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Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, the purpose was to determine whether sufficient topically applied sunscreen penetrated into human viable epidermis to put the local keratinocyte cell populations at risk of toxicity.
The penetration and retention of the test material in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration–human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation (DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreen causing toxicity.
GLP compliance:
not specified
Specific details on test material used for the study:
Chemical name in the publication : 2-ethylhexyl-p-dimethyl PABA (Padimate O) (ISP)
Radiolabelling:
no
Species:
other: Human epidermis in vitro
Vehicle:
other: mineral oil
Doses:
- Nominal doses: 2, 1 and 0.5 % in mineral oil
Details on study design:
- Method for preparation of dose suspensions: The test material was diluted to a 2.0 % w/w solution in mineral oil. This solution was sonicated for 10 minutes to hasten dissolution. Subsequent dilution of this was used to prepare 1.0 and 0.5 % mineral oil solutions.

VEHICLE
- Justification for use and choice of vehicle: Formulations in mineral oil were chosen as the majority of sunscreen products are formulated in oil/water emulsions, with the water component of these formulations evaporating following application to skin under ‘in use’ conditions. The 1 % sunscreen concentrations were also chosen to reflect the concentrations of sunscreen agents in commercial products, which are in the order of 0.5 to 3 %.
- Concentration (if solution): 2, 1 and 0.5 %
Details on in vitro test system (if applicable):
DOSING AND ANALYSIS OF HUMAN EPIDERMIS IN VITRO
Epidermal membranes, prepared by the heat separation technique using human female abdominal skin, were mounted stratum corneum (SC) uppermost in horizontal Franz-type diffusion cells (surface area approximately 1.3 cm^2 ) above a receptor phase consisting of 4 % bovine serum albumin in phosphate buffer pH 7.4 maintained at 35 °C and continuously agitated with a magnetic stirrer.
The epidermal membrane was dosed by placing two drops of the test material solutions onto the membrane surface. The average weights (± SEM) of applied solutions were 0.5 % solution 17.8 ± 0.24 mg, 1 % solution 17.6 ± 0.24 mg and 2 % solution 18.2 ± 0.20 mg.
After 24 hours of exposure, epidermal membranes were removed from the diffusion cells and samples taken of tissue levels either by method A or B. Method A involves washing the surface with 0.5 mL 1: 1 DMSO:water, blotting dry, removing 2 tape strips from the surface of the epidermis, removing the viable epidermis using the enzyme digestion technique outlined below, assaying the remaining epidermal membrane following enzyme digestion and analysing the amount penetrating into the receptor phase. Method B involves the same procedure without enzyme digestion of the viable epidermis but analysis of the entire epidermal membrane as a whole following surface washing drying and tape stripping. The amount of test material in each level was determined by HPLC with UV detection as described in a previous publication and expressed as a percent of the applied dose.

ISOLATION OF VIABLE EPIDERMAL CELLS AFTER IN VITRO DIFFUSION EXPERIMENTS
Trypsin solution was prepared (0.25 % w/v) in PBS pH 7.4, and 50 µL was placed on the underside of the epidermal membrane (approx. 1.3 cm^2). The tissue was allowed to digest for 15 min at room temperature. The area immediately under the site of exposure was swabbed with a piece of pre-weighed filter paper. The filter paper and attached tissue sample were then assayed for test material content. The remaining epidermis was wiped to remove excess tissue, and the remaining test material was then extracted with acetonitrile for HPLC analysis for test material content.

TOXICITY TESTING IN HUMAN KERATINOCYTE CULTURES
- KERATINOCYTE CULTURE
Human epidermal keratinocytes (HEKs) were isolated from neonatal foreskins and cultured. Briefly, HEKs were isolated enzymatically on exposure to dispase and then trypsin, after which they were maintained in standard culture medium without epidermal growth factor. This medium was replaced with one containing epidermal growth factor and subsequent passages were maintained in serum-free keratinocyte medium with penicillin/streptomycin (10 kU/mL), gentamycin (10 µg/mL) and bovine pituitary extract (protein 1 mg/mL). Plates were kept in 5 % CO2 atmosphere at 37 °C. Cultures were used in this study prior to becoming confluent.

- DNA SYNTHESIS ASSAY
Proliferation of HEK cultures was determined by the incorporation of [3H]-thymidine ([3H]-methyl-thymidine) 5.0 mCi/mmol. HEKs were grown in 25 mm wells and both control and treatment cultures were incubated with 2.5 µCi/mL of [3H]-thymidine for 3 hours at 37 °C. Cells were washed with cold phosphate-buffered saline and then incubated with acid alcohol (90 % ethanol/10 % acetic acid) for 1 hour at 4 °C. The fixed HEKs were solubilised with 0.2 M NaOH at 37 °C for 30 minutes and neutralised with 0.5 M HCl. Aliquots were taken for LSC and determination of protein content. The radioactivity of aliquots was determined by mixing with Ultima Gold LSC, and counting in a Packard LSC with background subtraction.

