5-methyl-2-[(2-nitrophenyl)amino]-3-thiophenecarbonitrile

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-05-02 to 1996-05-25

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:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-300-1

EC Name:

-

Cas Number:

138564-59-7

Molecular formula:

C12H9N3O2S

IUPAC Name:

5-methyl-2-[(2-nitrophenyl)amino]thiophene-3-carbonitrile

Test material form:

solid: particulate/powder

Details on test material:

97.7%
Item code QA405K
Lilly lot no: 356MO1

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor and 356M01
- Purity test date: 1995-11-10

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A primary stock solution of the test substance in methanol was prepared to a concentration of 0.65 mg test ingredient/ml
- Final dilution of a dissolved solid, stock liquid or gel: Diluted to the test concentrations using the pH 4, 7, or 9 buffer

FORM AS APPLIED IN THE TEST (if different from that of starting material)
0.65 ug test ingredient/ml

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: Intiation, Day 1, and Day 5
- Sampling method: Triplicate vessels at each pH were removed from the bath (Time 0 samples were not placed in the bath) and samples of the test solutions were analyzed for the test substance at Time 0, Time 24 hours, and on Day 5 by HPLC.
- Sampling intervals/times for pH measurements: Intiation, Day 1, and Day 5

Buffers:

- pH: 4
- Type of buffer: Aqueous buffer
- Composition of buffer: 0.01 M sodium acetate adjusted to pH 4.0 with 0.1 M acetic acid.

- pH: 7
- Type of buffer: Aqueous buffer
- Composition of buffer: 0.067 M NaH2PO4 mixed with 0.067 M K2HPO4 adjusted to pH 7 .0 with a small amount of 0.1 M phosphoric acid, then diluted 10-fold and pH readjusted to 7.0.

- pH: 9
- Type of buffer: Aqueous buffer
- Composition of buffer: 0.035 M Na2B4O7 adjusted to pH 9.0 with 0.1M acetic acid.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 50 ml Erlenmeyer flasks with ground glass stoppers
- Sterilisation method: Autoclaving at 124°C for a minimum of 1 hour
- Lighting: Excluded from light
- Measures taken to avoid photolytic effects: Exclusion from light and capped
- Measures to exclude oxygen: The flask was capped with a ground glass stopper.
- If no traps were used, is the test system closed/open: Closed

TEST MEDIUM
- Volume used/treatment: 1 L
- Kind and purity of water: NANOpure (Barnstead, Boston, MA) water system
- Preparation of test medium: Solution of the test substance in methanol was prepared at 0.65 mg/mt. For the study, 1.0 mL of the stock solution in methanol was added to each of three 1-L volumetric flasks. The flasks were filled to volume with either pH 4, 7, or 9 buffer.
- Renewal of test solution: Once a day.
- Identity and concentration of co-solvent: Methanol, 0.1% conc.

OTHER TEST CONDITIONS
- Adjustment of pH: None was undertaken during the study
- Dissolved oxygen: Not monitored.

Duration of test

Duration of test 1

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

ca. 0.577 mg/L

Duration of test 2

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

ca. 0.622 mg/L

Duration of test 3

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

ca. 0.624 mg/L

Number of replicates:

9 for each pH

Positive controls:

no

Negative controls:

no

Statistical methods:

For each pH, the mean and standard deviation of the initial concentration (C0) and the concentra­tion measured after 2.4 hours (C2.4hrs) and 5 days (C5) was calculated. The percent hydrolysis was determined by the change in the the test substance concentration in samples analyzed on Day 0 and after 2.4 hours and 5days at 50°C. The equation used was as follows:
Percent of the Initial test substance Hydrolyzed = ((C0-Ct)/C0) * 100
where Ct is the concentration of the test article at time point, t.

Results and discussion

Preliminary study:

No preliminary study was done.

Transformation products:

no

Details on hydrolysis and appearance of transformation product(s):

None

Total recovery of test substance (in %)

Total recovery of test substance (in %) 1

% Recovery:

ca. 2.08

pH:

4

Temp.:

50 °C

Duration:

ca. 5 d

Total recovery of test substance (in %) 2

% Recovery:

ca. 1.13

pH:

7

Temp.:

50 °C

Duration:

ca. 5 d

Total recovery of test substance (in %) 3

% Recovery:

ca. 9.56

pH:

9

Temp.:

50 °C

Duration:

ca. 5 d

Dissipation DT50 of parent compound

Key result

pH:

9

Temp.:

20 °C

DT50:

> 1 yr

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

Results with reference substance:

No reference substance was used.

Any other information on results incl. tables

There was a second study conducted for the hydrolysis rate at pH 9 of the test substance. Its results have been averaged with the results from the first study, as the conditions were the same as the first.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study validly assessed the hydrolysis potential of the test substance. The results suggest that hydrolysis of the test substance will not likely occur in aqueous solution at pH 4 and 7, and would occur extremely slowly at pH 9, with an estimated hydrolysis half-life of greater than 1 year at environmentally relevant temperature.

Executive summary:

Testing was carried out in accordance with EU Method C7.Over a 5-day period at 50°C, the test substance was stable at pH 4 and 7 and hydrolyzed slightly at pH 9. For the first study, the percent of the test substance hydrolyzed by Day 5 was 2.08%, 1.13%, and 12.50%, for pH 4, 7, and 9, respectively. For the second study, the percent of initial test substance hydrolyzed by Day 5 was 6.28% of the initial Time 0 concentration at pH 9. The combined results for the pH 9 systems from the two studies yielded a value of 9.56% hydrolysis. These results suggest that hydrolysis of the test substance will not likely occur in aqueous solution at pH 4 and 7, and would occur extremely slowly at pH 9, with an estimated hydrolysis half-life of greater than 1 year at environmentally relevant temperature.