4,4'-isopropylidenediphenol

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Scientific Committee on Consumer Safety (SCCS). Basic Criteria for the In Vitro Assessment of Dermal Absorption of Cosmetic Ingredients. SCCS/1358/10, 22 June 2010.

GLP compliance:

yes

Test material

Radiolabelling:

yes

Test animals

Species:

human

Strain:

not specified

Sex:

male/female

Administration / exposure

Type of coverage:

other: in vitro

Vehicle:

other: phosphate buffered saline solution (PBS)

Duration of exposure:

24h

Doses:

300, 60, 12, 2.4mg/l

No. of animals per group:

4 donors

Control animals:

no

Results and discussion

Percutaneous absorption

Dose:

300 mg/l

Parameter:

percentage

Absorption:

> 2 %

Remarks on result:

other: 24 h

Remarks:

Overall, the majority of the applied radioactivity was associated with epidermis samples (10.66%) compared to dermis (3.28%) and receptor fluid (1.98%) samples.

Any other information on results incl. tables

As part of the safety evaluation of Bisphenol A (BPA), a study was required by the European Chemicals Agency (ECHA) to assess the rate and extent of absorption and metabolism of BPA following topical application to human skin.

 

A recent Community Rolling Action Plan (CoRAP) decision regarding the REACH dossier submission on BPA has required the completion of in vitro dermal penetration studies to provide more information on the dermal exposure pathway. The CoRAP decision noted the need to account for (potential) dermal metabolism of BPA. In order to fulfil this requirement, the study described herein was conducted.

 

The study was conducted according to the OECD principles of Good Laboratory Practice as incorporated into the United Kingdom Statutory Instrument for Good Laboratory Practice and as accepted by Regulatory Authorities throughout the European Union, United States of America (FDA and EPA) and Japan (MHLW, MAFF and METI) and other countries that are signatories to the OECD Mutual Acceptance of Data Agreement. All routine activities performed during the conduct of the study are detailed in Charles River Standard Operating Procedures.

 

The abdominal skin was obtained fresh from surgery from 4 different donors (3 female, 1 male). The metabolic competence for phase I metabolism of the skin was confirmed by assessment of MTT reduction. The mean results are summarised in the following table.

 

	Concentration of MTT Formazan Metabolite (mg/mL)
Donor No.	Viable Skin	Heat Deactivated Skin
Donor 1 (0622)	0.080	0.017
Donor 2 (0659)	0.077	0.023
Donor 3 (0668)	0.088	0.039
Donor 4 (0667)	0.071	0.024

 

The metabolic activity was higher in the viable skin discs than heat deactivated skin discs. It was noted that heat deactivated skin produced MTT formazam. This was potentially due to the incomplete destruction of metabolic activity within the skin by heating or that chemicals within the skin may have been direct MTT reducers. This observation is in line with historic data for this assay within this laboratory. The greater reduction in the viable skin compared with the controlstill demonstrated evidence of active MTT mitochondrial reductase enzymes.

 

For three donors, the metabolism of BPA by human skin disks in vitro was assessed in 12 and 96‑well plates. All samples from both viable and heat deactivated skin samples at 0 h after dosing demonstrated no metabolism. The results from the viable skin samples at 24 h demonstrated that metabolism of BPA was possible in the skin. The level of metabolic transformation was less than 25% in all cases. Metabolites observed in the radio-chromatograms had retention consistent with the BPA-glucuronide and BPA‑sulfate. It was noted that the level of transformation was up to 18% for Donors 2 and 4 for the 24 h heat deactivated skin control samples. These results were unexpected and were potentially due to the incomplete destruction of metabolic activity within the skin from the heating process.

 

Split‑thickness human skin membranes were mounted into flow through diffusion cells (0.64 cm², n=4 per dose) and the receptor fluid was pumped underneath the skin at a flow rate of 0.75 mL/h ± 0.15 mL/h. The skin surface temperature was maintained at 32 °C ± 1 °C throughout the experiment. Electrical resistance barrier integrity test was performed and any skin sample exhibiting a resistance lower than 10.9 kΩ was excluded from subsequent absorption measurements with the exception of two samples (Cell 37 and Cell 40) from Donor 3. The barrier integrity results for Cell 37 and Cell 40 were 5.857 kΩ and 4.960 kΩ, respectively. There was no skin remaining from Donor 3 to replace these cells, therefore, these cells were used on this study. The lower electrical resistance observed in these samples indicates poorer barrier integrity and hence potential for greater absorption therefore, including these samples was the conservative approach for a risk assessment. 

