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Diss Factsheets

Toxicological information

Basic toxicokinetics

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Administrative data

basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
The Enzymatic Formation and Chemical Reactivity of Quinone Mathides Correlate with Alkylphenol-Induced Toxicity in Rat Hepatocytes
Judy L.Bolton, Luis G. Valerio Jr. and John A. Thompson
Bibliographic source:
Chem.Res.Toxicol. 1992, 5, 816-822

Materials and methods

Objective of study:
other: The effects of o-alkyl substituents on both the cytochrome P450-catalyzed oxidation of phenols to p-quinone methides (QM’s) and on the rates of nucleophilic additions to the 4-methylene carbon of QM’s
Test guideline
no guideline followed
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

other: in vitro test in rat liver microsomes and in isolated rat hepatocytes (Sprague-Dawley male rats)

Results and discussion

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:
Chemical oxidation of 2,4,6-trimethylphenol (TMP) to related p-quinone methides (TMP-QM) by cytochrome P450.
It was also investigated the conjugate formation rate of of TMP-QM with glutathione GSH (TMP-SG) by liver Microsomes and isolated hepatocytes

Applicant's summary and conclusion

Quinone methides have been described as resonancestabilized carbocations. The electronic distribution of any particular quinone methide should depend on the ability of substituent groups and solvent molecules to support charge separation. Large, hydrophobic alkyl substituents adjacent to the oxo group provide no stabilizing influence and, in addition, effectively prevent solvent interactions with the carbonyl oxygen. BHT-QM, therefore, should exist mainly in the uncharged form and undergo normal Michael additions of nucleophiles. Although the bulky substituents of BHTOH-QM also hinder access of solvent to the oxo group, the side-chain hydroxyl forms an intramolecular hydrogen bond with the carbonyl oxygen, thereby encouraging charge separation. The result of this interaction is a more reactive electrophile relative to BHT-QM, but reactivity may be diminished somewhat by competitive hydrogen bonding of the hydroxyl group to water. A small substituent adjacent to the oxo group, such as methyl, allows intermolecular hydrogen bonding with water to stabilize charge separation. In aqueous media, therefore, compounds such as BDMP-QM and TMP-QM are expected to behave more like carbocations (compared with BHT-QM), and this proposal is supported by rapid rates of nucleophile addition.
In addition to structural factors responsible for altering quinone methide reactivity, data on the enzymatic conversion of phenolic compounds to these electrophilic metabolites have been obtained with rat liver microsomes and isolated rat hepatocytes. For the series of alkylphenols investigated, results demonstrate that tert-butyl substitution enhances P450-dependent oxidation of the aromatic T electron system. Whether this finding is due to the relatively high lipophilicity of phenols containing a tertbutyl substituent or to the blockade of alternative oxidation pathways (i-e., altered P450 regiospecificity) by bulky alkyl groups is not known.
Three alkylphenols, BMP, BDMP, and BHTOH, completely destroyed hepatocyte viability within 1-2 h, as measured by the loss of plasma membrane integrity, but BHT, TMP, and DMP had little or no effect. Hepatocytes were partially protected from alkylphenol-induced toxicity by an inhibitor of P450-catalyzed metabolism. Cell death was preceded by GSH depletion, and lowering of intracellular GSH levels with DEM exacerbated alkylphenol toxicity. These results support the proposal that electrophilic quinone methides mediate alkylphenol-induced destruction of hepatocytes by forming covalent bonds to critical nucleophilic sites.