Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Aqueous solution
Details on test material:
Batch number: 0705071467
Description: Aqueous solution of sodium chlorite
Purity: 30.8 % (w/w)
Radiolabelling:
no

Test animals

Species:
other: Human skin and rats
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Human: The skin samples were obtained from human donors (male and female) post-mortem and were supplied by the International Institute for the Advancement of Medicine.
Rat: Male rats; Charles River UK Ltd, Margate, Kent, UK
- Age at study initiation:Human: 52, 60 and 70 years old
- Weight at study initiation:Rat: Bodyweight at sacrifice: 101-109 g;
Estimated age at sacrifice: 29-31 days

Administration / exposure

Doses:
High dose level: 390 g/L (30.8 %)
Low dose level: 44 g/L (3.5 %)
Seven static diffusion cells were prepared at each dose level.
Volume applied: 10 µL/cm2 exposed skin area (9.5 µL of dose, unoccluded)
No. of animals per group:
Human: dermatomed skin from 3 subjects
Rat: dermatomed skin from 3 rats
Control animals:
yes
Details on in vitro test system (if applicable):
SKIN PREPARATION
Human skin: Prior to use, human skin samples were thawed to room temperature. Each full thickness skin membrane was then swabbed with 70 % v/v ethanol/water to remove residual fat and blood, wiped dry and re-hydrated with distilled water prior to dermatoming.
Rat skin: Each rat was sacrificed by cervical dislocation. After sacrifice, the body was shaved with electric clippers, the skin removed and stored at <-15 ºC. Prior to use, skin samples were thawed to room temperature. The skin was then prepared in the same manner as human skin.Dermatomed skin: The full thickness skin sample was pinned out on a dermatome board (cork board with raised rubber cutting surface) and a mini-dermatome was used to cut slices of skin which contained epidermis and some dermis (dermatomed skin was 200 - 400 μm thick, measured using a digital calliper). Only skin from the dorsal region of the rat was dermatomed.

SIZE OF TEST SITEStatic glass diffusion cells were used providing an exposure area of approximately 0.95 cm2.

EXPOSURE PERIOD
The skin samples were exposed to the test material for 8 hours, after which time the remaining dose was washed off the skin with a mild detergent solution.

SAMPLING TIME
The receptor fluid was taken from each diffusion cell and replenished with fresh receptor fluid (3 mL) at 2, 4, 6 and 8 hours after dose application. The receptor fluid taken at these times and that removed at the end of the experiment (24 hours) were retained for analysis.

SAMPLES
Receptor fluid samples were collected at 0, 2, 4, 8 and 24 hours after dosing. At the end of the experiment, the skin samples were tape stripped to remove residual surface dose and stratum corneum.

CONTROLS
A sample of fresh 50 % v/v ethanol : water was analysed as the zero hour receptor fluid. Untreated diffusion cells containing skin membranes were also set up to assess background levels of sodium chlorite (control cells) and to provide samples of matrices for the determination of procedural recoveries through the analytical method (procedural recovery cells).

Results and discussion

Absorption in different matrices:
HIGH DOSE LEVEL:
Thinning of the epidermis in both human and rat skin samples was observed at 2 hours post dose. The test membranes appeared to have regions of thinning of the skin; these regions were of a circular, bluish appearance. During the swabbing procedure at 8 hours, some disruption of the stratum corneum was observed in all rat skin samples; this disruption was most pronounced in cells 11, 12 and 14 where areas of stratum corneum were removed with the cotton swab. At the end of the experiment (24 hours post dose) all human cells had areas of disrupted epidermis and the stratum corneum of each cell was very thin and dry in appearance. At 24 hours post dose all rat cells showed extensive damage to the stratum corneum with either very little stratum corneum remaining (cells 11, 12 and 14) or fragmentation of the stratum corneum (cells 8, 9, 13 and 16).Following a single application of the high level formulation of sodium chlorite to dermatomed human skin, 9.41 % of the applied dose was recovered in the receptor fluid over the 0 - 24 hour period. The skin swabs taken at 8 hours contained most of the applied dose (42.41 %), with 0.45 % removed in the surface tape strips taken after 24 hours. Tape strips taken to remove the stratum corneum contained 0.29 % of the dose. The remaining skin contained 0.20 % whilst the material remaining on the receptor chamber of the diffusion cell accounted for 0.03 % dose. The material recovered in the skin swabs, surface tape strips and that remaining on the donor chamber of the diffusion cell was considered non-absorbed and accounted for 43.19 % of the applied dose. The overall mean recovery of the dose was 53.12 %.Following a single application of the high level formulation of sodium chlorite to dermatomed rat skin, 67.03 % of the applied dose was recovered in the receptor fluid over the 0 - 24 hour period. The skin swabs taken at 8 hours contained 7.47 % of dose, with 0.01 % removed in the surface tape strips taken after 24 hours. The mean amount of material on the surface tape strips in the tape strips taken to remove the stratum corneum and on the receptor chamber of the diffusion cell was not calculable as greater than 50 % of samples contained levels of sodium chlorite of <0.01 % of the dose. The remaining skin contained 1.03 % of the dose. The material recovered in the skin swabs, surface tape strips and that remaining on the donor chamber of the diffusion cell was considered non-absorbed and accounted for 7.59 % of the applied dose. The overall mean recovery of the dose was 75.71 %.The mean steady-state absorption rates for sodium chlorite at the high dose level were 51.88 µg/cm2/hr and 1182 µg/cm2/hr in human and rat skin, respectively.

