Registration Dossier

Administrative data

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: In vitro experimental study, published in peer reviewed literature.

Data source

Reference
Reference Type:
publication
Title:
Oxidative DNA damage by minor metabolites of toluene may lead to carcinogenesis and reproductive dysfunction
Author:
Murata, M.
Year:
1999
Bibliographic source:
Biochem Biophys Res Commun. 1999 Aug 2;261(2):478-83.

Materials and methods

Principles of method if other than guideline:
The ability of methylhydroquinone to induce DNA damage was evaluated by using 32P-5'-end-labeled DNA fragments obtained from human genes. In addition, 8-oxodG formation was measured in the presence and absence of NADP and/or Cu(II).
GLP compliance:
not specified
Type of assay:
other: oxidative DNA damage

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylhydroquinone
EC Number:
202-443-7
EC Name:
2-methylhydroquinone
Cas Number:
95-71-6
Molecular formula:
C7H8O2
IUPAC Name:
2-methylbenzene-1,4-diol
Details on test material:
- Name of test material (as cited in study article): methylhydroquinone (MHQ)

Method

Target gene:
P53 tumorsuppressor gene and c-Ha-ras-1 protooncogene
Test concentrations with justification for top dose:
5, 10, 20 µM
Details on test system and experimental conditions:
Detection of DNA damage by MHQ
- Reaction mixture: the DNA fragments, sonicated calf thymus DNA, the test substance, CuCl2, and NADH in sodium phosphate buffer containing DTPA.
- After incubation at 37°C.
- The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the Maxam-Gilbert procedure using a DNA-sequencing system (LKB 2010 Macrophor).
- The relative amounts of oligonucleotides from the treated DNA fragments were measured with a laser densitometer (LKB 2222 UltroScan XL).

Analysis of 8-oxodG formation: DNA fragments from calf thymus were incubated with MHQ, CuCI2 and NADH at 37°C. After ethanol precipitation, DNA was enzymatically digested to the nucleosides with nuclease P, and calf intestine phosphatase and analyzed by HPLC-ECD.

Results and discussion

Additional information on results:
GENOTOXICITY:
- MHQ caused DNA damage in the presence of Cu(II), although MHQ alone did not. Using other metal ions (Mn(III), Fe(II), and Fe(III)) MHQ caused no DNA damage. MHQ plus NADH induced piperidine-labile sites frequently at thymine and cytosine residues in the presence of Cu(II). When denatured DNA was used, preferential damage occurred more frequently at guanine residues.
- 8-oxodG formation was increased. However, without Cu(II) or NADH, MHQ did not induce the increase of 8-oxodG formation.

Applicant's summary and conclusion