Reaction mass of 1,5-dihydroxy-4-nitro-8-(phenylamino)anthraquinone and 1,8-dihydroxy-4-nitro-5-(phenylamino)anthraquinone

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 04 April 2016 and 23 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC No. 440/2008, 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT 92504/C
Appearance/Physical state: Blue solid
Batch: BOP 01-15
Purity: 86.4 %
Expiry date: 18 November 2020
Storage conditions: Approximately 4 °C, in the dark

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Analysis of the Sample Solutions
The sample solutions were taken from the waterbath at various times and the pH of each solution recorded.
The concentration of the sample solution was determined by high performance liquid chromatography (HPLC).

Buffers:

Specification of Buffer Solutions

Buffer solution(pH) Components Concentration (mmol dm-3)
4 Citric acid 6
Sodium chloride 4
Sodium hydroxide 7
7 Disodium hydrogen
orthophosphate (anhydrous) 3
Potassium dihydrogen orthophosphate 2
Sodium chloride 2
9 Disodium tetraborate 1
Sodium chloride 2
These solutions were subjected to ultrasonication and degassing with nitrogen to minimize dissolved oxygen content

Details on test conditions:

Performance of the Test
Preparation of the Test Solutions
Sample solutions were prepared in stoppered glass flasks at a nominal concentration of 1.0E-4 g/L in the three buffer solutions. A 1% co-solvent of acetonitrile (methanol for preliminary testing) 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 1
Sample solutions at pH 4, 7 and 9 were maintained at 50.0 ± 0.5 °C for a period of at least 120 hours.

Tier 2
Results from the Preliminary test/Tier 1 showed it was necessary to undertake further testing at pH 4, pH 7 and pH 9

Duration:

120 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

ca. 0.1 mg/L

Duration:

120 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

ca. 0.1 mg/L

Duration:

120 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

ca. 0.1 mg/L

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Preliminary study:

The test item concentrations at the given time points are shown in the following tables:
pH 4 at 50.0 ± 0.5 ºC
Time (Hours) Concentration (g/L) % of Mean Initial Concentration
A B A B
0 7.15 x 10-5 7.52 x 10-5 - -
24 4.93 x 10-5 5.35 x 10-5 67.2 72.9
48 5.25 x 10-5 4.99 x 10-5 71.5 68.0
120 3.92 x 10-5 2.34 x 10-5 53.4 31.9
168 3.41 x 10-5 3.21 x 10-5 46.5 43.8
Result: The extent of hydrolysis indicated that a further test (Tier 2) was required to estimate the rate constant and half-life at 25 °C.

pH 7 at 50.0 ± 0.5 ºC
Time (Hours) Concentration (g/L) % of Mean Initial Concentration
A B A B
0 9.27 x 10-5 9.29 x 10-5 - -
24 9.14 x 10-5 9.16 x 10-5 98.5 98.7
48 8.60 x 10-5 8.52 x 10-5 92.7 91.8
120 8.39 x 10-5 8.36 x 10-5 90.4 90.1
192 8.21 x 10-5 8.26 x 10-5 88.4 89.0
264 9.09 x 10-5 9.05 x 10-5 97.9 97.5
Result: The extent of hydrolysis indicated that a further test (Tier 2) was required to estimate the rate constant and half-life at 25 °C. Testing with acetonitrile under the same conditions confirmed that further test (Tier 2) was required.

pH 9 at 50.0 ± 0.5 ºC
Time (Hours) Concentration (g/L) % of Mean Initial Concentration
A B A B
0 9.34 x 10-5 9.31 x 10-5 - -
24 5.45 x 10-5 5.57 x 10-5 58.5 59.8
48 4.56 x 10-5 4.57 x 10-5 48.9 49.0
120 3.28 x 10-5 3.26 x 10-5 35.1 35.0
144 3.12 x 10-5 3.13 x 10-5 33.5 33.6
168 3.17 x 10-5 3.22 x 10-5 34.0 34.5
Result: The extent of hydrolysis indicated that a further test (Tier 2) was required to estimate the rate constant and half-life at 25 °C.

Transformation products:

no

Remarks on result:

not determinable because of methodological limitations

Remarks on result:

not determinable because of methodological limitations

Details on results:

No significant peaks were observed at the approximate retention time of the test item on analysis of any matrix blank solutions. From the purity of the test item, 86.4%, the small peaks eluting after the main peak were considered to be due to impurities. The hydrolysis was carried out at elevated temperatures (50, 60 and 70 °C), where first order loss of test item was not observed. Due to the inconsistencies with the loss of test item with temperature and pH additional testing was carried out at 25 °C. Hydrolysis of the test item at 25 °C produced second order loss of test item at all pH’s. The test item showed hydrolysis to a cut off value, then no significant change in the hydrolysis. At pH 4 the test item reduced to approximately 70 % of the initial concentration, approximately 90 % at pH 7 and approximately 50 % at pH 9. The test item is likely therefore to not hydrolyse but show a tautomeric relationship between the keto-enol and the amino-imine and thus after an initial loss of the test item a tautomeric equilibration. Due to the mechanism observed the half-life determination was beyond the scope of the method and therefore no hydrolysis rate could be calculated.

Validation

Linearity

The linearity of the detector response with respect to concentration was assessed over the nominal concentration range of 0.2 to 10 mg/L (n = 8).  The correlation curve was satisfactory with a first order correlation coefficient (r) of 0.9999 being obtained.  Linearity standards were prepared in methanol not acetonitrile as main method.  No changes in peak area or peak shape were seen, initial preliminary testing was carried out in methanol, however metalation of the test item was seen during hydrolysis, therefore co-solvent was changed to acetonitrile.  Therefore the linear range is still valid with solvent change.

Specificity

Standard blank and sample blank solutions were injected with the samples and no peak was detected at the sample retention time of the test item peak.

Accuracy / Precision

The accuracy / precision of the analytical method was assessed at a nominal concentration of 0.10 mg/L (n = 5).  The results were satisfactory with mean % recovery (accuracy) results of 97.9 % at pH 4, 97.0 % at pH 7 and 98.5 % at pH 9 and relative standard deviation (RSD) (precision) results of 1.68 % at pH 4, 2.65 % at pH 7 and 0.906 % at pH 9. All accuracy and precision used standards prepared in methanol and final step of extraction in methanol, not acetonitrile as main method.  No changes in peak area or peak shape were seen, initial preliminary testing was carried out in methanol, however metalation of the test item was seen during hydrolysis, therefore co-solvent was changed to acetonitrile.

Validity criteria fulfilled:

not applicable

Conclusions:

FAT 92504/C is likely to not hydrolyse but show a tautomeric relationship between the keto-enol and the amino-imine and thus after an initial loss of the test item a tautomeric equilibration.

Executive summary:

Assessment of hydrolytic stability was carried out using a procedure designed to be compatible with Method C.7 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. The test item is likely to not hydrolyse but show a tautomeric relationship between the keto-enol and the amino-imine and thus after an initial loss of the test item a tautomeric equilibration. Due to the mechanism observed the half-life determination was beyond the scope of the method and therefore no hydrolysis rate could be calculated.

Description of key information

Assessment of hydrolytic stability was carried out using a procedure designed to be compatible with Method C.7 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. The test item is likely to not hydrolyse but show a tautomeric relationship between the keto-enol and the amino-imine and thus after an initial loss of the test item a tautomeric equilibration. Due to the mechanism observed the half-life determination was beyond the scope of the method and therefore no hydrolysis rate could be calculated.