4,4'-Methylene-bis-(3-chloro-2,6-diethylaniline)

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 1996 - April 1997

Reliability:

2 (reliable with restrictions)

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:

1997

Report date:

1997

Materials and methods

Objective of study:

other: In vitro investigation of N-hydroxylation of the test substance P5376 in guinea pig liver microsomes and comparison with that of the reference substance MbOCA

Test guideline

Qualifier:

according to guideline

Guideline:

other: In-House

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-130-7

EC Name:

-

Cas Number:

106246-33-7

Molecular formula:

C21 H28 Cl2 N2

IUPAC Name:

4-[(4-amino-2-chloro-3,5-diethylphenyl)methyl]-3-chloro-2,6-diethylaniline

Test material form:

solid: crystalline

Details on test material:

- Name: LONZACURE® M-CDEA
- Internal substance code: P5367
- Storage: Keep container tightly closed. Keep in a dry, cool and well-ventilated place.
- Aggregate state/appearance: Crystalline solid
- Colour: white to off-white
- Odour: Odourless

Radiolabelling:

no

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

male

Details on test animals or test system and environmental conditions:

Three young adult male pirbright guinea pigs with a starting body weight of about 400-500 g were housed in the animal facility of the test lab. The guinea pigs were housed in standard cages (macrolon type 4) on softwood bedding at 22°C, at a relative humidity of 25-45%, and a 12 hour light/dark cycle. The animals were acclimatised to laboratory conditions for 8 days prior to sacrifice. A pelleted standard chow and tap water via water bottles were offered ad libitum.
The animals were not fasted prior to necropsy. Upon sacrifice, the animals were bled under carbon dioxide anaesthesia. The livers were immediately chopped, weighed and frozen in liquid nitrogen. Frozen liver samples were stored at -80°C until processed.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Control animals:

no

Details on dosing and sampling:

Subcellular fractionation
Microsomal and cytosolic liver fractions from 3 guinea pigs were prepared individually. The subcellular fractionation was performed at 4°C. The frozen liver samples were thawed to 0°C within 30 min and homogenised in 10 mmol/L TrislHCI buffer, pH 7.5, containing
250 mmol/L sucrose to give a 20% (w/v) homogenate. Microsomal and cytosolic liver fractions were obtained by centrifugation of 12'000g (15 min) supernatants for 1 hour at 100'000g. Microsomes were washed once by resuspending the 100'000g pellets in 50 mmol/L. TrislHCI buffer, pH 7.5, and centrifugation at 100'000g for 1 hour. The washed microsomal pellets were resuspended in 50 mmollL TrislHCI buffer, pH 7.5, to a final concentration corresponding to 0.5 g liver equivalents per mL. Microsomal and cytosolic fractions were frozen in suitable aliquots in liquid nitrogen and stored at -80°C. Protein contents of liver microsomal fractions were determined according to Smith et al. (1985)

Microsomal incubations
The N-hydroxylation of the test article P5367 as well as of the reference compound MOCA was investigated in liver microsomal fractions from guinea pigs in the presence of an NADPH regenerating system. The incubations typically consisted of liver microsomal fractions (diluted to yield a final protein concentration in the range of about 0.5 to 4 mg/mL) in 0.1 mol/L TrislHCI buffer, pH 7.4, in a total volume of 1100 uL. An NADPH-regenerating system was used, containing final concentrations of 1 U/L of glucose-6-phosphate-dehydrogenase (Boehringer, Mannheim No. 127663), 1 mmol/L of NADP (Boehringer No. 128058), 5 mmol/L of glucose-6-phosphate (Boehringer, No. 127647) and 5 mmol/L ofMgCb. The test article P5367 as well as the reference compound MOCA were added as solutions in methanol to receive final concentrations of 2.5 to 100 JlmollL (final methanol content of the incubation mixture: 1% v/v). The incubations were performed at 37°C in a water bath for incubation times between 0 and 120 min. The incubations were stopped by two extractions of the parent compound and the N-hydroxylamines with one volume of methylene chloride. The methylene chloride extract was evaporated to dryness on a SpeedVac, the residue was dissolved in 50 flL acetonitrile and 40 flL were used for quantificatioon by reversed phase HPLC with UV detection. Additionally, 10 flL of the reconstituted extract were analysed by LC/MS. The recoveries for the parent compounds and its N-hydroxylamines and the limit of quantification for the NOH-P5367 were determined by analyses of microsomal incubations immediately after addition of known amounts of the investigated compounds. Yields of 100 and 93% (P5367, n=2), 95 and 89% (NOH-P5367, n=2), 77±1O% (MOCA, n=4) and 66±8% (NOH-MOCA, n=4) were obtained. The limit of quantification for NOH-P5367 was determined to be about 0.2 flmollL. The same limit of quantification was assumed for NOHMOCA.

Results and discussion

Any other information on results incl. tables

Following incubation of both substances under optimal conditions (100 umol/litre substrate, 2mg/ml microsomal protein), formation of the N-hydroxylamine metabolite from the test substance P5367 was shown to occur.  The maximal amount of N-hydroxylamine formed was about 5-fold less than the equivalent metabolite produced for the reference
substance MbOCA.  The formation of the N-hydroxylamine metabolite was proportional with time up to 10 minutes incubation for both substances.  In the case of P-5367, at 15 minutes, N-hydroxylamine formation reached a plateau at around 5umol/litre which was maintained for a further 15 mins.  This was then followed by a rapid decline over the remaining 90 minutes of the experiment such that at 120 minutes the concentration of NOH-P5367 was about 0.45 uml/l. In marked contrast to P-5367, the concentration of NOH-MbOCA continued to increase up to 30 mins to a level of around 20umol/l at 120 minutes (i.e. essentially steady state was obtained between 30 and 120 minutes).

The formation of the N-hydroxylamine metabolites for both substances was proportional to the concentration of microsomal protein over a 15-minute incubation time up to 1 mg/ml of protein but was found to plateau with increasing protein concentration.  The initial N-hydroxylamine rate over 10 minutes was linearly dependent upon the substrate concentration for both substances, with formation of N-hydroxylamine metabolite considerably greater at lower substrate concentrations for MbOCA than for P5367.  So, for up to 50umol/l of parent substrate about 3-6% of P-5367 was found to convert to NOH-P5367 whereas 30-36% of MbOCA was converted at these substrate concentrations. Overall, therefore, the formation of NOH-P5367 under optimal conditions in this assay was significantly lower than the formation of NOH-MbOCA and this difference was found to be even greater at substrate concentrations of 50umol/l or less.  Under optimal conditions although NOH-P5367 was formed over the first 30 minutes, levels declined markedly over to subsequent 90 suggesting either more rapid conversion to a further metabolite and/or inactivation of P450.

Applicant's summary and conclusion

Conclusions:

The data suggest that less N-hydroxylamine is metabolically available from P5367 than from MOCA indicating a lower genotoxic potential ofP5367. Indeed, in contrast to MOCA, P5367 was negative in vivo as well as in vitro in various short term tests for genotoxicity.