2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one

Adsorption / desorption

Link to relevant study record(s)

Reference

Endpoint:

adsorption / desorption: screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-28 until 2016-11-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 121 (Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC))

Version / remarks:

January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of method:

HPLC estimation method

Specific details on test material used for the study:

CA3747 tech., 2-amino-6-methyl-5-oxo-4,5-dihydro-s-triazole-(1,5-a)-pyrimidine

Radiolabelling:

no

Test temperature:

25 °C

Details on study design: HPLC method:

EQUIPMENT
- Apparatus: Thermo Scientific Dionex HPLC equipped with Ultimate 3000 UV-detector
- Type, material and dimension of analytical column: Zorbax CN, 250 x 4.6 mm, 5 µm, Agilent Technologies with guard column (12.5 x 4.6 mm) of the same material
- Detection system: UV

MOBILE PHASES
- Type: methanol : 0.01 M Tris-HCl buffer pH 8 (50:50, v:v) or methanol : 0.01 M citrate buffer pH 4 (50:50, v:v) (additional with methanol:water (50:50, v:v))
- Experiments with additives carried out on separate columns: no
- pH: 4, 8
- Solutes for dissolving test and reference substances: acetonitrile (stock solutions of reference compounds), methanol:water (50:50, v:v) (stock solution of test item), methanol:buffer pH 4 (50:50, V,v), methanol:buffer pH 8 (50:50, v:v) or methanol:water (50:50, v:v) (test item solution, reference compound solutions)

DETERMINATION OF DEAD TIME
- Method: by inert substances which are not retained by the column (formamide)

REFERENCE SUBSTANCES
- Identity: acetanilide, atrazine, isoproturon, linuron, phenol, triadimenol A

DETERMINATION OF RETENTION TIMES
- Quantity of test substance introduced in the column: 2.5 µg (50 µL of a 50 µg/mL solution)
- Quantity of reference substances: 0.5 µg of acetanilide, atrazine and phenol (50 µL of 10 µg/mL solutions), 1.0 µg of linuron (50 µL of a 20 µg/mL solution) and 2.5 µg of isoproturon and triadimenol A (50 µL of 50 µg/mL solutions)
- Intervals of calibration: All reference compounds were injected before (injection A) and after (injection B) the test substance to confirm that retention times had not drifted.

REPETITIONS
- Number of determinations: Two replicates (injections) per reference compounds and test item

EVALUATION
- Calculation of capacity factors k': k' and log k' were calculated using the measured retention times
- Determination of the log Koc value: correlation graph was produced by plotting the log k' value versus the log Koc value for each reference compound; literature log Koc values were obtained from OECD Guideline 121. A log Koc value for the test item was then extrapolated from the correlation graph.

Type:

Koc

Value:

< 32

Details on results (HPLC method):

Although the retention times of CA3747A at the two pH-conditions pH 4 and pH 8 were different, both retention times of CA3747A were generally shorter compared to the retention times of all the reference substances used. At pH 8 CA3747A was un-retained and had a shorter retention time than formamide which was used to determine the dead time of the column. The reference substances included acetanilide and phenol which have the lowest log k’ values and log Koc values listed in the OECD guideline 121. This means that CA3747A is below the calibration range of the recommended reference compounds. In the guideline it is specified that only log Koc values ranging from 1.5 to 5.0 can be determined. For this reason, the adsorption coefficient of CA3747A can be specified to be log Koc < 1.5 and Koc < 32.

An additional run using methanol:water (50:50, v:v) as mobile phase confirm the result that the adsorption coefficient of CA3747A can be specified to be log Koc < 1.5 and Koc < 32.

Capacity factor (k') and adsorption coefficient (Koc) values for CA3747A of a HPLC-run using methanol:buffer pH 8 as mobile phase

Compound	Retention Time UV-detector of each injection (min)	Average^1Retention Time UV-detector (min)	Capacity Factor k’	Log10k’	Log10Koc
Formamide	2.96	2.965	0.000	Unretained	Unretained*
	2.97
Acetanilide	4.21	4.21	0.420	-0.377	1.25*
	4.21
Phenol	4.49	4.50	0.516	-0.287	1.32*
	4.50
Atrazine	5.63	5.64	0.901	-0.046	1.81*
	5.64
Isoproturon	6.45	6.46	1.177	0.071	1.86*
	6.46
Triadimenol A	9.15	9.15	2.086	0.319	2.40*
	9.15
Linuron	11.85	11.87	3.002	0.477	2.59*
	11.88
CA3747A	2.33	2.33	-0.214	#	#
	2.33