LOW DOSE LEVEL:Following application of the low level formulation of sodium chlorite to dermatomed human skin, 3.63 % of the applied dose was recovered in the receptor fluid over the 0 - 24 hour period. The skin swabs taken at 8 hours contained 0.57 % of the dose with a further 4.20 % of the dose removed in the surface tape strips taken at 24 hours. Tape strips taken to remove the stratum corneum contained a mean of 2.74 % of the dose. The remaining skin contained 1.20 % of the dose whilst the material remaining on the receptor chamber of the diffusion cell accounted for 0.08 % dose. The material recovered in the skin swabs, surface tape strips and that remaining on the donor chamber of the diffusion cell was considered to be non-absorbed and accounted for 11.05 % of the applied dose. The overall mean recovery of the dose was 18.70 %.Following a single application of the low level formulation of sodium chlorite to dermatomed rat skin, 18.18 % of the applied dose was recovered in the receptor fluid over the 0 - 24 hour period. The skin swabs taken at 8 hours contained 1.06 % of the dose, with a further 0.05 % removed in the surface tape strips taken after 24 hours. Tape strips taken to remove the stratum corneum contained 0.31 % of the dose, whilst the remaining skin contained 0.33 % of the dose. Material remaining in the receptor chamber of the diffusion cell accounted for 0.06 % dose. The material recovered in the skin swabs, surface tape strips and that remaining on the donor chamber of the diffusion cell was considered non-absorbed and accounted for 1.35 % of the applied dose. The overall mean recovery of the dose was 20.23 %.The mean steady-state absorption rates for sodium chlorite at the low dose level were 1.48 µg/cm2/hr and 21.02 µg/cm2/hr in human and rat skin, respectively.The distribution of sodium chlorite following the application of sodium chlorite to dermatomed human and rat skin is summarised in Tables 1 and 2.

OTHER EFFECTS:Following the application of the high dose sodium chlorite solution (390 g/L) to human and rat skin in vitro, membrane damage was noted during the course of the first sampling time point (2 hours). The test membranes appeared to have regions of thinning of the skin; these regions were of a circular, bluish appearance. No disruption of the stratum corneum was noted up to swabbing at 8 hours. At this point some rat skin cells had areas of stratum corneum that were inadvertently removed during this process with the remaining rat cells less affected but still showing some degree of disruption.Upon termination of the experiment, the extent of this damage was more pronounced in the rat skin than in human skin. The severity of this damage ranged from complete disruption of the stratum corneum observed in most of the rat skins to a ‘thinning’ of the stratum corneum and the partial disruption of the underlying epidermis in the human skin.
Percutaneous absorptionopen allclose all
Key result
Dose:
390 g/L
Parameter:
percentage
Absorption:
9.7 %
Remarks on result:
other: Maximum proportion of the high dose that is absorbed through human skin.
Key result
Dose:
44 g/L
Parameter:
percentage
Absorption:
5.1 %
Remarks on result:
other: Maximum proportion of the low dose that is absorbed through human skin.

Any other information on results incl. tables

Table 1   Distribution of sodium chlorite following application of the high dose solution (390 g/L) to dermatomed human and rat skin

The results are expressed as both percent applied dose and µg of sodium chlorite. The normalized results have been adjusted for method recoveries and include the proportion of dose that was degraded.

