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Physical & Chemical properties

Stability in organic solvents and identity of relevant degradation products

Administrative data

Endpoint:
stability in organic solvents and identity of relevant degradation products
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 October 2016 to 17 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Formulations prepared on 04 Oct 2016 at target concentrations of 10 and 220 mg/mL were analysed on the day of preparation. Aliquots of the formulations were stored at room temperature for 6 and 10 days and analysed to assess test substance stability.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Light brown, slightly viscous, liquid
- Storage conditions: Room temperature in the dark

Results and discussion

Test substance stable:
yes
Transformation products:
no

Any other information on results incl. tables

RESULTS

- Under the described chromatographic conditions, the retention time of the test substance was approximately 7.1 minutes.

- Typical chromatograms of a calibration standard, a processed QC sample, a processed formulation sample, and a processed vehicle blank sample are shown in Figure 1 to Figure 4.

- The total analysis time required for each run was 10.0 minutes.

 

SPECIFICITY / SELECTIVITY

- As shown in Figure 4 (attached), and in contrast to the chromatograms shown in Figure 1, Figure 2, and Figure 3 (attached), assay specificity/selectivity was confirmed when HPLC/UV analysis of processed vehicle samples revealed no significant peaks (with S/N > 10) at or near the retention time for the test substance (approximately 7.1 minutes).

 

ASSAY VALIDATION – CALIBRATION REPRODUCIBILITY

- During each of 3 validation sessions, triplicate calibration standards at 5 concentrations were prepared and analysed.

- Single injections were made of each calibration standard. The resulting test item peak area versus theoretical test item concentration data were fit to the linear function using least-squares regression analysis.

- Results of the regression analyses were used to back-calculate the corresponding concentrations from the peak area data.

- As per the protocol, the reproducibility of the calibration curve data was considered valid when 1) the inter-session variability, expressed as RSD, of the back-calculated concentrations at each calibration level was10% RSD, except at the lowest calibration level where15% was acceptable; and 2) the mean back-calculated concentrations at each calibration level were within±10% of the theoretical values (%RE within±10%), except at the lowest calibration level where %RE within±15% was acceptable. The back-calculated concentrations and the associated intra- and inter-session statistics for the test item assay calibration standards are summarised in Table 1 (attached).

- The inter-session variability (RSD) of the back-calculated concentrations ranged from 0.18% to 0.60% RSD. The inter-session mean concentrations had %RE values ranging from -1.0% to 0.32%. Based on the stated criteria, the reproducibility of the calibration data was acceptable.

 

ASSAY VALIDATION – PRECISION AND ACCURACY

- During each of 3 validation sessions, triplicate QC samples at 3 concentrations were prepared and analysed.

- Single injections were made of each processed QC sample. The results of the regression analyses were used to calculate the corresponding concentrations from the QC peak area data.

- The variability (RSD) of the calculated QC concentration data was used as a measure of assay precision, and the difference between the theoretical and calculated mean QC concentrations (%RE) was used as a measure of assay accuracy.

- According to the protocol, the precision of the method was considered acceptable when the inter-session RSD of the calculated concentrations at each QC level was15%, and the accuracy of the method was considered acceptable when the inter-session calculated mean concentration at each QC level had a %RE value within±15%.

- The calculated concentrations and the associated intra- and inter-session statistics for the test item assay QC samples are summarized in Table 2 (attached). The inter-session variability (RSD) of the calculated concentrations of each QC sample (precision) ranged from 0.60% to 1.1% RSD.

- The inter-session mean concentrations of the QC samples had %RE values (accuracy) ranging from -0.76% to -0.39%. Based on the stated criteria, the precision and accuracy of the test item assay were acceptable.

 

ASSAY RUGGEDNESS

- Assay ruggedness, as required by SOP, was successfully demonstrated for this method because at least 2 of the 3 validation sessions were performed by different analysts.