¹Mean retention time of two injections per sample

* Reference Compound Literature values taken from OECD Guideline 121

# Not possible to calculate; test substance was unretained using methanol:buffer pH 8

 Equation of calibration plot: y = 0.6084x – 1.1123

Coefficient of determination of calibration plot = 0.9893

Capacity factor (k') and adsorption coefficient (Koc) values for CA3747A of a HPLC-run using methanol:buffer pH 4 as mobile phase

Compound	Retention Time UV-detector of each injection (min)	Average^1Retention Time UV-detector (min)	Capacity Factor k’	Log10k’	Log10Koc
Formamide	2.95	2.96	0.000	Unretained	Unretained*
	2.96
Acetanilide	4.21	4.22	0.426	-0.370	1.25*
	4.22
Phenol	4.51	4.51	0.526	-0.279	1.32*
	4.51
Atrazine	5.63	5.64	0.909	-0.042	1.81*
	5.65
Isoproturon	6.42	6.42	1.171	0.069	1.86*
	6.41
Triadimenol A	9.09	9.10	2.078	0.318	2.40*
	9.10
Linuron	11.90	11.90	3.025	0.481	2.59*
	11.89
CA3747A	3.11	3.11	0.052	-1.280	-0.35†
	3.11

¹Mean retention time of two injections per sample

* Reference Compound Literature values taken from OECD Guideline 121

† Calculated from equation of calibration plot (see below) although log k’ of CA3747A is below the log k’ values of all reference substances (below calibration)

 Equation of calibration plot: y = 0.6033x – 1.0998

Coefficient of determination of calibration plot = 0.9895

Validity criteria fulfilled:

yes

Conclusions:

The method for the estimation of the adsorption coefficient by HPLC was not suitable for the test item CA3747A since the test item has a log Koc value <1.5. According to OECD 121, only log Koc values ranging from 1.5 to 5.0 can be determined by the method for the estimation of the adsorption coefficient by HPLC.

However, CA3747A can be classified as “highly” mobile.

Executive summary:

STUDY DESIGN

The adsorption coefficients (Koc) of CA3747A were estimated according to the OECD Guideline for Testing of Chemicals, 121, Estimation of the Adsorption Coefficient (Koc) on Soil and on Sewage Sludge using High Performance Liquid Chromatography (HPLC), January 2001.

The test item was chromatographed on a cyanopropyl solid phase HPLC column concurrently with 6 reference compounds of known adsorption coefficient values.

Because the test item had a weak acidic pKa of 6.7 at 25°C, buffered mobile phase at two pH conditions were used during this study to estimate the adsorption coefficients for both ionised and non-ionised forms of the test item: One HPLC run was carried out using methanol:buffer pH 8 (50:50, v:v) and a second HPLC run was performed using methanol:buffer pH 4 (50:50, v:v) as mobile phase. Following chromatography, a correlation graph for each HPLC run was produced by plotting the Log k’ value versus the Log Koc value for each compound. Literature Log Koc values were obtained from OECD Guideline 121.

Log Koc values for the test item were then extrapolated from the correlation graphs.

RESULTS

Good correlations between Log k’ and Log Koc were observed for the reference compounds and the pH-conditions tested with coefficients of determination r² of 0.9893 and 0.9895 at pH 8 and pH 4, respectively.

Although the retention times of CA3747A at the two pH-conditions pH 4 and pH 8 were different, both retention times of CA3747A were generally shorter compared to the retention times of all the reference substances used. At pH 8 CA3747A was un-retained and had a shorter retention time than formamide which was used to determine the dead time of the column. The reference substances included acetanilide and phenol which have the lowest log k’ values and log Koc values listed in the OECD guideline 121. This means that CA3747A is below the calibration range of the recommended reference compounds. In the guideline it is specified that only log Koc values ranging from 1.5 to 5.0 can be determined. For this reason, the adsorption coefficient of CA3747A can be specified to be log Koc < 1.5 and Koc < 32.

CONCLUSION

The method for the estimation of the adsorption coefficient by HPLC was not suitable for the test item CA3747A since the test item has a log Koc value < 1.5.

Log Koc of CA3747A < 1.5

Koc of CA3747A < 32

However, CA3747A can be classified as "highly" mobile.

Description of key information

The log Koc of the substance was found to be <1.5 in a valid and reliable study according to OECD TG 121.