Salicylonitrile

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In vitro study on 6 test substances (nitriles), published in a peer-reviewed journal, according to scientific standards. Experimental details well documented, but no individual analytical data on 2-cyanophenol presented. Negative result.

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance (4 mM) was incubated with liver microsomes from phenobarbital-induced rats with an NADPH-generating system, at 37°C for 30 min., quenched with ethanol, filtered and analyzed for metabolites with HPLC and TLC.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals were used only as donors of liver subcellular fractions, in particular microsomes, after phenobarbital induction
- Actual metabolic testing was then performed in vitro
- Age at study initiation: no data
- Weight at study initiation: 210 - 220g
- Fasting period before study: overnight before sacrifice
- Water: 0.1% w/v sodium phenobarbital solution in water, ad libitum, in place of drinking water
- Induction period: 5 days

PREPARATION OF LIVER SUBCELLULAR FRACTIONS
- Four rats killed by decapitation
- Livers excised and rinsed with 0.15 M KCl buffer, then homogenized in 0.15 M KCl buffer
- Homogenate centrifuged at 600g for 20 minutes (to remove the nuclear pellet)
- 600g supernatant centrifuged at 9000g for 20 minutes (to remove the mitochondrial pellet)
- 9000g supernatant centrifuged at 105000g for 60 minutes (to obtain the microsomal pellet)

Route of administration:

oral: drinking water

Vehicle:

water

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDY
- Metabolism of test substance studied in vitro, upon addition of phenobarbital-induced rat liver microsomes
- Test substance (final concentration 4 mM) dissolved in 5% ethanol before addition into the incubation flask
- Microsomes: final protein concentration 6.7 mg protein / mL
- From how many animals: microsomes from 4 rats (pooled)
- NADPH-generating system: 2.5 mM glucose 6-phosphate, 2 mM NADP, 4 mM MgCl2, 5 units glucose 6-phosphate per 200 mg (w/w) microsomes
- Buffer: 0.1 M potassium phosphate, pH 7.4
- Aerobic incubation (by leaving the vessel open)
- Controls: treated in the same way as the samples, except that liver microsomes were boiled for 5-10 min prior to use
- Incubation temperature: 37°C
- Incubation time: 30 min in a shaking waterbath
- Termination of incubation: addition of an equal volume of ethanol, followed by freezing on dry ice
- Filtering of the quenched incubation mixtures, evaporation of filtrate
- Trituration of precipitate with methanol, or extraction of aqueous phase with ethyl acetate, and reconstitution of evaporation residue with methanol
- Method type(s) for identification (HPLC-UV (254 nm), TLC)
- HPLC conditions: isocratic elution with methanol / acetic acid / water (60:20:20, v/v)
- TLC conditions: 20x20 silica-gel GF 250 analytical plates, CH2Cl2 (90:10 v/v)
- Limits of detection and quantification: no data


TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):

Type:

metabolism

Results:

No evidence of metabolites in an incubation of 2-cyanophenol with rat liver microsomes

Metabolites identified:

no

Details on metabolites:

No evidence of metabolites in an incubation of 2-cyanophenol with rat liver microsomes was detectable with the analytical tools described, TLC and HPLC. In a parallel experiment, metabolism of 4-cyanophenol was studied under the same conditions, and formation of 3,4-dihydroxybenzonitrile was seen as a prominent HPLC peak not present in the control. This peak was identified by comparison of the HPLC retention time with an authentic reference, as well as by TLC. It is not clear why the metabolism of 2-cyanophenol is different from that of 4-cyanophenol.

Conclusions:

In vitro study suitable for use as supporting information. No metabolism observed under the conditions of the test.

Description of key information

Short description of key information on bioaccumulation potential result: 
Metabolism in vitro, rat microsomes: no metabolism detectable

Additional information

No evidence of metabolites was detected in an aerobic incubation of 2-cyanophenol with phenobarbital-induced rat liver microsomes, in the presence of an NADPH-generation system, for 30 minutes at 37°C. This finding is in contrast to 4-cyanophenol, which was metabolized to 3,4-dihydroxybenzonitrile under the same conditions. The reason for this difference remains unclear. No in vivo metabolism data was available.