
Duration:
5 d
pH:
4
Temp.:
50 °C
Initial conc. measured:
20 g/L
Duration:
5 d
pH:
7
Temp.:
50 °C
Initial conc. measured:
20 g/L
Duration:
5 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
20 g/L
Test performance:
The temperature of the water bath used to control the temperature of the test solutions ranged from 49.9 to 50.3°C. Measurements of pH recorded in the test solutions showed that the buffering capacity had not been decreased.

The hydrolysis was performed in pH 4 (0.05 M acetate), pH 7 (0.05 M phosphate) and pH 9 (0.05 M borate) sterile aqueous buffer solutions at an application rate of 20 mg/L (20 ppm). At selected intervals, duplicate samples were analysed directly by high performance liquid chromatography with ultraviolet detection (HPLC/UV) to quantify the test substance present in the aqueous samples.

Concentrations of BPA-TA measured during the hydrolysis test showed that less than 10% degradation of BPA-TA was observed after 5 days incubation at 50 ± 0.5°C in sterile pH 4, pH 7 and pH 9 buffer samples. The test substance was considered to be hydrolytically stable (half-life greater than 1 year) and no additional testing was performed.

Analysis of QC samples resulted in measured concentrations that were consistent with the acceptable range. Based on these results, it was established that the appropriate precision and quality control was maintained during the analysis of the test solutions.
Transformation products:
no
Key result
pH:
4
Temp.:
50 °C
DT50:
> 1 yr
Remarks on result:
other: Less than 10 % degradation in 5 days
Key result
pH:
7
Temp.:
50 °C
DT50:
> 1 yr
Remarks on result:
other: Less than 10 % degradation in 5 days
Key result
pH:
9
Temp.:
50 °C
DT50:
> 1 yr
Remarks on result:
other: Less than 10 % degradation in 5 days

Concentrations of BPA-TA measured by HPLC/UV in the test and QC samples at 50ºC during the hydrolysis test

Sample

Day/ Sample No.

Target

Concentration

(mg a.i./L)

Area

Concentration

(mg a.i./L)

% of Nominal

pH 4 buffer

Day0

20.0

2614263

19.395

96.98

Day0

20.0

2714379

20.133

100.67

Average

98.82

Day5

20.0

2665233

19.776

98.88

Day5

20.0

2672121

19.827

99.14

Average

99.01

QC Sample

1114QC

20.0

2607271

19.344

96.72

1119QC

20.0

2655726

19.706

98.53

pH 7 buffer

Day0

20.0

2653228

19.683

98.42

Day0

20.0

2697684

20.010

100.05

Average

99.23

Day5

20.0

2733794

20.282

101.41

Day5

20.0

2738655

20.317

101.59

Average

101.5

QC Sample

1114QC

20.0

2673616

19.833

99.17

1119QC

20.0

2716701

20.155

100.78

pH 9 buffer

Day0

20.0

2674464

19.839

99.20

Day0

20.0

2691034

19.961

99.81

Average

99.50

Day5

20.0

2713129

20.129

100.65

Day5

20.0

2716123

20.151

100.76

Average

100.70

QC Sample

1114QC

20

2545432

19.961

99.81

1119QC

20

2688286

19.946

99.73

Note: Samples were incubated at 50 ± 0.5°C for 5 days before HPLC analysis.

Validity criteria fulfilled:
yes
Conclusions:
Less than 10% degradation of the parent was observed after 5 days (half-life greater than 1 year) in the sterile pH 4, pH 7 and pH 9 buffer samples. Based on the results of this study, the BPA-TA was considered to be hydrolytically stable and no additional testing is required.
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
yes
Remarks:
It was not feasible to determine the effect of the high co-solvent concentrations used (> 1%) on the hydrolysis of BPA-DA.
GLP compliance:
yes
Remarks:
The study plan was not fully executed because of low water solubility and rapid hydrolysis.
Analytical monitoring:
yes
Buffers:
pH 2 solution: Aqueous HCl was added to deionized water (DIW) to a pH of 2 (measured by pH meter)

pH9 solution: Aqueous NaOH was added to DIW to a pH of 9 (measured by pH meter)
Details on test conditions:
A hydrolysis standard solution of the BPA-DA hydrolysis product 4Ac (1.079 mg/mL) was prepared in acetonitrile and aqueous NaOH and was stored at approximately 22 °C for 24 hours before analysis by HPLC-MS. The solution was acidified with concentrated HCl.

Standard solutions of BPA-DA (1 mg/mL and approximately 0.5 mg/mL) were prepared in acetonitrile. Minor amounts of BPA-DA remained undissolved in these solutions.

Calibration standards of BPA-DA ranging in nominal concentration from 0 to 520 µg/mL were prepared in acetonitrile by dilution of the 0.5 mg/mL standard solutions.

Test solutions were prepared in DIW (neutral samples), the pH 2 solution (acidic samples) and the pH 9 solution (basic samples) by dilution of the 0.5 mg/mL standard solution. The pH of the DIW used was between 5 and 6. Concentration ranges were as follows: Acidic, 312 to 515 µg/mL, neutral, 302 to 499 µg/mL, and basic, 302 to 499 µg/mL.

