4-benzyloxy-2-nitro-beta-pyrrolidinostyrene

Additional physico-chemical information

Administrative data

Endpoint:

other: Half-life in various aqueous media

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 03/09/2018 to 06/09/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Principles of method if other than guideline:

The stability of the test item in aqueous media was determined by measuring the test item and its degradation products concentration over time, using a validated HPLC method.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pyrrolidine, 1-[2-[2-nitro-4-(phenylmethoxy)phenyl]ethenyl]-

Cas Number:

99474-22-3

Molecular formula:

C19H20N2O3

IUPAC Name:

Pyrrolidine, 1-[2-[2-nitro-4-(phenylmethoxy)phenyl]ethenyl]-

Results and discussion

Results:

The standard in all media showed insufficient stability. After 4 hours at the latest, more than half of the initial concentration of the test item had decomposed. Only in the hydrolysis buffer pH 9 and in the alga test medium a low signal (concentration < LOQ) of the test item could be detected after 24 h.
To perform a half-life calculation, at least 3 measured values are required, therefore only the half-life of the test item in the media demineralized water and hydrolysis buffer solution pH 9 can be calculated.
The half life of the test item was determined considering a first order kinetics.

The calculated half-life of the test item in demineralized water is:
t1/2 (demin. H2O; 25 °C) = 1.2 ± 0.7 h

The calculated half-life of the test item in hydrolyse buffer pH 9 is:
t1/2 (demin. H2O; 25 °C) = 3.1 ± 0.2 h

Any other information on results incl. tables

RESULTS

Stability in demineralized water

Nominal Concentration [mg/L]		Dilution factor		Peak Area [mAU*min]		Concen- tration [mg/L]		Mean Concentration [mg/L]		Stability [%]
50 t = 0 h		none		6.2270		39.18		36.58		100
			5.9970		37.76
			5.6509		35.62
			5.3531		33.78
50 t = 0.5 h		none		4.2755		27.12		25.83		70.6
			4.1483		26.33
			3.9402		25.05
			3.9038		24.82
50 t =4 h		none		1.5530		10.29		9.58		26.2
			1.5415		10.22
			1.3235		8.88
			1.3297		8.91
50 t = 24 h		none		n.a.		n.a.		n.a.		0.0
			n.a.		n.a.
			n.a.		n.a.
			n.a.		n.a.

 

The test item showed a very low stability in demineralized water. In addition to the decomposition of the test item, degradation products (DP) could be detected. The following table reports the retention times and the relative areas at the corresponding points in time of the test item and the degradation products.

Sample point [h]	Test item [%]	DP 1 [%]	DP 2 [%]	DP 3 [%]	DP 4 [%]	DP 5 [%]	DP 6 [%]
Retention time [min]	1.92	0.77	0.99	1.26	0.56	1.04	1.39
0	94.74	n.a	5.26	n.a	n.a	n.a	n.a
0.5	89.46	n.a	10.54	n.a	n.a	n.a	n.a
4	54.92	4.85	37.32	2.91	n.a	n.a	n.a
24	n.a	9.82	32.03	11.54	18.85	24.04	3.73

 

Stability in hydrolysis buffer solution pH 4

The test item immediately decomposed with the addition of the hydrolysis buffer solution pH 4. No signal of the test object could be detected in the chromatogram, only degradation products (DP) could be detected. The following table reports the retention times and the relative areas at the corresponding points in time of the degradation products.

Sample point [h]	Test item [%]	DP 1 [%]	DP 2 [%]	DP 3 [%]	DP 4 [%]
Retention time [min]		0.78	0.99	1.26	0.58
0	n.a	12.34	87.66	n.a	n.a
0.5	n.a	12.97	87.03	n.a	n.a
4	n.a	11.81	81.41	6.09	n.a
24	n.a	9.78	67.95	13.35	8.92

 

Stability in hydrolysis buffer solution pH 7

The test item immediately decomposed with the addition of the hydrolysis buffer solution pH 7. No signal of the test object could be detected in the chromatogram, only degradation products (DP) could be detected. The following table reports the retention times and the relative areas at the corresponding points in time of the degradation products.

