Acrylonitrile

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23rd June-17th August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Acrylonitrile

EC Number:

203-466-5

EC Name:

Acrylonitrile

Cas Number:

107-13-1

Molecular formula:

C3H3N

IUPAC Name:

prop-2-enenitrile

Radiolabelling:

yes

Remarks:

[14C]acrylonitrile

Test animals

Species:

other: rat and human skin, in vitro

Administration / exposure

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Duration of exposure:

1 hour

Doses:

The application volume was 10 µL/cm2

Details on in vitro test system (if applicable):

Charles River UK supplied five samples of full-thickness skin obtained from male CD® rats (Rat/IGS (Crl: CD®(SD) (IGS BR)) aged 6-8 weeks old, weighing 200 to 250 g.

Five samples of full-thickness human skin (2 breast, 1 abdomen, 1 abdomen/breast and 1 abdomen/arms/back) were obtained from donors aged 23 to 53 years old. Samples were cleaned of subcutaneous fat and connective tissue using a scalpel blade. The skin samples were washed in cold running water and dried using tissue paper. The skin samples were then cut into smaller pieces (where appropriate), wrapped in aluminium foil, placed into self sealing plastic bags and stored in a freezer set to maintain a temperature of 20°C until they were used in the study.

Human and rat skin samples were removed from storage and allowed to thaw at ambient temperature. The fur of the rat skin was trimmed using electric clippers. The thickness of the full thickness skin membranes were measured using a micrometer. Split thickness membranes were prepared by pinning the full thickness skin, stratum corneum uppermost, onto a raised cork board and cutting at a setting equivalent to 200 400 µm depth using a dermatome.

An automated flow through diffusion cell apparatus was used. The flow through diffusion cells were placed in a steel manifold heated via a circulating water bath to maintain the skin surface temperature at 32C. The cells were connected to multi-channel peristaltic pumps from their afferent ports with the receptor fluid effluent dropping via fine bore tubing into scintillation vials on a fraction collector. The surface area of exposed skin within the cells was 0.64 cm2. The receptor chamber volume was 0.25 mL. The peristaltic pumps were adjusted to maintain a flow-rate of 1.5 mL/h ± 0.15 mL/h.
Phosphate buffered saline (PBS) was used as the receptor fluid. The receptor fluid was degassed by sonication prior to use. Acrylonitrile has a water solubility of 7.3% (w/w) at 20ºC in water. For an application of 10 µL/cm2 over a 0.64 cm2 application area, this would be 5.16 mg/0.64 cm2 of Acrylonitrile (density = 806 kg/m3). If 100% of Acrylonitrile was absorbed in 1 h (1.5 mL), then 5.16 mg/1.5 mL is 3.44 g/L. Therefore, this receptor fluid was not considered to be rate limiting for solubility and was accepted for use.

Sections of split thickness skin membrane 1.5 x 1.5 cm were cut out, positioned on the receptor chamber of the diffusion cell, which contained a magnetic flea and the donor chamber was tightened into place. The prepared cells were then placed in the heated manifold and connected to the peristaltic pump. The magnetic stirrer was switched on to mix the contents of the receptor chamber. An equilibration period of 15 minutes was allowed while receptor fluid was pumped through the receptor chambers at 1.5 mL/h ± 0.15 mL/h. The effluent was then collected for 30 minutes and retained as blank samples for use in the tritiated water barrier integrity assessment.

Tritiated water (250 µL, 100,000 dpm) was applied to the surface of each skin sample and the donor chamber occluded. Penetration of tritiated water was assessed by collecting receptor fluid for 1 hour and analysing the sample by liquid scintillation counting. The mean dpm applied for the tritiated water was calculated from the seven mock tritiated water samples taken at the time of dosing. Absorption was then calculated for each skin sample from the receptor fluid sample collected. Any human and rat skin sample exhibiting greater than 0.6% absorption was excluded from subsequent absorption measurements. At the end of the 1 hour period, residual tritiated water was removed from the skin surface by rinsing with water (1 2 mL). The skin was then dried with tissue paper. An equilibration period of up to 3 hours was allowed prior to collection of the pre dose sample which was collected for 0.5 hours.

