Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 February to 16 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Radiolabelling:
yes

Test animals

Species:
other: in vitro rabbit skin
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
Rabbit skin was obtained from Bioreclamation LLC, USA. Skin was from 2 male and 2 female New Zealand White rabbits approximately 3 months of age

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
24 hours
Doses:
approx 10mg/cm2 (or 7mg/0.64cm2)
No. of animals per group:
Triplicate skin samples each from 4 rabbits
Control animals:
no
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Bioreclamation LLC, USA
- Type of skin: rabbit

- Preparative technique:
On the day of experiment, skins were thawed at room temperature and underlying fat removed. Excessive hairs were removed with an electric clipper and skin sections (samples) of appropriate thickness (200-500 microns) were prepared. Prepared skin membrane from each subject was placed in labelled petri dishes, one for each individual rabbit subject, containing cell culture medium at room temperature while the other skin sections were being processed. Skin sections free of blemishes and of the appropriate thickness were chosen from each subject. Three disc shaped pieces, large enough to fit 0.64cm2 flow-through diffusion cells, were punched out from each skin sample and rinsed with water. the thickness of all skin pieces was measured with a digimatic micrometer before placing each piece epidermis side up in the diffusion flow-through cells.

- Thickness of skin (in um): 200-500

- Membrane integrity check:
Each piece of skin received and infinite dose of 3H-H20 (~200ul, exact dose determined gravimetrically) for 20 minutes in order to determine if the barrier integrity of the skin had been compromised. The 3H-H20 was diluted to obtain specific gravity of ~ 0.000893mCi/g (1982 disintegrations per minute/mg, DPM/mg) using analytical grade water.

The 3H-H20 dose was removed by blotting the skin surface after 20 minutes of exposure and skin was rinsed with reverse osmosis water (analytical grade water) and dried by gentle blotting. Aliquots of the receptor fluid were continued to be collected for an additional 60 minutes after the dose was removed. Radioactivity content in the receptor fluid was determined by liquid scintillation analysis.

- Storage conditions: shipped on dry ice and stored frozen at -20 degrees C

PRINCIPLES OF ASSAY
- Receptor fluid: HBSS-Hank's Balanced Salt Solution with 0.6% HEPES, 0.005% Gentamicin and 4% Bovine Serum Albumin was mixed, the pH was adjusted to 7.3 - 7.5 with 1N NaOH and then filtered through a 0.45 micron filter. The receptor fluid was pumped beneath the skin samples through the receptor compartment for 24 hours at a flow rate between 2.0 - 2.5 ml/hr and collected using a fraction collector.
- Solubility of test substance in receptor fluid: Approximately 10.0 ml of the receptor fluid containing 4% Bovine Serum Albumin was placed in a specially designed flask with a sampling port at the bottom. The 14C test article (10mg) was spiked onto the surface of the receptor fluid. Receptor fluid aliquots were taken from the bottom of the flask via the sampling port at 1 and 2 hours and radioactivity content analysed by liquid scintillation counter. Data showed that 14C test article migrated from the surface into the receptor fluid reaching a concentration of 0.57 ppm in 2 hours. This concentration represents >1000-fold increase in solubility relative to the water solubility of trifluoropropylmethylcyclotrisiloxane (0.0013 ppb). Also, 0.19% of applied 14C test article was recovered in receptor fluid with 2 hours demonstrating that solubility of the test article was higher than the amount of test article penetrated into the receptor fluid over 24 hours of skin exposure (average 0.098% of applied dose).

- Flow-through system: Bronaugh flow-through apparatus
- Test temperature: not stated
- Humidity: not stated
- Occlusion: no

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Percutaneous absorption
Dose:
10 mg/cm2
Parameter:
percentage
Absorption:
0.098 %
Remarks on result:
other: entire 24 hour period

Any other information on results incl. tables

The average dose applied to the skin sample was 0.00742g by syringe dose measurement (Table 1).

