Diallyl hexahydrophthalate

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2018 to 23 April 2018 (Experimental start to experimental end)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test item name: MDAC (Diallyl hexahydrophthalate)
- Source of test material: Osaka Soda Co., Ltd. Japan
- Lot/batch No.of test material: 40201
- Expiration date of the lot/batch: 26 January 2019
- Purity test date: 26 September 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled at room temperature, protected from light
- Stability under test conditions: Assumed stable for the durationj of the test
- Solubility and stability of the test substance in the solvent/vehicle: Not relavent
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not relavent

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
- Sampling method: 250 mL sterile solutions were prepared (50 μg/mL of test item concentration in each buffer with 1% ACN content). The solutions were ultrasonicated and filtered on 0.22 μm filter. The pH of each buffer solution was checked with a calibrated pH meter.

Solutions were transferred into screw cap tubes. Three from each test solution and one control tubes were prepared. The tubes were thermostated at 49.5 – 49.9°C. The reaction solutions were analysed at the start of the test with five replicate samples and after five days with two replicate samples per tubes. The 5 day samples were cooled to room temperature. Samples for the start were diluted with eluent 10 fold and for the
end 5 fold, then they were analysed with the above presented HPLC-UV method. The control samples were analysed directly, without any dilution.

- Sampling intervals/times for pH measurements: Experimental satrt and at 5 days
- Sampling intervals/times for sterility check: Not specified
- Sample storage conditions before analysis: None
- Other observation, if any (e.g.: precipitation, color change etc.): None specified

Preliminary study:

RESULTS OF THE METHOD VALIDATION (17/277-316AN)
Selectivity: No interfering component was observed
Reinjection repeatability (7 injections): CV% ≤ 0.7%
Linear range: 0.5 – 50 μg/mL
Limit of Quantification (LOQ): 0.5 μg/mL
Recovery of the test item from pH 4 buffer (50 mg/L): 99%
Recovery of the test item from pH 7 buffer (50 mg/L): 94%
Recovery of the test item from pH 9 buffer (50 mg/L): 92%
Precision in pH 4 buffer: 1.2%
Precision in pH 7 buffer: 2.7%
Precision in pH 9 buffer: 1.2%
Stability of the samples in the autosampler: At least 97 hours
Stock solution stability at 5 ± 3°C: At least 4 days

TEST CONDITIONS
250 mL sterile solutions were prepared (50 μg/mL of test item concentration in each buffer with 1% ACN content). The solutions were ultrasonicated and filtered on 0.22 μm filter. The pH of each buffer solution was checked with a calibrated pH meter.

Solutions were transferred into screw cap tubes. Three from each test solution and one control tubes were prepared. The tubes were thermostated at 49.5 – 49.9°C. The reaction solutions were analysed at the start of the test with five replicate samplesand after five days with two replicate samples per tubes. The 5 day samples were cooled to room temperature. Samples for the start were diluted with eluent 10 fold and for the end 5 fold, then they were analysed with the above presented HPLC-UV method. The control samples were analysed directly, without any dilution.

Transformation products:

no

Key result

pH:

4

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

7

Remarks on result:

hydrolytically stable based on preliminary test

Key result

pH:

9

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

Test temperature: 49.5 – 49.9°C
Hydrolysis was examined at three different pH values: 4, 7 and 9 in the dark.

Buffer solutions:
pH 4.0: 1 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Potassium hydrogen
phthalate was diluted to 500 mL with ultrapure water
pH 7.0: 73.9 mL 0.2M Sodium hydroxide and 125 mL 0.2M Potassium dihydrogen
phosphate was diluted to 500 mL with ultrapure water
pH 9.0: 53.5 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Boric acid and Potassium
chloride was diluted to 500 mL with ultrapure water

These sterile buffer solutions were prepared using reagent grade chemicals and ultrapure,
sterile water.
The pH of each buffer solution was checked with a calibrated pH meter.
The hydrolysis reaction was carried out using a dark thermostat to avoid photolyticeffects.
Nitrogen was bubbled into the water before the preparation of the solutions in
order to exclude oxygen.

Calibration: The calibration series was prepared from stock solution (~0.5 mg Test Item / 1 mL acetonitrile) and working solution (50 μg/mL) with eluent. It was measured at every analytical occasions. Concentrations of the calibration samples were 0.5, 1, 2, 5, 10, 25 and 50 μg/mL.