 

For six additional skin samples barrier integrity was also performed at 0 h and 24 h post dose, using the method described above. This assessment was conducted using skin from Donor 2 only. All electrical resistance values at 0 h and 24 h were >10.9 kΩ. This confirmed the chosen receptor fluid did not interfere with the integrity/barrier function of skin.

 

[¹⁴C]‑BPA was incorporated into phosphate buffered saline solution (PBS) to produce four test preparations at final BPA concentrations of ca 300 mg/L, 60 mg/L, 12 mg/L and 2.4 mg/L for Test Preparations 1-4, respectively. The test preparations were applied (10 µL/cm²) to human split‑thickness skin membranes.

 

Percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (DMEM) containing ethanol (ca 1%, v/v), Uridine 5’‑diphosphoglucuronic acid (UDPGA, 2 mM) and 3’‑phosphoadenosine‑5’‑phosphosulfate (PAPS, 40 µM)) at 0, 1 h and then in two hourly fractions from 2 to 24 h post dose, with the exception that for Donor 1 (0622), where the receptor fluid was collected at 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h post dose.

 

At 24 h post dose, exposure was terminated with a concentrated commercial handwash soap, rubbed in with a tissue swab, followed by rinsing with a dilute 2% (v/v) soap solution and drying the skin surface with tissue paper (tissue swabs); this process was repeated. The receptor chamber was emptied and rinsed with receptor fluid. The cell was dismantled and the donor chamber and receptor chamber retained separately in the pre‑weighed pots containing ethanol. The skin was removed from the cells and the stratum corneum was removed by tape stripping. The unexposed skin was cut away from the exposed skin. The exposed epidermis was separated from the dermis using a scalpel. Twelve skin samples from each test preparation were solubilised with Solvable^® tissue solubiliser. These samples were analysed by liquid scintillation counting. The remaining solubilised skin samples and receptor fluid samples obtained from four cells from each Donor were analysed by HPLC‑UV.

 

A summary of the mean results is provided in the following table.

 

Test Preparation*	1	2	3	4
Target Concentration (mg/L)	300	60	12	2.4
	(% Applied Dose)
Total Dislodgeable Dose	72.34±5.64	70.91±6.20	71.95±7.98	71.57±9.11
WholeStratum Corneum	10.25±5.44	9.25±4.30	7.31±3.33	7.70±4.92
Total Unabsorbed Dose	82.61±8.37	80.31±6.92	79.33±9.97	79.37±9.91
Epidermis	10.66±6.40	10.45±5.73	10.38±5.36	11.91±4.86
Dermis	3.28±2.44	3.97±1.99	6.19±4.28	4.51±3.73
Total Absorbed Dose	1.98±1.42	1.68±1.20	2.72±1.95	3.62±1.69
Dermal Delivery	15.92±8.14	16.10±7.01	19.28±8.54	20.04±6.24
Mass Balance	98.53±1.99	96.41±1.45	98.62±2.18	99.40±6.54
	ng equiv./cm^2
Total Dislodgeable Dose	2326.98±204.66	452.46±36.03	89.64±9.41	17.96±1.90
WholeStratum Corneum	330.28±177.24	59.08±27.96	9.14±4.28	1.96±1.31
Total Unabsorbed Dose	2657.95±299.42	512.53±41.60	98.88±12.31	19.95±2.31
Epidermis	343.27±209.71	67.14±37.98	12.93±6.71	3.04±1.35
Dermis	104.05±75.82	25.55±13.11	7.76±5.41	1.13±0.92
Total Absorbed Dose	63.27±45.29	10.71±7.84	3.38±2.46	0.91±0.43
Dermal Delivery	510.60±263.45	103.41±46.83	24.07±10.81	5.07±1.70
Mass Balance	3168.54±122.76	615.94±23.90	122.95±2.90	25.02±1.88

 

*Each Test Preparation was applied to a total of 12 samples of skin from 4 donors (3 skin samples per donor). The same donors were used for each test preparation.

 

Total Dislodgeable Dose = skin wash + tissue swabs + pipette tips + donor chamber wash

Unabsorbed Dose = dislodgeable dose + wholestratum corneum (all tape strips+ unexposed skin

Absorbed Dose = receptor fluid + receptor chamber wash + receptor rinse

Dermal Delivery = epidermis + dermis + absorbed dose

Mass balance = dermal delivery + unabsorbed dose

 

[¹⁴C]‑Bisphenol A in Test Preparation 1 (ca 300 mg/L) was applied to human split‑thickness skin in vitro. At 24 h post dose, the mean total dislodgeable dose was 72.34% of the applied dose. The stratum corneum retained a mean of 10.25% of the applied dose, with a mean of 2.46% being removed with the first 2 tape strips. The mean total unabsorbed dose was 82.61% of the applied dose. The total absorbed dose and dermal delivery accounted for a mean of 1.98% and 15.92% of the applied dose, respectively.