Calculations: The concentrations (C) of BPA-DA in the test solutions were calculated as follows: C = (S-b)/F, where: S = area of BPA-DA HPLC peak, m = slope of calibration curve, and b = y-intercept of the calibration curve. The relative concentrations of the intermediate and final hydrolysis products (2Ac and 4Ac, respectively) were calculated as BPA-DA using the BPA-DA calibration curve.

The hydrolysis rate constant (k) was calculated using the following equation: A = Ai *E-kt, where Ai = initial concentration (M) of BPA-DA, and A = observed BPA-DA concentration (M) at time t (hours)

The half-life (t1/2) was calculated as t1/2= 0.693/k

Transformation products:
no
Key result
pH:
2
Temp.:
23 °C
Remarks on result:
other: hydrolytically unstable
Key result
pH:
5.5
Temp.:
23 °C
Remarks on result:
other: hydrolytically unstable
Key result
pH:
9
Temp.:
23 °C
Remarks on result:
other: hydrolytically unstable
Details on results:
The HPLC-UV calibration curve showed a linear relationship with a correlation coefficient of 0.997, indicating acceptable method precision for the analysis of the test material in acetonitrile.

Chromatographic results showed decreasing BPA-DA concentrations and increasing tetraacid (4Ac) terminal hydrolysis product concentrations with increasing aqueous concentrations. The intermediate diacid (2Ac) hydrolysis product was observed to increase and then decrease with increasing aqueous concentrations.

Using a first order kinetic model, the half-lives of BPA-DA in acetonitrile/aqueous were estimated at 1.5 hours (h), 2.0 h and 2.5 h at 23 °C under basic, acidic and neutral conditions, respectively.

Given that the estimated BPA-DA half-lives were obtained at high acetonitrile cosolvent concentration, and that the hydrolysis rates should be faster in the absence of cosolvent (due to the higher concentration of water), BPA-DA should be considered hydrolytically unstable at pH 4, 7, and 9 at 23 °C, based on the OECD stability criterion of hydrolysis half-life greater than 1 year at 25 °C (OECD, 2004).

Observed Concentrations of 4Ac, 2Ac, and BPA-DA in Test Solutions

Sample Conc. (µg/mL)

4Ac Conc. (µg/mL)

2Ac Conc.(µg/mL)

BPA-DA Conc. (µg/mL)

Aq. Conc. (v/v%)

Basic Test Solutions

0

499

479

454

403

302

8

40

44

118

662

1030

8

240

2263

412

280

8

8

771

760

528

34

8

40

1

5

10

20

40

Acidic Test Concentrations

0

515

494

468

416

312

8

35

49

200

1025

1267

8

259

316

572

208

8

8

1071

972

571

8

8

40

1

5

10

20

40

Neutral Test Concentrations

0

499

479

454

403

302

8

35

49

193

905

998

8

262

277

464

101

8

8

1083

726

394

8

8

40

1

5

10

20

40

Estimated BPA-DA Hydrolysis Half-lives (t1/2) using a First-Order Kinetic Model

pH

Initial Nominal BPA-DA Conc.

Time (h)

Observed BPA-DA Conc.

Kinetic Parameters

µg/mL

M

µg/mL

M

k

t1/2

Acidic

312

5.99E-4

12.5

4

7.69E-6

0.34

2.0 hr

Neutral

302

5.81E-4

16.1

4

7.69E

0.28

2.5 hr

Basic

302

5.81E-4

9.0

4

7.69E

0.48

1.5 hr

Conclusions:
The test material should be considered hydrolytically unstable at pH 2, 5-6, and 9 at 23 °C, based on the OECD stability criterion of hydrolysis half-life greater than 1 year at 25 °C (OECD, 2004).

Description of key information

The registered substance was determined to be hydrolytically unstable at pH 2, 5-6 and 9 at 23 °C.

Key value for chemical safety assessment

Additional information

The hydrolytic stability of the test material was investigated in accordance with OECD 111 under GLP conditions, though the study plan was not fully executed because of low water solubility and rapid hydrolysis. Alternative methods were therefore used (Vizon SciTec Inc., 2005). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

First order half-lives of the test material in acetonitrile:aqueous solution (60: 40, v/v) at 23 °C are 1.5, 2.0 and 2.5 h at pH 4, 7 and 9, respectively. Given that the estimated half-lives were obtained at high acetonitrile co-solvent concentration, and that the hydrolysis rates should be faster in the absence of co-solvent (due to the higher concentration of water), the test material should be considered hydrolytically unstable at pH 2, 5 -6 and 9 at 23 °C, based on the OECD stability criterion of hydrolysis half-life greater than 1 year at 25 °C (OECD, 2004).

The half-life of the primary hydrolysis product was extrapolated from a preliminary test at 50 °C and was considered to be > 1 year at pH 4, 7 and 9 at ambient conditions.

 

The hydrolytic stability of the primary hydrolysis product BPA-TA (4,4-Bisphenol A Tetra-Acid, CAS No. 38103-05-8) was investigated in accordance with OECD 111 under GLP conditions (Springborn Smithers Laboratories, 2009). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Less than 10 % degradation of the substance was observed after 5 days (half-life greater than 1 year) in the sterile pH 4, pH 7 and pH 9 buffer samples. Based on the results of this study, the test material was considered to be hydrolytically stable and no additional testing was required.