Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Additional physico-chemical information

Administrative data

Endpoint:

other: Validation of an Analytical Method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd November to 5th December 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No guideline but specifically developed to be used in line with the test material for various endpoints. Done to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

no guideline required

Principles of method if other than guideline:

Analytical Method Validation

Linearity of Response:
Approximately 50 mg of WRS-2390 was accurately weighed into a 100 ml volumetric flask then dissolved and made to volume using acetonitrile to give a ca 500 mg.l-1 solution. Sonication was used to aid dissolution. The ca 500 mg.l-1 solution was diluted by adding 2.5 ml to a 50 ml volumetric flask and made to volume using 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) to give a ca 25 mg.l-1 solution. This solution was then diluted as detailed in the following table:
Standard Identity Concentration of Stock Solution used (mg.l-1) Volume Diluted (ml) Final Volume (ml) *Nominal Concentration (mg.l-1)
B1 25 4 50 1.42
B2 25 3 50 1.07 B3 25 2 50 0.71
B4 25 1 50 0.36
B5 25 1 100 0.18
B6 2 5 100 0.071
B7 2 2.5 100 0.036
* corrected for test item purity (71.2 %)

Each standard was made to volume using 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v). Each standard solution was analysed, in duplicate, according to the chromatographic conditions detailed in Analytical Method No. 4275.

System Precision:
Aliquots (n=10) of WRS-2390 in 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) at a concentration of ca 1.43 mg.l-1 (prepared as detailed as above) were analysed according to the chromatographic conditions detailed in Analytical Method No. 4275.

Assay Accuracy:
Six aliquots (ca 50 mg) of WRS-2390 were accurately weighed into separate 100 ml volumetric flasks then dissolved and made to volume using acetonitrile to give ca 500 mg.l-1 solutions. Sonication was used to aid dissolution. The ca 500 mg.l-1 solutions were then individually diluted by adding 2.5 ml to separate 50 ml volumetric flasks and made to volume using 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) to give ca 25 mg.l-1 solutions. These solutions were then individually diluted by adding 2 ml to separate 50 ml volumetric flasks and made to volume using 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) to give ca 1 mg.l-1 (0.71 mg.l-1 purity corrected) solutions. The ca 1 mg.l-1 (0.71 mg.l-1 purity corrected) solutions were analysed, in duplicate, according to the chromatographic conditions detailed in Analytical Method No. 4275.

Aqueous Assay Accuracy Samples:
Aliquots (9 x ca 50 mg) of WRS-2390 were accurately weighed into separate 100 ml volumetric flasks. Triplicate aliquots were dissolved and made to volume using Milli RO water and algae and daphnia media. Each solution was sonicated to ensure complete dissolution then diluted using the corresponding aqueous media to give ca 1 mg.l-1 (ca 0.71 mg.l-1, purity corrected concentration) solutions.
In addition, an aliquot of each stock solution was diluted using 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) to give ca 1 mg.l-1 (ca 0.71 mg.l-1, purity corrected concentration) solutions.
The ca 1 mg.l-1 (ca 0.71 mg.l-1, purity corrected concentration) solutions were analysed, in duplicate, according to the chromatographic conditions detailed in Analytical Method No. 4275.

Stability of WRS-2390 in Solution:
The stability of WRS-2390 in 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, V/V) at concentrations of ca 1.43 mg.l-1, ca 0.71 mg.l-1 ca 0.036 mg.l-1 was investigated.
The stability of WRS-2390 in Milli RO water and algae and daphnia media at a concentration of ca 0.71 mg.l-1 (purity corrected concentration) was investigated after 24 h and 72 h storage at ambient laboratory temperature.
The stability of WRS-2390 prepared in Milli RO water and algae and daphnia media then diluted to ca 0.71 mg.l-1 using 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) was investigated.
The samples were analysed according to the chromatographic conditions detailed in Analytical Method No. 4275 after 24 h and 72 h storage at ambient laboratory temperature.

System Specificity:
Aliquots of blank Milli RO water and algae and daphnia media were directly injected onto the HPLC column and analysed under the chromatographic conditions detailed in Analytical Method No. 4275.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.

Results and discussion

Results:

Analytical Method Validation:
The analytical method was successfully validated over the purity corrected concentration range 1.432 mg.l-1 to 0.036 mg.l-1 (Analytical Method No. 4275).

Linearity of Response:
The correlation coefficient was determined to be 0.99992 over the range 1.432 mg.l-1 to 0.036 mg.l-1 indicating that a linear response had been achieved.

System Precision:
Replicate injections (n=10) of WRS-2390 in 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) at a concentration of 1.432 mg.l-1 were performed. The coefficient of variation (CV) was calculated to be 0.23 %.

Assay Accuracy:
The overall mean percentage recovery from the purity corrected nominal concentration for the assay accuracy samples was calculated to be 101.62 % (CV = 1.18 %).

