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Reference
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
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).γ))]]
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 γ 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

γ

-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.

Description of key information

Under the conditions of the dermal absoprtion 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.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
1
Absorption rate - inhalation (%):
100

Additional information

No experimental studies of the absorption, distribution, metabolism or elimination of SpeedCure EHA/2-ethylhexyl 4-(dimethylamino)benzoate(CAS number:21245-02-3) in mammals are available. However, the physical chemical properties and the existing toxicology studies on the substance and on the structurally-similar substance SpeedCure EDB/ethyl-4-(dimethylamino)benzoate(CAS number: 10287-53-3) have been used to infer as far as possible, its potential toxicokinetics.

 

The substance SpeedCure EHA/2-ethylhexyl 4-(dimethylamino)benzoateisa mono-constituent substance. It is a pale yellow liquid, with a bulk density of 0.994 g/cm3at 20°C and molecular weight (MW) of 277.4g/mol. The substance is very poorly water-soluble (0.1 mg/L at 20°C), and its partition coefficient (Log Kow/Log Pow) is greater than 6.2. The vapour pressure was determined to be 5 × 10-4Pa at 25°C. The substance is readily biodegradable.

 

Absorption

The very low water-solubility and high Log Pow would limit the rate of absorption from the gut; the molecular weight greater than 200 also indicates that passage through aqueous pores or transport through the epithelial barrier by the bulk passage of water are unlikely. However, micellar solubilisation may be of particular importance for absorption mainly in the small intestine. The results of the acute oral studies, with the LD50 much higher than 2000 mg/kg, confirm a limited absorption however, under repeated oral dosing regimen, unspecific toxicity, evidenced by a reduction in body weight and effects on testes/epididymides, spleen and liver were observed. Similarly, with the structurally-similar substance SpeedCure EDB/ethyl-4-(dimethylamino)benzoate(CAS number: 10287-53-3), effects on testes and male fertility were observed in an OECD 421 study at 175 and 600 mg/kg/day. Based on this information, it is clear that significant oral absorption is likely, but the exact extent cannot be determined, and so, in accordance with ECHA guidance, is assumed to be 50% for risk assessment purposes.

 

The very low vapour pressure (5 × 10-4Pa at 25°C) indicates that the substance cannot generate an inhalable vapour. Also for inhalation, micellar solubilisation may be of particular importance for absorption. In the absence of any quantitative data, and in accordance with ECHA guidance, for risk assessment purposes absorption by inhalationofSpeedCure EHA/2-ethylhexyl 4-(dimethylamino)benzoateis assumed to be 100%.

 

Although SpeedCure EHA/2-ethylhexyl 4-(dimethylamino)benzoate is not an excessively large molecule, the extremely low water solubility(0.1 mg/L at 20°C) and high Log Pow (>6.2) indicate that dermal uptake will be low, and the rate of transfer between the stratum corneum and the epidermis will be slow. The substance is neither irritating to the skin, nor possesses any skin sensitising potential. In an in vitro dermal absorption study conducted with the registered substance, following 24-h exposure of human skin to 0.5, 1 and 2% solutions in mineral oil no more than 0.1% was recovered in the receptor fluid and approximately 0.35% in the epidermis. Based on these results, and as worst-case, 1% dermal absorption will be used for risk assessment purposes.

 

Distribution

Repeat-dose oral toxicity studies identified the liver, red blood cells and testes as the target organs. The relatively low molecular weight of the substance would suggest wider distribution although this might be countered by the low water solubility. The Log Pow value suggests that the intracellular concentration may be higher than extracellular concentration. There was however no evidence of bioaccumulation from the available repeat-dose toxicity studies.

 

Metabolism and Excretion

No information on metabolism of SpeedCure EHA/2-ethylhexyl 4-(dimethylamino)benzoate is available, and no evidence of genotoxicity was determined in in vitro studies performed with the substance. The low molecular weight would suggest prevalence of urinary excretion in case conjugated metabolites are formed. Given the high Log Pow, it is expected that any substance, which has penetrated the stratum corneum but not penetrated the viable epidermis may be sloughed off with skin cells.

A dermal absoprtion 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 study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

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