Summary of Standard Curve Parameters:
Analytical occasion Intercept Slope Correlation Coefficient
18 April 2018 0.195 3.78 1.0000
23 April 2018 0.444 3.71 1.0000

The hydrolysis test was performed at 49.5 – 49.9°C, at pH 4, 7 and 9.

pH	Sampling time (day)	Concentration of Test Item (μg/mL)			Mean of the measured pH
		Results of the separate test vessels	Mean with the 95% confidence intervals (μg/mL)	End/Start (%)
4.0	0 (start)	Control buffer		-	4.01
		50.1	49.8 ± 0.35	-	4.01
		49.9
		49.6
		49.9
		49.4
	5	Control buffer		-	4.05
		50.1	50.2 ± 0.16	101	4.05
		50.1
		50.1
		50.1
		50.5
		50.2
7.0	0 (start)	Control buffer		-	6.99
		50.3	50.4 ± 0.24	-	6.99
		50.3
		50.7
		50.4
		50.3
	5	Control buffer		-	7.03
		51.5	50.4 ± 0.63	100	7.05
		50.1
		50.0
		49.9
		50.5
		50.1
9.0	0 (start)	Control buffer		-	9.00
		50.5	50.2 ± 0.30	-	9.00
		50.0
		49.9
		50.4
		50.3
	5	Control buffer		-	9.15
		29.1	27.7 ± 0.70	55	9.21
		27.6
		27.3
		27.4
		27.5
		27.6

Validity criteria fulfilled:

yes

Conclusions:

In the course of the preliminary test the observed hydrolysis of MDAC was less than 10% after 5 days at 50 ± 0.5°C at pH 4 and 7, therefore it is considered to be hydrolytically stable under these conditions.

However, 45% of hydrolysis was observed at pH 9 under the same conditions, therefore further additional testing is required for the determination of hydrolysis of MDAC at pH 9 under Study code: 17/277-336AN.

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 July to 23 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

yes

Remarks:

They had no effect on the result of the study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

They had no effect on the result of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Osaka Soda / 40201
- Expiration date of the lot/batch: 26 Jan 2019
- Purity test date: 26 Sept 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature, protected from light
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: no data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: none
- other information: none

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Three or two sample vials were removed with one control sample and analysed at each analytical occasion. pH of the samples was checked. At the end of the test three tubes were submitted for sterility confirmation.

Buffers:

Buffer solution pH 9: 428 mL of 0.2 M Sodium hydroxide and 1000 mL of 0.2 M Boric acid and 0.2 M Potassium chloride was diluted to 4000 mL with ultrapure water.

Details on test conditions:

Test solution: Approximately 250 mg of Test Item was dissolved in 50 mL of ACN. 30 mL of this stock solution (~ 5 mg/mL in ACN) was filled up to 3000 mL with pH 9 buffer solution (~ 50 mg/L in pH 9 buffer with 1% ACN content.) Test solution was sterilised by filtering on a 0.22 µm Millipore filter (Steritop). The pH of the solutions was checked with a calibrated pH meter.

Duration:

742 h

pH:

9

Temp.:

25 °C

Initial conc. measured:

50.7 other: µg/ml

Duration:

504 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

50.7 other: µg/ml

Duration:

161 h

pH:

9

Temp.:

67 °C

Initial conc. measured:

50.7 other: µg/ml

Number of replicates:

1

Positive controls:

no

Negative controls:

no

Transformation products:

no

% Recovery:

83

pH:

9

Temp.:

25 °C

Duration:

742 h

% Recovery:

14

pH:

9

Temp.:

50 °C

Duration:

504 h

% Recovery:

8

pH:

9

Temp.:

67 °C

Duration:

161 h

Key result

pH:

9

Temp.:

25 °C

DT50:

97.8 d

Type:

second order

Key result

pH:

9

Temp.:

50 °C

DT50:

7.6 d

Type:

second order

Key result

pH:

9

Temp.:

67 °C

DT50:

1.7 d

Type:

second order

Validity criteria fulfilled:

not specified

Conclusions:

Based on the results of Tier 1, the hydrolysis rate of MDAC was investigated at pH 9 at 3 different temperatures. The Kobs values and the half-lives of the reaction were determined at 25, 50 and 67°C. The half-lives of the hydrolysis of MDAC at pH 9 were 2348 hours at 25°C, 183 hours at 50°C and 41 hours at 67°C.

Description of key information

This substance is hydrolytically stable.

Key value for chemical safety assessment

Half-life for hydrolysis:

97.8 d

at the temperature of:

25 °C

Additional information

Based on the results of Tier 1, the hydrolysis rate of MDAC was investigated at pH 9 at 3 different temperatures. The k_obsvalues and the half-lives of the reaction were determined at 25, 50 and 67°C. The half-lives of the hydrolysis of MDAC at pH 9 were 2348 hours at 25°C, 183 hours at 50°C and 41 hours at 67°C.