Table 1: determination of Dose (Syringe dose gravimetric)

Trial

Syringe + Dose Solution weight (g)

Empty Syringe weight (g)

Dose weight (g)

1

8.3017

8.2943

0..074

2

8.3033

8.2954

0.0079

3

8.3030

8.2957

0.0073

4

8.3031

8.2958

0.0073

5

8.3031

8.2960

0.0071

6

8.3030

8.2957

0.0073

7

8.3034

8.2959

0.0075

8

8.3032

8.2959

0.0073

9

8.3032

8.2957

0.0075

10

8.3037

8.2961

0.0076

Mean
SD
%RSD

 

 

0.00742
0.00022
2.97

A small amount of 14C-test article penetrated through the skin into the receptor fluid (0.098% of applied dose +/- 0.01) over the 24 hour exposure period (Table 2). The mean cumulative penetration of radioactivity over the 24 hour period was 30009.51 +/- 2954.86 DPM/cm2 of skin and 1.125 +/- 0.111 µg equivalent cm2 skin (Table 3). Permeability coefficient (Kp) was estimated to be 0.000122 cm/hr based on steady state flux established between 1 and 5 hours.

The percent of applied 14C-test article recovered from all analysed samples was 92.63% +/- 4.2 (Table 2). The majority of the applied dose was found on the skin surface from the exposure site (62.55% +/- 5.27) and in the skin (29.36% +/- 6.97). Only 0.629% +/- 0.223 of applied dose was found on the PorPak-P tubes and the part of the apparatus specifically designed to hold them. The total percent dose absorbed was estimated to be 29.45% +/- 6.67 of applied dose with virtually all of the absorbed 14C retained in the skin after washing and tape stripping of the exposure site.

Table 2: in vitro disposition of radioactivity following application of 14C-test article to rabbit skin

Dose Solution

Skin Surface (%)

Volatilized Dose (%)

Skin
(%)

Penetrated
(%)

Absorbed (%)

Total Dose Recovery (%)

14C

62.55

0.629

29.36

0.098

29.45

92.63

SEM

5.27

0.223

6.97

0.01

6.43

4.2

Table 3: Cumulative penetration of 14C-test article through rabbit skin

Cell No.

Skin subject

Cumulative DPM/cm2

skin

Mean cumulative DPM/cm2 skin

SD

SEM

Cumulative

µg equiv./cm2skin

Mean Cumulative

µg equiv./cm2skin

SEM

SD

1

A

28894.16

 

 

 

10.83

 

 

 

2

A

30508.84

 

 

 

11.43

 

 

 

3

A

33384.05

30929.01

2274.24

1313.04

12.51

11.59

0.49

0.85

4

B

28234.72

 

 

 

10.58

 

 

 

5

B

28358.89

 

 

 

10.63

 

 

 

6

B

38419.14

31670.91

5844.46

3374.30

14.40

11.87

1.26

2.19

7

C

43482.07

 

 

 

16.29

 

 

 

8

C

22911.55

 

 

 

8.59

 

 

 

9#

C

 

33196.81

14545.55

10285.26

 

12.44

3.85

5.45

10

D

16354.17

 

 

 

6.13

 

 

 

11

D

44819.91

 

 

 

16.80

 

 

 

12

D

14737.16

25303.75

16920.82

9769.24

5.52

9.48

3.66

6.34

Mean
SD
SEM

 

30009.51
9800.15
2954.86

 

 

 

11.25
3.67
1.11

 

 

 

# Cell No. 9 was excluded due to skin integrity test.

Applicant's summary and conclusion

Conclusions:
The study demonstrated that large amounts of 14C-trifluoropropylmethylcyclotrisiloxane was recovered from the skin surface (62.55% of applied dose) after a 24 hour exposure period or remained in the skin after washing and tape stripping (29.36 % of applied dose). Only a small amount of dose (0.63% of applied dose) volatilized from the skin surface. The percent of applied dose of 14C-trifluoropropylmethylcyclotrisiloxane recovered from all analysed samples was 92.63%. Little (0.098%) of the applied dose penetrated through the skin into the receptor fluid with the mean cumulative penetration (+/- SEM) over 24 hours being only 11.25 +/- 1.11 µg equivalents 14C-trifluoropropylmethylcyclotrisiloxane per cm2 exposed skin.