Results and discussion

Signs and symptoms of toxicity:

not specified

Remarks:

: not applicable

Dermal irritation:

not specified

Remarks:

: not applicable

Percutaneous absorption

Percutaneous absorption 1

Parameter:

percentage

Absorption:

1.3 %

Remarks on result:

other: 24 hours

Remarks:

Human skin

Percutaneous absorption 2

Parameter:

percentage

Absorption:

0.76 %

Remarks on result:

other: 24 hours

Remarks:

Rat skin

Any other information on results incl. tables

Distribution of radioactivity

Species:	Human		Rat
Application rate:	8015.89 µg equiv./cm2 (10 µL/cm2)		8026.48 µg equiv./cm2 (10 µL/cm2)
Distribution	(% Applied Dose)	(µg equiv./cm2)	(% Applied Dose)	(µg equiv./cm2)
Dislodgeable Dose 1 h:	27.10	2160.19	47.98	3824.99
Total Dislodgeable Dose:	27.35	2180.01	48.30	3850.48
Unabsorbed Dose:	27.74	2210.86	48.55	3870.84
Absorbed Dose:	1.05	83.88	0.55	43.59
Dermal Delivery:	1.30	103.47	0.76	60.32
Mass Balance:	29.04	2314.33	49.31	3931.15
Volatile loss after 1 h post dose:	26.80	2136.28	47.78	3809.76
Total volatile loss:	26.97	2149.68	48.07	3832.69

Applicant's summary and conclusion

Conclusions:

Following the topical application of [14C]-acrylonitrile to human and rat skin membranes in vitro, the primary route of loss was via volatilisation. This factor is reflected in the low mass balance for both rat and human skin. The absorbed dose was 1.05% (83.88 µg equiv./cm2) and 0.55% (43.59 µg equiv./cm2) in human and rat skin membranes, respectively. Dermal delivery was 1.30% (103.47 µg equiv./cm2) and 0.76% (60.32 µg equiv./cm2), respectively.

The mass balance for human and rat skin membranes was 29.04% and 49.31%, respectively. The low mass balances were attributed to the volatility of acrylonitrile; findings suggest that nearly all of the lost acrylonitrile had volatilised within the first hour after application and that the filter and trap were not effective at collecting all of this volatilised material. Given the high and variable rates of volatile loss, absorption rates could not be reliably compared between the species. However the results indicate that 1.30% in human and 0.76% in rats of the applied dose was either retained in the skin or had been absorbed. The results of the study indicate that systemic absorption resulting from occupational dermal exposure to acrylonitrile is likley to be be limited due to the volatile nature of the substance.

Executive summary:

The dermal absorption of 14C-radiolabeled acrylonitrile was investigated in vitro in human and rat skin membranes. Split‑thickness human and rat skin membranes were mounted into flow‑through diffusion cells using phosphate- buffered saline as receptor fluid. The skin surface temperature was maintained at 32°C ± 1°C throughout the experiment.  A tritiated water barrier integrity test was performed prior to the main study to ensure the integrity of the skin membranes.

14C-acrylonitrile was applied to the topical surface of the skin membranes at an application volume of 10 µL/cm2. Immediately after application, the donor chamber of the cells was occluded with a trap containing an activated charcoal filter intended to collect any volatile fraction. Percutaneous absorption was assessed by collecting receptor fluid in hourly fractions from 0-8 hours post‑application and in 2‑hourly fractions from 8-24 hours post‑application.  At 1 hour post application, the volatile trap was removed and exposure was terminated by washing the skin surface with water and drying with tissue swabs.  A new trap was then used to occlude the cells. At 24 hours post application, the underside of the skin membranes and the receptor chamber were rinsed with receptor fluid. The trap was collected, the skin was removed from the diffusion cells, dried and the stratum corneum was removed with 20 successive tape strips.  The remaining skin was divided into exposed and unexposed skin and solubilised. All samples were analysed by liquid scintillation counting.

The mass balance for human and rat skin membranes was 29.04% and 49.31%, respectively. The low mass balances were attributed to the volatility of acrylonitrile; findings suggest that nearly all of the lost acrylonitrile had volatilised within the first hour after application and that the filter and trap were not effective at collecting all of this volatilised material. Given the high and variable rates of volatile loss, absorption rates could not be reliably compared between the species. However the results indicate that 1.30% in human and 0.76% in rats of the applied dose was either retained in the skin or had been absorbed. The results of the study indicate that systemic absorption resulting from occupational dermal exposure to acrylonitrile is likley to be limited by the volatile nature of the substance.