 

[¹⁴C]‑Bisphenol A in Test Preparation 2 (ca 60 mg/L) was applied to human split‑thickness skinin vitro. At 24 h post dose, the mean total dislodgeable dose was 70.91% of the applied dose. Thestratum corneumretained a mean of 9.25% of the applied dose, with a mean of 2.27% being removed with the first two tape strips. The total unabsorbed dose accounted for a mean of 80.31% of the applied dose. The absorbed dose and dermal delivery accounted for a mean of 1.68% and 16.10% of the applied dose, respectively.

 

[¹⁴C]‑Bisphenol A in Test Preparation 3 (ca 12 mg/L) was applied to human split‑thickness skin in vitro. At 24 h post dose, the mean total dislodgeable dose was 71.95% of the applied dose. The stratum corneum retained a mean of 7.31% of the applied dose, with a mean of 1.79% being removed with the first two tape strips. The total unabsorbed dose accounted for a mean of 79.33% of the applied dose. The absorbed dose and dermal delivery accounted for a mean of 2.72% and 19.28% of the applied dose, respectively.

 

[¹⁴C]‑Bisphenol A in Test Preparation 4 (ca 2.4 mg/L) was applied to human split‑thickness skin in vitro. At 24 h post dose, the mean total dislodgeable dose was 71.57% of the applied dose. The stratum corneum retained a mean of 7.70% of the applied dose, with a mean of 2.29% being removed with the first two tape strips. The total unabsorbed dose accounted for a mean of 79.37% of the applied dose. The absorbed dose and dermal delivery accounted for a mean of 3.62% and 20.04% of the applied dose, respectively.

 

The dose‑response relationship observed for Test Preparations 1‑4 was linear (R²≥ 0.995). The mean total absorbed dose is between 1.68% and 3.62% of the applied dose and dermal delivery is between 15.92% and 20.04% of the applied dose. Therefore, extrapolation within this dose range is possible for this formulation type. 

 

Metabolism was only investigated for the Test Preparation 1 group (300 mg BPA/L). Metabolism was not investigated in samples dosed with Test Preparations 2‑4 since the levels of radioactivity in these samples were too low to allow detection. From dermal delivery, the majority of the radioactivity was associated with epidermis samples compared to dermis and receptor fluid samples. No metabolism was observed in any of the epidermis samples, However metabolism was observed in dermis and receptor fluid samples (0-14%). The metabolites observed in the radio-chromatograms had retention consistent with the BPA-glucuronide and BPA‑sulfate. The level and distribution of metabolites found in the receptor and dermis samples varied among donors. However, overall the cumulative level of metabolism in the receptor fluid plus dermis was reasonably consistent (12-17%).

 

In conclusion, following topical application of [¹⁴C]‑Bisphenol A in Test Preparation 1, Test Preparation 2, Test Preparation 3 and Test Preparation 4 to fresh human skin in vitro, the mean absorbed dose was 1.98% (63.3 ng equiv./cm²), 1.68% (10.7 ng equiv./cm²), 2.72% (3.38 ng equiv./cm²) and 3.62% (0.91 ng equiv./cm²) of the applied dose, respectively. The mean dermal delivery was 15.92% (511 ng equiv./cm²), 16.10% (103 ng equiv./cm²), 19.28% (24.1 ng equiv./cm²) and 20.04% (5.07 ng equiv./cm²) of the applied dose, respectively. The mean mass balance was 98.53% (3169 ng equiv./cm²), 96.41% (616 ng equiv./cm²), 98.62% (123 ng equiv./cm²) and 99.40% (25.0 ng equiv./cm²) of the applied dose, respectively. 

 

Metabolism was investigated for the Test Preparation 1 group only. Metabolism was not investigated in samples dosed with Test Preparations 2‑4 since the levels of radioactivity in these samples were too low to allow detection. From dermal delivery, the majority of the applied radioactivity was associated with epidermis samples (10.66%) compared to dermis (3.28%) and receptor fluid (1.98%) samples.

