Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Diphenylthiourea, a common rubber chemical, is bioactivated to potent skin sensitizers.
Author:
Samuelsson K., Bergström MA., Jonsson CA, Westman G., and Karlberg AT.
Year:
2011
Bibliographic source:
Chem.Res.Toxicol.,24: 35-44.

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aim of this study was to investigate if DPTU is a prohapten that can be activated by skin metabolism. This metabolic activation and covalent binding of 14C-labeled DPTU to proteins were tested using a skinlike cytochrome P450 cocktail containing the five most abundant P450s found in human skin (CYP1A1, 1B1, 2B6, 2E1 and 3A5) and human liver microsomes.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
14C-DPTU was synthesized by Isotope Chemistry at AstraZeneca R&D Mölndal (Moölndal, Sweden).
Radiolabelling:
yes
Remarks:
14C-DPTU

Test animals

Species:
other: the skinlike P450 cocktail or human liver microsomes

Administration / exposure

Route of administration:
other: in vitro study
Details on exposure:
A mixture of 14C6-DPTU and unlabeled DPTU with a specific activity of 4.51 kBq/nmol was used for the microsomal incubations.
Duration and frequency of treatment / exposure:
60 minutes
Details on study design:
The metabolism of 14C6-DPTU was assessed, and metabolites were detected after incubations with the skinlike P450 cocktail or human liver microsomes. The metabolites and trapped adducts were separated using reversed-phase HPLC and online RAD for quantification. MS/MS was used for structure elucidation.

Results and discussion

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
The total conversion of DPTU after 60 min of incubation in human liver microsomes and the P450 cocktail was 29 and 12%, respectively. A loss of approximately 10% of parent was observed in the control experiments without NADPH and is considered to be due to decomposition of DPTU. This was confirmed in an additional control experiment run in the absence of metabolizing enzymes.
In total, six metabolites were detected in the human liver mi¬crosome incubations without trapping agents. Four of the metabolites were detected by MS and are denoted Ml-M4. In the P450 cocktail incubations, M1-M4 were detected, however, the nonidentified peaks from the human liver microsome incubations were not seen (Figure 2A). Amounts of metabolites formed and masses of metabolites are presented in Table 3. M1, a monooxygenated metabolite, was the major metabolite formed in incubations with both the P450 cocktail and the human liver microsomes. The minor metabolites M2-M4 were formed via monooxygenation (M2), desulfuration (M4), and combined oxygenation and desulfuration (M3).
The m/z given for the compounds is the unlabeled MH+. The MS/MS spectrum of DPTU (MH+ at m/z 229) contains product ions at m/z 195, 136, and 94. Proposed structures of the DPTU product ions are shown in the Supporting Information. M1 and M2 have pseudomolecular ions of m/z 245 corresponding to a net mass gain of +16 Da and are thus monooxygenated metabolites. Both M1 and M2 produced product ions at m/z 227, which indicates that both compounds lose the mass of one water molecule upon fragmentation of the parent at m/z 245. Hydroxylation on aromatic rings is a common metabolic pathway, but loss of water is unlikely to occur for phenols when subjected to collision-induced dissociation. Thus, on the basis of MS/MS interpretation, M1 and M2 are postulated to be the sulfenic acid (S-OH) and hydroxylamine (N-OH) of DPTU, respectively. S- and N-oxidations are catalyzed both by microsomal P450 and FMO (25). S-oxidation produces sulfenic (S-OH), sulfinic(SO2H), and sulfonic acids (SO3H). Thiourea sulfoxides are unstable and difficult to isolate and identify. For this reason, only the sulfenic acid of DPTU (M1) was identified in the metabolic mixture. Using a synthetic standard, metabolite M3 at m/z 213 was identified as diphenyl urea (DPU). M3 produced the same MS/MS spectrum and had the same retention time as the reference compound DPU (data not shown). Metabolite M4 was the most nonpolar metabolite identified. This compound was found in relatively low amounts and only in incubates without trapping agents. The protonated molecular ion had a m/z of 197 and is proposed to be diphenyl formamidine. Its MS/MS product ions at m/z 104 and 94 correspond to C2H6N and protonated aniline.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no data
DPTU was mainly metabolically activated to reactive sulfoxides resulting in desulfurated adducts in both enzymatic systems used. Also, phenylisothiocyanate and phenylisocyanate were found to be metabolites of DPTU.
Executive summary:

Diphenylthiourea (DPTU) is a known skin sensitizer commonly used as a vulcanization accelerator in the production of synthetic rubber, for example, neoprene. The versatile usage of neoprene is due to the multifaceted properties of the material; for example, it is stretchable, waterproof, and chemical- and abrasion-resistant. The wide application of neoprene has resulted in numerous case reports of dermatitis patients allergic to DPTU. The mechanism by which DPTU works as a contact allergen has not been described; thus, the aim of the present study was to investigate if DPTU is a prohapten that can be activated by skin metabolism. The metabolic activation and covalent binding of 14C-labeled DPTU to proteins were tested using a skinlike cytochrome P450 (P450) cocktail containing the five most abundant P450s found in human skin (CYP1A1, 1B1, 2B6, 2E1, and 3A5) and human liver microsomes. The incubations were carried out in the presence or absence of the metabolite trapping agents glutathione, methoxylamine, and benzylamine. The metabolism mixtures were analyzed by LC-radiochromatography, LC-MS, and LC-MS/MS. DPTU was mainly metabolically activated to reactive sulfoxides resulting in desulfurated adducts in both enzymatic systems used. Also, phenylisothiocyanate and phenylisocyanate were found to be metabolites of DPTU.