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Endpoint:
basic toxicokinetics, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
See enclosed files
Objective of study:
absorption
distribution
excretion
metabolism
Qualifier:
according to guideline
Guideline:
other: REACH Guidance on QSARs R.6
Qualifier:
according to guideline
Guideline:
other: REACH Guidance on IR&CSA, Chapter R.14, Occupational exposure assessment Update to change the scope of the guidance from exposure estimation to exposure assessment
Version / remarks:
August 2016
Principles of method if other than guideline:
pkCSM uses graph-based signatures to develop predictive models of central ADME properties. pkCSM performs as well or better than current methods.
Specific details on test material used for the study:
SMILES: N(c(cccc1)c1)C(=S)Nc(cccc2)c2
Type:
absorption
Results:
Intestinal absorption (human): 88.602%
Type:
distribution
Results:
VDss (human) (log L/kg): 0.341
Type:
distribution
Results:
Fraction unbound (human) : 0.058
Type:
distribution
Results:
BBB permeability (log BB): 0.418
Type:
distribution
Results:
CNS permeability (log PS): -1.489
Type:
excretion
Results:
Renal OCT2 substrate: yes
Type:
excretion
Results:
Total Clearance (log ml/min/kg): -0.205
Details on absorption:
According to the model "Intestinal absorption (human)", 88% of the substance is absorbed after oral exposure.

Details on distribution in tissues:
According to the model "VDss (human)", the volume of distribution (VD, i.e. theoretical volume that the total dose of a drug would need to be uniformly distributed to give the same concentration as in blood plasma) is moderate (Log between -0.15 and 0.45).
According to the model "Fraction unbound (human)", 5.8 % of the absorbed dose is unbound in the plasma.
According to the model "BBB permeability", the substance is readily cross the blood-brain barrier (Log BB > 0.3).
According to the model "CNS permeability",the substance is considered to penetrate the Central Nervous System (CNS)(log PS > -2).
Details on excretion:
According to the model "Renal OCT2 substrate", the substance is a OCT2 substrate. The substance is transported by this renal transporter.
According to the model "Total clearance" , the predicted total clearance (hepatic & renal clearance) is of 1.6 ml/min/kg (log(ml/min/kg) 0,205) corresponding to the very low clearance (below 6 ml/min/kg).
Metabolites identified:
no

Property

Model Name

Predicted Value

Unit

Absorption

Water solubility

-3.882

Numeric (log mol/L)

Absorption

Caco2 permeability

1.58

Numeric (log Papp in 10-6cm/s)

Absorption

Intestinal absorption (human)

88.602

Numeric (% Absorbed)

Absorption

Skin Permeability

-2.507

Numeric (log Kp)

Absorption

P-glycoprotein substrate

No

Categorical (Yes/No)

Absorption

P-glycoprotein I inhibitor

No

Categorical (Yes/No)

Absorption

P-glycoprotein II inhibitor

No

Categorical (Yes/No)

Distribution

VDss (human)

0.341

Numeric (log L/kg)

Distribution

Fraction unbound (human)

0.058

Numeric (Fu)

Distribution

BBB permeability

0.418

Numeric (log BB)

Distribution

CNS permeability

-1.489

Numeric (log PS)

Metabolism

CYP2D6 substrate

No

Categorical (Yes/No)

Metabolism

CYP3A4 substrate

YES

Categorical (Yes/No)

Metabolism

CYP1A2 inhibitior

YES

Categorical (Yes/No)

Metabolism

CYP2C19 inhibitior

YES

Categorical (Yes/No)

Metabolism

CYP2C9 inhibitior

YES

Categorical (Yes/No)

Metabolism

CYP2D6 inhibitior

No

Categorical (Yes/No)

Metabolism

CYP3A4 inhibitior

No

Categorical (Yes/No)

Excretion

Total Clearance

-0.205

Numeric (log ml/min/kg)

Excretion

Renal OCT2 substrate

YES

Categorical (Yes/No)