Sample point [h]	Test item [%]	DP 1 [%]	DP 2 [%]	DP 3 [%]	DP 4 [%]
Retention time [min]		0.78	0.99	1.39	0.56
0	n.a	12.10	87.90	n.a	n.a
0.5	n.a	12.91	87.09	n.a	n.a
4	n.a	10.60	81.41	8.00	n.a
24	n.a	7.75	57.03	13.21	22.01

 

Stability in hydrolysis buffer solution pH 9

Nominal Concentration [mg/L]	Dilution factor	Peak Area [mAU*min]	Concen- tration [mg/L]	Mean Concentration [mg/L]	Stability [%]
50 t = 0 h	none	7.5369	47.28	46.69	100
		7.6106	47.73
		7.1660	44.98
		7.3281	45.99
50 t = 0.5 h	none	6.7903	42.66	41.76	89.8
		6.7398	42.35
		6.4459	40.53
		6.5996	41.48
50 t =4 h	none	2.8297	18.18	18.01	38.7
		2.8882	18.55
		2.7213	17.51
		2.7640	17.78
50 t = 24 h	none	n.a.	0.00	< LOQ	0.0
		n.a.	0.00
		0.5887	< LOQ
		0.5818	< LOQ

 

The test item showed a low stability in hydrolysis buffer solution pH 9. In addition to the decomposition of the test item, degradation products (DP) could be detected. The following table reports the retention times and the relative areas at the corresponding points in time of the test item and the degradation products.

Sample point [h]	Test item [%]	DP 1 [%]	DP 2 [%]	DP 3 [%]	DP 4 [%]	DP 5 [%]
Retention time [min]	1.92	0.75	1.00	1.21	0.56	1.04
0	99.12	n.a	0.88	n.a	n.a	n.a
0.5	97.46	n.a	2.54	n.a	n.a	n.a
4	64.49	3.06	26.68	5.77	n.a	n.a
24	9.29	8.54	n.a.	10.18	10.47	61.52

 

Stability in algal test medium

Nominal Concentration [mg/L]	Dilution factor	Peak Area [mAU*min]	Concen- tration [mg/L]	Mean Concentration [mg/L]	Stability [%]
50 t = 0 h	none	7.5662	47.46	44.26	100
		7.3644	46.21
		6.6201	41.61
		6.6467	41.77
50 t = 24 h	none	0.0838	< LOQ	< LOQ	0.0
		0.0847	< LOQ
		0.0839	< LOQ
		0.0818	< LOQ
50 t = 72 h	none	n.a.	n.a.	n.a.	0.0
		n.a.	n.a.
		n.a.	n.a.
		n.a.	n.a.

 

The test item showed a low stability in algal test medium. In addition to the decomposition of the test item, degradation products (DP) could be detected. The following table reports the retention times and the relative areas at the corresponding points in time of the test item and the degradation products.

 

Sample point [h]	Test item [%]	DP 1 [%]	DP 2 [%]	DP 3 [%]	DP 4 [%]	DP 5 [%]	DP 6 [%]
Retention time [min]	1.92	0.70	0.95	1.26	0.53	1.04	1.39
0	77.52	19.49	2.63	n.a	0.35	n.a	n.a
24	2.24	45.34	21.81	15.03	11.17	n.a	4.41
72	n.a	30.14	10.32	18.50	28.70	4.00	5.32

 

Stability in daphnia test medium

Nominal Concentration [mg/L]	Dilution factor	Peak Area [mAU*min]	Concen- tration [mg/L]	Mean Concentration [mg/L]	Stability [%]
50 t = 0 h	none	7.2693	45.62	44.21	100
		7.1750	45.04
		6.9313	43.53
		6.7850	42.63
50 t = 24 h	none	n.a.	n.a.	n.a.	0.0
		n.a.	n.a.
		n.a.	n.a.
		n.a.	n.a.
50 t = 48 h	none	n.a.	n.a.	n.a.	0.0
		n.a.	n.a.
		n.a.	n.a.
		n.a.	n.a.

