Registration Dossier

Administrative data

monitoring data
Type of information:
other: publication
Adequacy of study:
disregarded due to major methodological deficiencies
4 (not assignable)

Data source

Reference Type:

Materials and methods

Principles of method if other than guideline:
not applicable
GLP compliance:
no information available

Test material

Test material form:
not specified
Details on test material:
Supplier: not specified.
Specific details on test material used for the study:
The livers of adult Coho Salmon were pooled and microsomes were extracted and microsomes were extracted with 0.25 M sucrose buffer to remove cytosolic factors and the final pellet was resuspended in 0.05 M Tris-CL buffer (pH 7.5) containing 0.1 M KCL, 0.1 mM EDTA, 0.01 mM phenylmethane-sulfonyl fluoride, 0.01 mM dithiothreitol and 20% glycerol. The suspended microsomes contained 19.1 mg/mL of protein and a P-450 concentration of 0.630 µM . The microsomes were stored at - 30°C until used.

Study design

Details on sampling:
preparation of incubation mixtures in a 2 mL volume: micosomal preparation, 0.05 M Tris-Cl buffer (pH 7.5), 0.1 M KCl, 1.0 mM NADPH and 20% glycerol. After addition of of 10 µL cyclohexane, the mixtures were incubated in a shaking water bath at constant temperature. The samples were extracted with 1.0 mL methylene chloride containing the internal standard (1-hexanol).

Results and discussion

Details on results:
Optimum conditions for the production of cyclohexanol via hydroxylation by salmon liver microsomal system are: 20°C; pH 8.0 to 8.5, ionic strength: 0.026.

Any other information on results incl. tables

Formation rate cyclohexanol by Soho Salmon microsomes is linear for the first 60 min with a gradual increase in the rate from 60 to 90 min. The optimum temperature range for the hydroxylation occurs between 15 and 25 °C with a significant decrease outside this range. Maximum rate at 20 °C and pH 8.0 to 8.5, highly ionic strength dependant. In the range of 0.026 to 1.40 the reaction rate increased dramatically below 0.50 with a maximum at 0.026.

Applicant's summary and conclusion

Optimum conditions for the production of cyclohexanol via hydroxylation by salmon liver microsomal system are: 20 °C; pH 8.0 to 8.5, ionic strangth: 0.026.
Executive summary:

Conditions of the microsome incubation were varied systematically to determine the optimum temperature, pH and ionic strength for cyclohexanol production. Cyclohexanol was quantified by capillary column gas chromatography. Maximum cyclohexanol formation was achieved at 20 °C, a pH of 8.0 to 8.5 and an ionic strength of 0.026. A linear rate of cyclohexanol formation is seen from 0 to 60 min of incubation and there is an apparent decrease in the rate from 60 to 90 min. Poor stability of the microsomal preparation from the species studied was also identified and several stability studies have been undertaken using cyclohexane metabolism as a monitor.