Aqueous Assay Accuracy Samples:
The overall mean percentage recovery from the purity corrected nominal concentration for samples prepared and diluted using Milli RO water and algae and daphnia media was calculated to be 83.87 % (CV = 1.92 %), 82.94 % (CV = 2.39 %) and 81.32 % (CV = 9.33 %), respectively.
The overall mean percentage recovery from the purity corrected nominal concentration for samples prepared in Milli RO water and algae and daphnia media then diluted using 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) to give a purity corrected concentration of ca 0.71 mg.l-1 was calculated to be 101.78 % (CV = 2.23 %), 100.47 % (CV = 4.02 %) and 97.52 % (CV = 1.96 %), respectively.

Stability of WRS-2390 in Solution:
Solutions of WRS-2390 in 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) at concentrations of ca 1.43 mg.l-1, ca 0.71 mg.l-1 and ca 0.036 mg.l-1 were analysed after 24 h and 72 h storage at ambient laboratory temperature. The mean percentage recoveries from the purity corrected nominal concentration were determined to be 103.19 % (CV = 3.46 %) and 101.39 % (CV = 2.82 %) after 24 h and 72 h storage, respectively.
WRS-2390 in 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) was considered to be stable for at least 72 h at ambient laboratory temperature.
The stability of WRS-2390 in aqueous media was determined after 24 h and 72 h storage at ambient laboratory temperature.

Mean percentage recoveries from the T0 concentrations were determined to be 100.80 % (CV = 2.49 %), 98.29 % (CV = 2.16 %) and 96.86 % (CV = 2.62 %) for samples prepared and diluted in Milli RO water and algae and daphnia media, respectively, after 24 h storage at ambient laboratory temperature.
Mean percentage recoveries from the T0 concentrations were determined to be 101.78 % (CV = 1.06 %), 101.28 % (CV = 0.89 %) and 101.28 % (CV = 1.37 %) for samples prepared in Milli RO water and algae and daphnia media then diluted with 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v), respectively, after 24 h storage at ambient laboratory temperature.

Mean percentage recoveries from the T0 concentrations were determined to be 98.86 % (CV = 2.25 %), 98.73 % (CV = 2.75 %) and 96.90 % (CV = 1.63 %) were achieved for samples prepared and diluted in Milli RO water and algae and daphnia media, respectively, after 72 h storage at ambient laboratory temperature.

Mean percentage recoveries from the T0 concentrations were determined to be 101.93 % (CV = 0.51 %), 101.95 % (CV = 0.65 %) and 102.40 % (CV = 0.37 %) for samples prepared in Milli RO water and algae and daphnia media then diluted with 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v), respectively, after 72 h storage at ambient laboratory temperature.

WRS-2390 was considered to be stable in aqueous media and aqueous media diluted with 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v) for at least 72 h at ambient laboratory temperature.

System Specificity:
Analysis of blank Milli RO water and algae and daphnia media indicated that there was no significant interference from these media with test item.

Limit of Quantification:
The limit of quantification (LOQ) is defined as the concentration that gives a signal to noise ratio of ca 10. A typical LOQ was calculated to be 0.009 mg.l-1.

Limit of Detection:
The limit of detection (LOD) is defined as the concentration that gives a signal to noise ratio of ca 3. A typical LOD was calculated to be 0.003 mg.l-1.

Tail Factor and Column Efficiency:
A typical tail factor was calculated to be 1.38. A typical column efficiency was determined to be 199779.

Any other information on results incl. tables

Analytical Method No.4275 summary: 

Solutions of WRS-2390 are assayed by high performance liquid chromatography. Detection is by uv and external standardisation is used. Other instrumentation may be used providing adequate accuracy and precision can be achieved.

Applicant's summary and conclusion

Conclusions:

The analytical method was successfully validated over the purity corrected concentration range 1.42 mg.l-1 to 0.036 mg.l-1. The analytical method was determined to be suitable for the analysis of WRS-2390 in 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v). The analytical method is also suitable for analysis of test item prepared in Milli RO water and algae and daphnia media then diluted with 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v).

WRS-2390 was determined to be stable in 0.1% phosphoric acid in Milli Q water/acetonitrile (9:1, v/v), Milli RO water and algae and daphnia media at ambient laboratory temperature for at least 72 h.

Executive summary:

An analytical method to determine the concentration of WRS-2390 in aqueous media has been successfully validated.

Initially, the analytical method was validated and found suitable and that the system response was linear over teh concentration range tested. System Precision was established and the coefficient of variation calculated to be 0.23%. During the determination of the assay accuracy the mean percentage from nominal was 101.62%. The method was determined as being suitable for use with Milli rO water and media used in aquatic toxicity tests,as during validation work mean percentage recoveries were determined to be 97.52% to 101.78%. the substance was found to be stable for at least 72h under laboratory conditions in Milli RO water and appropriate media used for aquatic toxicity tests on algae and daphnia.