 

ASSAY ACCEPTABILITY

- In addition to the experimental samples, each analytical session consisted of (but was not limited to) calibration standards at 5 concentrations and triplicate QC samples prepared at each of 3 concentrations. In this study, the formulations were prepared at target concentrations of 10 and 220 mg test item361302

/mL, and the QC samples were prepared at nominal concentrations of 10.0, 100, and 220 mg test item/mL.

- According to SOP, for an analytical session to be considered valid, at least two-thirds of the calculated QC concentrations with at least 1 sample at each concentration had to be 85% to 115% of the nominal QC concentration. All reported results were from analytical sessions that met the acceptance criteria.

 

TEST SUBSTANCE STABILITY IN CALIBRATION STANDARDS

- Calibration standards prepared at 5.00 and 100μg/mL and analysed were stored at room temperature for 12 days before re-analysis to assess test substance stability. The mean post-storage concentrations were 100% and 98.8% of the pre-storage values (see Table 3, attached).

- Results met the protocol-specified requirement for stability i.e., the mean post-storage concentration was not < 90% of the pre-storage value.

 

TEST SUBSTANCE STABILITY IN PROCESSED SAMPLES

- Quality control samples prepared at nominal test substance concentrations of 10.0 and 220 mg/mL were processed and analyzed. The processed samples were stored at room temperature for 12 days before re-analysis to assess test substance stability.

- The mean post-storage concentrations were 99.9% and 98.8% of the pre-storage values (Table 3, attached).

- Results met the previously stated protocol-specified requirement for stability.

 

TEST SUBSTANCE HOMOGENEITY AND RESUSPENSION HOMOGENEITY – ASSESSMENT OF FORMULATIONS

- Duplicate samples from the top, middle, and bottom strata of the formulations prepared on 04 Oct 2016 at target test substance concentrations of 10 and 220 mg/mL were analysed to assess test substance homogeneity.

- The formulations that remained after sampling were divided into aliquots as would be used for daily dispensation.

- Representative aliquots were stored at room temperature for 6 and 10 days, at which time the test substance was resuspended by stirring and/or heated sonication.

- Duplicate samples were collected from the top and bottom strata of the aliquots and analysed to assess resuspension homogeneity.

- The homogeneity assessment of the 04 Oct 2016 formulations met the protocol-specified requirement, i.e., the RSD for the mean concentration was10% at a concentration within the acceptable limits (within 85% to 115% of target concentration). The resuspension homogeneity

assessments of the 04 Oct 2016 formulations met the protocol-specified requirement, i.e., the RSD for the mean concentration was10%.

- Results of the homogeneity and resuspension homogeneity analyses are presented in Tables 4, 5 and 6 (attached) and overall statistics are summarised below.

- The homogeneity assessment of the 04 Oct 2016 formulations met the protocol-specified requirement, i.e., the RSD for the mean concentration was10% at a concentration within the acceptable limits (within 85% to 115% of target concentration). The resuspension homogeneity assessments of the 04 Oct 2016 formulations met the protocol-specified requirement, i.e., the RSD for the mean concentration was10%.

 

Homogeneity Assessment of the 04 Oct 2016 Formulations

 

Low group

(10 mg/mL)

High group

(220 mg/mL)

Mean concentration (mg/mL)

9.96

211

SD

0.39

3.8

RSD (%)

3.9

1.8

Mean concentration % of target

99.6

95.9

 

6-Day Room Temperature Storage Resuspension Homogeneity Assessment of the 04 Oct 2016 Formulations

 

Low group

(10 mg/mL)

High group

(220 mg/mL)

Mean concentration (mg/mL)

7.36

237

SD

0.22

4.2

RSD (%)

3.0

2.0

Mean concentration % of target

73.6

97.0

 

10-Day Room Temperature Storage Resuspension Homogeneity Assessment of the 04 Oct 2016 Formulations

 

Low group

(10 mg/mL)

High group

(220 mg/mL)

Mean concentration (mg/mL)

7.11

204

SD

0.41

1.6

RSD (%)

5.8

0.80

Mean concentration % of target

71.1

92.7

 

TEST SUBSTANCE STABILITY IN FORMULATIONS

- Formulations prepared on 04 Oct 2016 at target concentrations of 10 and 220 mg test item/mL

were analysed on the day of preparation.