Conclusions:
According to the QSAR pkCSM, the substance is well absorbed by oral route and well distributed into the body. Moreover, no hepatic and renal clearance is expected.
Endpoint:
basic toxicokinetics, other
Remarks:
G.I. human passive absorption
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source
Objective of study:
absorption
Guideline:
other: REACH Guidance on QSARs R.6
Principles of method if other than guideline:
Model to predict either high or low fraction absorbed for an orally administered, passively transported substance on the basis of a new absorption parameter. The model includes only two inputs: the octanol-water partition coefficient (Kow) and the dimensionless oversaturation number (OLumen). The latter is the ratio of the concentration of drug delivered to the gastro-intestinal (GI) fluid to the solubility of the compound in that environment.
Species:
other: Human
Route of administration:
oral: unspecified
Type:
absorption
Results:
Absorption from gastrointestinal tract for 1 mg dose: 100%
Type:
absorption
Results:
Absorption from gastrointestinal tract for 1000 mg dose: 90%
Conclusions:
Using a model to predict either high or low fraction absorbed for an orally administered, passively transported substance, the rates of absorption of DPTU were 100 and 90% for a dose of 1 and 1000 mg, respectively (Danish QSAR).
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:
guideline study
Objective of study:
metabolism
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
Specific details on test material used for the study:
14C-DPTU was synthesized by Isotope Chemistry at AstraZeneca R&D Mölndal (Moölndal, Sweden).
Radiolabelling:
yes
Remarks:
14C-DPTU
Species:
other: the skinlike P450 cocktail or human liver microsomes
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.
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 microsome 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.
Conclusions:
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.

Endpoint:
dermal absorption, other
Remarks:
QSAR
Type of information:
(Q)SAR
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Qualifier:
according to guideline
Guideline:
other: REACH Guidance on QSARs R.6
Qualifier:
according to guideline
Guideline:
other: REACH Guidance on IR&CSA, Chapter R.14, Occupational exposure assessment Update to change the scope of the guidance from exposure estimation to exposure assessment
Principles of method if other than guideline:
IH SkinPerm (v2.04) is a mathematical tool for estimating dermal absorption. The rate of mass build-up (or loss) on the skin comes from the deposition rate onto the skin minus the absorption rate into the Stratum Corneum (SC) and the amount evaporating from the skin to the air.
Species:
other: human
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on study design:
DATA INPUT
-temperature : 25°C
-Molecular weight : 288 g/mol
-Vapour pressure : 0,001 Pa
-Water solubility : 24,37 mg/l
-Log kow : 2.0
-Density : 300 mg/m3
-Melting point : 153,9 °C

SCENARIO PARAMETERS
- Instantaneous deposition
Deposition dose*: 1000 mg
Affected skin area**: 1000 cm²
Maximum skin adherence***: 2 mg/cm²
Thickness of stagnant air****: 1 cm
Weight fraction: 1
Timing parameters
. Start deposition: 0 hr
. End time observation: 8 hr
Report parameters
. Calculation (intervals/hr): 10000
. Report (intervals/hr): 100

- Deposition over time
Affected skin area**: 1000 cm²
Maximum skin adherence***: 1 mg/cm²
Dermal deposition rate: 2 mg/cm²/hr
Thickness of stagnant air****: 1 cm
Weight fraction: 1
Timing parameters
. Start deposition: 0 hr
. Duration of deposition: 8hr
. End time observation*: 8 hr
Report parameters
. Calculation (intervals/hr): 10000
. Report (intervals/hr): 100

*Default value defined according to the internal validation study
**Estimated skin surface of two hands of an adult.
***The skin adherence field is greyed out and a default of -1 is indicated if the substance is a liquid at 25°C. Smart logic is built into IH SkinPerm; the program recognizes whether a substance is a solid or liquid at standard temperature (25°C) based on the physicochemical properties. For substances
that are solids at 25°C a maximum adherence value up to 2 mg/cm² is allowed based on studies of soil-on-skin adherence. If the deposition rate results in an increase above the input figure (0.2-2 mg/cm²), it is assumed that the surplus disappears just by removal from the skin.
*** 3 cm if clothing involved, 1 cm if bare skin involved