 

The test item showed a very low stability in algal test medium. In addition to the decomposition of the test item, degradation products (DP) could be detected. The following table reports the retention times and the relative areas at the corresponding points in time of the test item and the degradation products.

Sample point [h]	Test item [%]	DP 1 [%]	DP 2 [%]	DP 3 [%]
Retention time [min]	1.93	0.70	0.96	1.21
0	77.20	20.04	2.76	n.a
24	n.a	51.20	36.09	12.72
48	n.a	45.22	43.50	11.28

 

Assessment of stability

The standard in all media showed insufficient stability. After 4 hours at the latest, more than half of the initial concentration of the test item had decomposed. Only in the hydrolysis buffer pH 9 and in the alga test medium a low signal (concentration < LOQ) of the test item could be detected after 24 h.

To perform a half-life calculation, at least 3 measured values are required, therefore only the half-life of the test item in the media demineralized water and hydrolysis buffer solution pH 9 can be calculated.

The half life of the test item was determined considering a first order kinetics.

 

The calculated half-life of the test item in demineralized water is:

t_1/2(demin. H2O; 25 °C) = 1.2 ± 0.7 h

 

The calculated half-life of the test item inhydrolyse buffer pH 9is:

t_1/2(demin. H2O; 25 °C) = 3.1 ± 0.2 h

Applicant's summary and conclusion

Conclusions:

The standard in all media showed insufficient stability. After 4 hours at the latest, more than half of the initial concentration of the test item had decomposed. Only in the hydrolysis buffer pH 9 and in the algal test medium a low signal (concentration < LOQ) of the test item could be detected after 24 h.
For the medias demineralised water and hydrolyse buffer pH 9 the half-life t1/2 was calculated:
t1/2 (demin. H2O; 25 °C) = 1.2 ± 0.7 h

t1/2 (hydrolyse buffer solution pH 9; 25 °C) = 3.1 ± 0.2 h

No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.

Executive summary:

The stability of the test item in aqueous media (demineralized water, Hydrolysis Buffer Solutions pH 4, pH 7, pH 9, Algal, and Daphnia test medium) was determined by measuring the concentration of the test item and its degradation products over time, using a validated HPLC method.

Due to the poor solubility of the test item, the medium / acetonitrile ratio must be 50 / 50 % (v/v).

The stability was determined by measuring a standard solution containing 50 mg/L :

-       in demineralized water, Hydrolysis Buffer Solutions pH 4, pH 7, pH 9 at the time levels 0 h, 0.5 h, 4 h and 24 h, stored in closed bottles at room temperature (24.2 – 25.8 °C).

-       in algal test medium at the time levels 0 h, 24 h and 72 h, stored in glass beakers covered with perforated plastic wrap at 21 – 23.3 °C.

-       in daphnia test medium at the time levels 0 h, 24 h and 48 h, stored in glass beakers covered with perforated plastic wrap at 22.2 ± 23.5 °C.

The standard in all media showed insufficient stability. After 4 hours at the latest, more than half of the initial concentration of the test item had decomposed. Only in the hydrolysis buffer pH 9 and in the algal test medium a low signal (concentration < LOQ) of the test item could be detected after 24 h.

For the medias demineralized water and hydrolysis buffer pH 9 the half-life t1/2 was calculated:       

t_1/2(demin. H2O; 25 °C) = 1.2 ± 0.7 h

 

t_1/2(hydrolysis buffer solution pH 9; 25 °C) = 3.1 ± 0.2 h

 

No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.