- Aliquots of the formulations were stored at room temperature for 6 and 10 days and analysed to assess test substance stability. The stability results are presented in Table 7 and Table 8 (attached).

- The mean post-storage concentrations ranged from 96.1% to 101% of the pre-storage values, which met the previously stated protocol-specified requirement for stability.

Applicant's summary and conclusion

Conclusions:
An HPLC/UV method for the determination of test item concentration in formulations containing peanut oil and test substance ranging in concentration from 10 to 220 mg/mL was validated in this study. Method specificity/selectivity, ruggedness, calibration reproducibility, precision, accuracy, and test substance stability in calibration standards and processed QC samples stored at room temperature for 12 days were assessed and validated, satisfying SOP and/or protocol-specified criteria. Formulations prepared at target test substance concentrations of 10 and 220 mg test item/mL met the protocol-specified requirement for homogeneity and, after 6 and 10 days of room temperature storage, resuspension homogeneity and stability.
Executive summary:

METHODS

 

An HPLC method using UV absorbance detection at a wavelength of 230 nm for the determination of test item concentration in formulations containing peanut oil and test substance ranging in concentration from 10.0 to 220 mg/mL was validated in this study. Also in this study, test substance was assessed in calibration standards and processed QC samples stored at room temperature for 12 days. Finally, test substance homogeneity and, following 6 and 10 days of room temperature storage, resuspension homogeneity and stability were assessed in formulations prepared at target concentrations of 10 and 220 mg test item/mL.

 

RESULTS

 

The test item assay procedure was validated in this study with 3 validation sessions. Quantitation was performed using calibration standards ranging in test substance concentration from 5.00 to 100 μg/mL. The mean back-calculated standard concentrations had inter-session variability ranging from 0.18% to 0.60%RSD and %RE ranging from -1.0% to 0.32% which met the protocol-specified acceptance criteria for calibration standards, i.e., RSD ≤ 10% and %RE within ± 10% (except at the lowest level where RSD ≤ 15% and %RE within ± 15% were acceptable). Assay precision and accuracy were verified by the analysis of QC samples prepared at 10.0, 100, and 220 mg test item/mL. The mean calculated QC concentrations had inter-session variability (precision) ranging from 0.60% to 1.1% RSD and %RE (accuracy) ranging from -0.76% to -0.39%. The results met the protocol-specified acceptance criteria for precision and accuracy, i.e., RSD ≤ 15% and %RE within ± 15%.

 

The test substance in calibration standards and processed QC samples stored at room temperature

for 12 days met the protocol-specified acceptance criteria for stability, i.e., the post-storage concentration was not < 90% of the pre-storage value.

 

The results of the test substance homogeneity assessment in formulations prepared at target

concentrations of 10 and 220 mg test item/mL met the protocol-specified acceptance criteria, i.e., the RSD for the mean concentration was ≤ 10% at a concentration within the acceptable limits (85% to 115% of target). Assessment of test substance resuspension homogeneity and stability in formulations prepared at target concentrations of test item/mL and following 6 and 10 days of room temperature storage met the protocol-specified acceptance criteria for resuspension homogeneity, i.e., the RSD for the mean concentration was ≤ 10%, and the previously stated protocol-specified criteria for stability.

 

CONCLUSION

An HPLC/UV method for the determination of test item concentration in formulations containing peanut oil and test substance ranging in concentration from 10 to 220 mg/mL was validated in this study. Method specificity/selectivity, ruggedness, calibration reproducibility, precision, accuracy, and test substance stability in calibration standards and processed QC samples stored at room temperature for 12 days were assessed and validated, satisfying SOP and/or protocol-specified criteria. Formulations prepared at target test substance concentrations of 10 and 220 mg test item/mL met the protocol-specified requirement for homogeneity and, after 6 and 10 days of room temperature storage, resuspension homogeneity and stability.