Time point:
8 h
Dose:
1000 mg
Parameter:
percentage
Absorption:
0.082 %
Remarks on result:
other: Instantaneous deposition
Time point:
8 h
Dose:
1 mg/cm²/h
Parameter:
percentage
Absorption:
0.005 %
Remarks on result:
other: Deposition over time for 8 hr
Conclusions:
The dermal absorption of DPTU is estimated to be low (< 10%).
Executive summary:

The dermal absorption of DPTU leads to the following results, obtained using the SkinPerm v2.04 model according to the input data:

 

Instantaneous deposition

 

Deposition over time

End time observation 8 hr

Total deposition (mg) or deposition rate (mg/cm²/hr)

1000

16000

Fraction absorbed (%)

0.0817

 0.00511

Amount absorbed (mg)

0.817

0.817

Lag time stratum corneum (min)

49.8

Max. derm. abs. (mg/cm²/h)

0.0000511

Description of key information

Based on the physical and chemical properties, the QSAR predictions and the toxicological data available, DPTU is considered as well absorbed by oral route and inhalation. However a low dermal route is predicted. DPTU is well distributed in the body and induced a thyroid toxicity.
A specific in vitro study on metabolism was found on DPTU. 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.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

Toxicokinetic of DPTU is estimated based on the available studies, the QSAR predictions and the physico-chemical properties.


Indeed, according to the REACH guidance document R7.C, information on absorption, distribution, metabolisation and excretion may be deduced from the physicochemical properties, including:


-Molecular weight: 228.3 g/mol


-Water solubility: 24.37 mg/L (20°C)


-Partition coefficient Log Kow: 2.0


-Vapour pressure: 0.001 Pa (25°C)


 


ORAL ABSORPTION


The physicochemical characteristics of DPTU (log Kow = 2) and the molecular mass (223.3 g/mol) are in the range suggestive of absorption from the gastro-intestinal tract subsequent to oral ingestion (with a molecular weight below 500, and a log Kow between -1 and 4).


Using a model to predict either high or low fraction absorbed for an orally administered, passively transported substance, the rates of absorption of DPTU were 100 and 90% for a dose of 1 and 1000 mg, respectively (Danish QSAR). According to the model "Intestinal absorption (human)" (pkCSN QSAR), 88% of the substance is absorbed after oral exposure.


This assumption of an oral absorption is confirmed in the repeated toxicity study in rats in which thyroid toxicity was observed at the doses of 250 and 1000 mg/kg after 28 days of exposure.


Based on these data, 100% of oral absorption is used for risk assessment.


 


DERMAL ABSORPTION


With a water solubility between 1 and 100 mg/L, dermal absorption is anticipated to be low to moderate.


According to the IH skin perm (QSAR), the dermal absorption of DPTU is estimated to be low (< 10%).


The dermal absorption is confirmed by the skin sensitisation data : DPTU is a strong skin sensitizer so DPTU is absorbed.


Based on these data, 10% of dermal absorption is used for risk assessment.


 


INHALATION ABSORPTION


DPTU is a solid with a low vapour pressure (< 1 Pa) : DPTU is not a volatile substance.


100% of inhalation absorption is used for risk assessment.


 


DISTRIBUTION


As a small molecule a wide distribution of DETU is expected. Moreover, the molecule is lipophilic (log Kow > 0), it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues.


Based on the predicted results obtained in QSAR (pkCSN), the volume of distribution (i.e. theoretical volume that the total dose of a drug would need to be uniformly distributed to give the same concentration as in blood plasma) is moderate ; 5.8 % of the absorbed dose is unbound in the plasma ; the substance is readily cross the blood-brain barrier and penetrates the Central Nervous System.


This assumption of well distribution is confirmed in the repeated dose toxicity studies following oral application in which liver and thyroid were identified as target organs of DPTU.


 


METABOLISM


One study on metabolism is available on DPTU (Samuelsson 2011) :


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.


 


ELIMINATION


Due to the small weight molecular, DPTU is probably excreted in the urines.


According to the model "Renal OCT2 substrate" (pkCSN QSAR), DPTU is a OCT2 substrate meaning that the substance is transported by this renal transporter.


A very low renal and hepatic clearance is expected according to the prediction of pkCSN QSAR.


 


 


 


 


Route: .live2