- PROTEIN ASSAY
Total protein was assayed in the digested cell solution with bicinchoninic acid. The assay was performed in 96-microwell plates, measuring UV absorbance in a Packard Spectra Count. A freshly prepared BSA standard curve, using duplicate samples in a working range 0.5 to 500 µg/mL, including reference solutions and linear regression, was used for calibration. Sample protein concentrations were defined using the calibration curve and absorbance obtained on each microwell plate. An inter-assay variability of less than 10 % was found between 5 full calibration curves conducted at different times during the study.

- DOSING OF HEK CULTURES
HEK cultures were exposed to initial concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 µg of test material per millilitre of media. Each plate contained controls and treatments in duplicate and was completed in triplicate. Due to the low solubility of test material in the media, test material solutions were initially prepared in DMSO with the requisite dose being spiked as 2 µL of solution per 1 mL of media, which was then placed on the HEKs. The chosen DMSO concentration was shown to have negligible effects on HEK proliferation. Gross morphology (shape changes, detachment and granulation) of treated HEKs were then assessed using light microscopy.

- ANALYSIS OF CELL PROLIFERATION DATA
The cellular uptake of [3H]-thymidine was quantified as disintegrations per minute (DPM) and normalised for amount of protein in each replicate (DPM/ µg). The rate of [3H]-thymidine uptake was then expressed as a percent of control and defined as DNA synthesis rates. Concentration-effect relationships were obtained by plotting DNA synthesis rates versus the log concentration (µM) of test material. The log concentration-effect relationships were then fitted with a sigmoid Hill function using GraphPad Prism (v. 3.00), used in the fitting to ensure a more Gaussian distribution of the independent parameter. The resulting three-parameter logistic function is given by:
f(log C) = Ebaseline ± [(Ebaseline – Emax) / [1+10^((logIC50 – logdose).¿))]]
where Ebaseline was the maximal level of DNA synthesis, Emax the greatest reduction observed in DNA synthesis, IC50 the treatment concentration causing 50 % maximal reduction and ¿ a shape factor (or Hill coefficient). Regression coefficient (r^2), parameter errors and residual analysis were used to assess goodness of fit.
Signs and symptoms of toxicity:
no effects
Dermal irritation:
not examined

Following 24 h exposure to epidermal membranes, detectable amounts of test material were present in the stratum corneum and viable epidermis. The balance of the test material (95 to 98 %) is recovered on the surface of the epidermis as non-penetrated material. It should be emphasised that these sets of data showed that more than 95 % of the sunscreen remains on the skin.

The results obtained in the keratinocyte toxicity assessment studies using thymidine uptake as a marker of DNA synthesis show the IC50 value for the test material to be 12.7 µM. This study quantified test material concentrations in the viable epidermis, where cells undergo division and differentiation, after topical application to human skin. The concentrations were then compared to equivalent IC 50 concentrations determined in keratinocyte culture following adjustment for the 40- fold difference in protein concentration between the two in vitro systems. This adjustment was necessary to normalise for the different concentrations of protein in the culture media and receptor phase solutions to allow comparison of the equivalent unbound fractions of sunscreen agent in each case. Increasing protein concentration can greatly reduce the sunscreen available to cause toxicity with apparent diminished sunscreen toxicity for an equivalent total concentration. In order to determine the free fraction in the viable epidermis, binding and partitioning in viable cells was also accounted for. Comparison of the values with IC 50 values determined from the cell culture studies shows that sunscreen levels in the viable epidermis after application to intact skin were well below levels required to cause toxicity in keratinocyte cultures.

In conclusion, epidermal permeation and retention of the test material was shown to occur following topical application to human skin. A comparison of the concentration of test material reaching the viable cells of the lower epidermis with that eliciting toxicity in cultured human keratinocytes suggests that the test material is unlikely to cause any significant effects in the viable epidermis after topical application to the skin.

Table 1: Summary of results

Parameter

Result

Emax (%)

82.6

IC50 (µM)

12.7

¿

-1.67

r^2

0.89

Receptor 0.1 % protein (µM)*

0.0043

VE:receptor partition coefficient

350

IC50 equivalent VE (µM)**

1.5

VE = Viable epidermis.

*Concentrations in receptor solution after penetration through human epidermis in vitro from 1 % (w/w) mineral oil vehicle adjusted for 40x difference in protein concentration between cell culture and receptor phase fluid composition.

**Equivalent unbound concentration in viable epidermis based on adjusted receptor phase concentrations (to 0.1 % protein) and free fraction partition coefficient between viable epidermis and receptor phase.

Conclusions:
Under the conditions of this study it is concluded that the human viable epidermal level of the test material in sunscreen is too low to cause any significant toxicity to the underlying human keratinocytes.
Executive summary:

The study was performed to determine whether sufficient topically applied sunscreen (test material) penetrated into human viable epidermis which would put the local keratinocyte cell populations at risk of toxicity.

The penetration and retention in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration-human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation (DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreens causing toxicity.

Following 24 h of human epidermal exposure, detectable amounts of the test material were present in the stratum corneum and viable epidermis. The concentration of sunscreen found in human viable epidermis after topical application, adjusting for skin partitioning and binding effects, was at least 5- fold lower, based on levels detected in viable epidermal cells, than those appearing to cause toxicity in cultured human keratinocytes.