 

No metabolism was observed in any of the epidermis samples, however limited levels of metabolism were observed in dermis and receptor fluid samples (0-14%) with formation of BPA-glucuronide and BPA-sulfate identified in supernatant from incubation of viable skin disks for 24 h (<25%). Metabolites with retention consistent with BPA‑glucuronide and BPA-sulfate, and also more polar components, were identified. It might be assumed, but is not analytically verified, that these polar compounds are mixed sulfate/glucuronide bis‑conjugate BPA metabolites.

 

Taking into account the skin disk experiments it can be concluded qualitatively that fresh human skin has some in vitro metabolic capacity but further experiments may be necessary to optimize the experimental conditions to quantify that metabolism.

Applicant's summary and conclusion

Executive summary:

The followingdermal absorption rates were reported in this study:

Target Concentration (mg/L)	300	60	12	2.4
	(% Applied Dose)
Total Dislodgeable Dose	72.34 ±5.64	70.91 ±6.20	71.95 ±7.98	71.57 ±9.11
WholeStratum Corneum	10.25 ±5.44	9.25 ±4.30	7.31 ±3.33	7.70 ±4.92
Total Unabsorbed Dose	82.61 ±8.37	80.31 ±6.92	79.33 ±9.97	79.37 ±9.91
Epidermis	10.66 ±6.40	10.45 ±5.73	10.38 ±5.36	11.91 ±4.86
Dermis	3.28 ±2.44	3.97 ±1.99	6.19 ±4.28	4.51 ±3.73
Total Absorbed Dose	1.98 ±1.42	1.68 ±1.20	2.72 ±1.95	3.62 ±1.69
Dermal Delivery	15.92 ±8.14	16.10 ±7.01	19.28 ±8.54	20.04 ±6.24
Mass Balance	98.53 ±1.99	96.41 ±1.45	98.62 ±2.18	99.40 ±6.54

 

There are two guidance documents to calculate the potential dermal absorption based on in vitro data.

a)EFSA Guidance on Dermal Absorption (EFSA Journal 2012. 10, 2665)

EFSA defines absorption= receptor fluid + receptor chamber washes + skin sample (excluding tape strips 1 and 2). The guidance indicates:“There is a general practise within EFSA PRAPeR9 meetings that the first 2 tape strips will represent material that will not become bioavailable due to desquamation. The Panel proposes to follow this approach. Thus, the first 2 tape strips can be excluded when calculating dermal absorption …If there is significant variation between replicates (i.e. the standard deviation is equal to or larger than 25% of the mean of the absorption as defined in section 5.6. and 5.8.) consideration should be given to using a value other than the mean or rejecting the study entirely. The preferred approach would be the addition of a standard deviation to the mean value. that the application site was swabbed to remove the test material before termination of the study.”

 

Applying the EFSA guidance leads to the following potential absorption values;bold values denote value to be used according to the EFSA Guidance.

Test Preparation	Mean Potentially Absorbable Dose	Mean+1SD	SD>25% of mean
300 mg/L	23.70	31.84	Yes
60 mg/L	23.09	29.13	Yes
12 mg/L	24.80	31.79	Yes
2.4 mg/L	25.42	30.79	No

 

b)SCCS Basic Criteria for the in vitro assessment of dermal absorption of cosmetic ingredients (SCCS/1358/1)

 

SCCS defines absorption as follows:“In a classicalin vitrodermal absorption setting, the amounts of penetrated substance(s) found in the receptor fluid are considered to be systemically available.Both the epidermis (except for the stratum corneum) and dermis are considered as a sink, wherefore the amounts found in these tissues are considered as absorbed and are added to those found in the receptor fluid.The amounts that are retained by the stratum corneum at the time of sampling are not considered to be dermally absorbed, and thus they are not expected to contribute to the systemic dose.

…

When studies correspond to all of the basic requirements of the SCCS, themean + 1SDwill be used for the calculation of the MoS… In case of significant deviations from the protocol and/or very high variability, themean + 2SDwill be used as dermal absorption for the calculation of the margin of safety.”

 

Applying the EFSA guidance leads to the following potential absorption values;no clear guidance is given on guideline to define if 1 or 2 SD should be used.

Test Preparation	Mean dermal delivery	Mean+1SD	Mean+2SD
300 mg/L	15.92	24.06	32.20
60 mg/L	16.10	23.11	30.12
12 mg/L	19.28	27.82	36.36
2.4 mg/L	20.04	26.28	31.59

 

Overall, a potentially bioavailable portion of BPA of 30% is taken to derive a corrected dermal starting point for DNEL derivation.