Under the conditions of this study it is concluded that the human viable epidermal level of the test material in sunscreen is too low to cause any significant toxicity to the underlying human keratinocytes.

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:
SMILE : O=C(OCC(CCCC)CC)c(ccc(N(C)C)c1)c1
Type:
absorption
Results:
Intestinal absorption (human): 96.46 %
Type:
distribution
Results:
VDss (human) (log L/kg): 0.341
Type:
distribution
Results:
Fraction unbound (human) : 0.097
Type:
distribution
Results:
BBB permeability (log BB): 0.512
Type:
distribution
Results:
CNS permeability (log PS): -2.022
Type:
excretion
Results:
Renal OCT2 substrate: no
Type:
excretion
Results:
Total Clearance (log ml/min/kg): 0.97
Details on absorption:
According to the model "Intestinal absorption (human)", 96 % 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. theoritical 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)", 9.7% of the absorbed dose is unbound in the plasma.
According to the model "BBB permeability", the substance is cross readily the blood-brain barrier (log BB > 0.3).
According to the model "CNS permeability", it is not possible to predict if the substance is unable or not to penetrate the CNS (-3
Details on excretion:
According to the model "Renal OCT2 substrate", the substance is not a OCT2 substrate. The substance is not transported by this renal transporter.
According to the model "Total clearance" , the predicted total clearance (hepatic & renal clearance) is of 9.3 ml/min/kg (log(ml/min/kg) 0.97) corresponding to a low clearance.
Metabolites identified:
no
Conclusions:
According to the QSAR pkCSM, the substance is well absorbed by oral route, and well distributed into the body. Moreover, a low total clearance is expected.
Endpoint:
basic toxicokinetics, other
Remarks:
G.I. human passive absorption
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
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:
According to this QSAR, 90-100% of the substance is absorbed after oral exposure.

Description of key information

No experimental toxicokinetic study is available on 2-ethylhexyl 4-(dimethylamino)benzoate.


However, as per REACH guidance document R7.C , information on absorption, distribution, metabolism and excretion may be deduced from the physical-chemical properties and QSAR predictions.


Based on the physical-chemical properties and QSAR predictions, the absorption of 2-ethylhexyl 4-(dimethylamino)benzoate is expected to be high by oral route and inhalation, but low by dermal route. A good distribution and excretion of the substance are expected.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

 


No experimental toxicokinetic study is available on 2-ethylhexyl 4-(dimethylamino)benzoate. However, as per REACH guidance document R7.C, information on absorption, distribution, metabolism and excretion may be deduced from the physical-chemical properties, including:


-Mean molecular weight: 277 g/mol


-Water solubility: 0.1 mg/L


-Partition coefficient Log Kow: 6.2


-Vapour pressure: 0.0005 Pa (25°C)


 


ABSORPTION


The high values of log Kow and the low water solubility of 2-ethylhexyl 4-(dimethylamino)benzoate are favorable for a low oral absorption. In the experimental studies, no clinical effects or mortality were observed after one single administration (2000 mg/kg) of 2-ethylhexyl 4-(dimethylamino)benzoate by gavage (oral route) in rats, however in the 28-day repeated toxicity study, clear systemic toxicity was noted at 1000 mg/kg/day. Using a model to predict either high or low fraction absorbed for an orally administered, passively transported substance, the rates of absorption of 2-ethylhexyl 4-(dimethylamino)benzoate were 100 and 90% for a dose of 1 and 1000 mg, respectively (Danish QSAR). According to the model "Intestinal absorption (human)", 96% of the substance is absorbed (pkCSM). 100% of oral absorption is taken into account for the risk assessment.


The high values of log Kow and the low water solubility of 2-ethylhexyl 4-(dimethylamino)benzoate are not favorable for a dermal absorption. In the experimental studies, no dermal toxicity was noted: 2-ethylhexyl 4-(dimethylamino)benzoate is not a skin sensitizer. According to the dermal absorption study, the dermal absorption of 2-ethylhexyl 4-(dimethylamino)benzoate is very low. That's why 10% of absorption is taken into account for the risk assessment.


Based on the low vapour pressure, 2-ethylhexyl 4-(dimethylamino)benzoate is considered to be not a volatile substance. However 100% of inhalation absorption is taken into account for the risk assessment (worst case).


 


DISTRIBUTION


No specific data is available on the distribution of 2-ethylhexyl 4-(dimethylamino)benzoate.


According to the QSAR pkCSM, the substance is well distributed into the body.


 


Specific organ toxicity on spleen and testes was observed in the 28 -day repeated toxicity study at the maximal dose of 1000 mg/kg/day confirming the good distribution of the substance into the body.


  


METABOLISM


There are no experimental study on metabolism of 2-ethylhexyl 4-(dimethylamino)benzoate.


 


ELIMINATION


Due to the low water solubility, the excretion of 2-ethylhexyl 4-(dimethylamino)benzoate in the urines is expected to be low. An excretion via bile and faeces is possible.


According to the QSAR pkCSM, a low total clearance (hepatic & renal) is expected.