 

 

Potential metabolism was also investigated in this study. No metabolism was observed in any of the epidermis samples, however limited levels of metabolism were observed in dermis and receptor fluid samples (0-14%) with formation of BPA-glucuronide and BPA-sulfate identified in supernatant from incubation of viable skin disks for 24 h (<25%). Metabolites with retention consistent with BPA‑glucuronide and BPA-sulfate, and also more polar components, were identified. It might be assumed, but is not analytically verified, that these polar compounds are mixed sulfate/glucuronide bis‑conjugate BPA metabolites. It can be concluded qualitatively that fresh human skin has somein vitrometabolic capacity but further experiments may be necessary to optimize the experimental conditions to quantify that metabolism.

Overall, as a conservative approach no metabolism was taken into account.

 

Comparison with available in vitro data:

EFSA (2015) summarized the available data as follows“In Demierre et al. (2012), the specific permeation kinetics with an initial high penetration rate and a subsequent low penetration rate are suggestive of effects arising from finite dosing (i.e. partial depletion of the dose on the skin surface) and/or evaporation of the aqueous vehicle (→ reduced hydration of the SC), which both are realistic conditions applicable to consumer exposure. In spite of differences in the diffusion-cell design, skin type, vehicle type and applied dose, the in vitro studies of Marquet et al. (2011), Mørck et al. (2010), and Kaddar et al. (2008) support the percutaneous penetration estimate of 8.6 % of Demierre et al. (2012), although tending to somewhat lower values: a rough calculation based on the comparison of permeability coefficients or the normalization of percutaneous penetration to 24 h incubation yielded estimates of 2.3 % (Marquet et al., 2011) and 6.5 % (Mørck et al., 2010) for human skin, and of 4.1 % (Kaddar et al., 2008) for pig skin.

…

For exposure scenarios with dermal contact to thermal paper, the CEF Panel used a conservative value of 10% dermal absorption. The CEF Panel did not consider skin metabolism (conservative decision). “

 

Comparison of the in vitro data between Mørck et al. (2010), Demierre et al. (2012) and Toner (2015)

Parameter	Mørck et al. (2010)^1	Demierre et al. (2012)^1	This study
Number of skin sections	human skin samples from breast surgery	dorsal part of the upper leg from 2 human cadavers.	abdominal skin obtained fresh from surgery from 4 different donors (3 female, 1 male)
Number of skin sections	11	7	12
Skin viability	non-viable	non-viable	Fresh skin
Skin Section thickness	800–1000 μm	200 μm	350-400 µm
Exposed area	2.12 cm2	0.64 cm2	0.64 cm2
Applied volume	32.6 μl	6 μl	6.4 µl
Applied volume per area	15.4 μl/cm2	9.4 μl/cm2	10 µl/cm2
Applied concentration	3995 mg/l (= 17.5 mM)	194 mg/l	4 different concentrations:300, 60, 12, 2.4 mg/l
Applied surface density	259 μg/cm2	1.82 μg/cm2	3, 0.6, 0.12, 0.04μg/cm2
Applied dose	452 μg	1.16 μg	1.9, 0.4, 0.08, 0.015 µg
Temperature	≈32 °C	30–32 °C	32°C ± 1°C
Method	static Franz diffusion cell OECD TG 428	flow-through Franz cell OECD TG 428	flow-through Franz cell OECD TG 428; SCCS
Skin integrity check	capacitance measurement	permeability coefficient within acceptance range	electrical resistance > 10.9 kΩ
donor solution (vehicle)	0.9% NaCl + 2% EtOH	water	Phosphate buffered saline (PBS)
receptor fluid	physiol. saline + BSA	physiological saline	DMEM;ca1%, v/v) + UDPGA (2 mM) + PAPS (40 µM).
Duration of incubation	48 h	24 h	24 h
Recovery	82.1 %	101.5 ± 1.6 %	96.4 – 99.4 %
Percutaneous penetration	13.0 ± 5.4 %	8.6 ± 2.1 %	1.7 – 3.6 %

  ¹: Information derived from EFSA (2015; page 575-576)

 

Taking into account the above mentioned 10 % dermal penetration taken by EFSA for their dermal risk assessment based on the ca. 10% BPA found in the receptor fluid in the Demierre et al. (2012) study a significantly lower penetration value into the receptor fluid was observed in the in vitro dermal penetration study using viable skin (1.7 – 3.6 %). The approach mentioned in the study request by ECHA to consider the dermis, epidermis (with stratum corneum) and receptor fluid for further calculations lead to substantially higher potential penetration values compared to the EFSA 2015 evaluation of BPA.