Skin type

% dose actual recoveriesa

% dose normalised recoveries*

-

Human

Rat

Human

Rat

TOTAL NON-ABSORBED

-

-

-

-

-

Skin surface (skin swabs + surface tape strips)

%

42.86

7.47

42.16

7.32

-

µg

1656

288.4

1628

282.9

Remaining on cell (donor chamber)

%

0.32

nc

0.32

nc

-

µg

12.52

-

12.52

-

Sub-total

%

43.19

7.59

42.49

7.32

-

µg

1667

292.8

1641

287.3

TOTAL ABSORBED

-

-

-

-

-

Receptor fluid

%

9.41

67.03

9.41

73.66

-

µg

363.3

2587

363.3

2843

Skin

%

0.20

1.03

0.22

1.18

-

µg

7.88

39.75

8.85

45.69

Remaining on cell (receptor chamber)

%

0.03

nc*

0.03

nc*

-

µg

1.03

1.83

1.03

1.83

Sub-total

%

9.64

68.11

9.66

74.84

-

µg

372.2

2629

373.2

2890

Stratum corneum

%

0.29

nc*

0.38

nc*

Degradation products

%

-

-

47.47

17.84

TOTAL RECOVERY

%

53.12

75.71

100.00

100.00

Absorption rate

µg equiv./cm2/hr

51.88

1182

51.88

1299

a Values quoted are the mean values of individual recoveries and not the sum of the individual components

* Corrected for sample matrix procedural recoveries.

nc Not calculable (>=50 % of samples were nd or less than the limit of detection)

nc* Not calculable (>=50 % of samples contained <0.01 % of dose)

Table 2   Distribution of sodium chlorite following application of the low dose solution (44 g/L) to dermatomed human and rat skin

The results are expressed as both percent applied dose and µg of sodium chlorite. The normalized results have been adjusted for method recoveries and include the proportion of dose that was degraded.

Skin type

% dose actual recoveriesa

% dose normalised recoveries*

-

Human

Rat

Human

Rat

TOTAL NON-ABSORBED

-

-

-

-

-

Skin surface (skin swabs + surface tape strips)

%

4.77

1.11

6.01

1.10

-

µg

15.94

3.74

20.09

3.72

Remaining on cell (donor chamber)

%

6.28

0.24

6.28

0.24

-

µg

20.98

0.79

20.98

0.79

Sub-total

%

11.05

1.35

12.29

1.34

-

µg

36.91

4.52

41.07

4.51

TOTAL ABSORBED

-

-

-

-

-

Receptor fluid

%

3.63

18.18

3.63

19.98

-

µg

12.13

60.71

12.13

66.71

Skin

%

1.20

0.33

1.35

0.38

-

µg

4.00

1.09

4.49

1.25

Remaining on cell (receptor chamber)

%

0.08

0.06

0.08

0.06

-

µg

0.26

0.19

0.26

0.19

Sub-total

%

4.91

18.56

5.06

20.42

-

µg

16.39

61.99

16.88

68.15

Stratum corneum

%

2.74

0.31

11.90

0.40

Degradation products

%

-

-

70.75

77.84

TOTAL RECOVERY

%

18.70

20.23

100.00

100.00

Absorption rate

µg equiv./cm2/hr

1.48

21.02

1.48

23.10

a Values quoted are the mean values of individual recoveries and not the sum of the individual components

* Corrected for sample matrix procedural recoveries.

Applicant's summary and conclusion

Conclusions:
Visual examination of the integrity of the skin samples post application of the high dose revealed that the membrane had been disrupted/damaged post dosing. There was a greater degree of membrane damage to the rat skin as opposed human skin. As such, the absorption rates and total absorbed data for the high dose are likely to be overestimates due to the membranes being compromised by the corrosive nature of the high concentration test material.Where possible, it is preferable to use a radiolabelled test material (usually carbon-14) for the conduct of in vitro skin absorption studies. Using a radiolabelled test material means that full mass balances are more easily achievable as any degradation products of the test material that still contain the radiolabel will also be quantified. As the test material for this study, sodium chlorite, does not contain any carbon it was not possible to take this approach. As such, analytical methodology was validated for the determination of sodium chlorite in the various sample types generated from this experiment. The mean recoveries for the receptor fluid and swabs were >90 %, the skin >87 % and the tape strips 77 %. Although some of the mean method recoveries were less than 90 %, these particular sample types actually contained the lower proportions of dose in the experimental results. As such, the low mass balance recoveries achieved are considered to be due to the degradation of the test material to chloride ions that were not quantifiable rather than incomplete method recoveries. As such, the proportions of sodium chlorite absorbed are considered to be valid even though a traditional mass balance of the sodium chlorite dosed was not observed.The absorption rates and total absorbed data are likely to be overestimates due to the skin membranes being compromised by the corrosive nature of a high concentration of the test material. From the above data is it proposed that the maximum proportion of high dose that is absorbed through human skin is 9.66 %.There was a considerable difference in the distribution of dose between human and rat skin membranes, with approximately a 7-fold greater proportion of dose absorbed through rat skin. The greater damage observed to the rat skin membranes by the test material may have contributed to the higher absorption results obtained with the rat skin.From the results obtained with the low dose solution of sodium chlorite (44 g/L) the maximum proportion of low dose that is absorbed through human skin is 5.06 %.There was a considerable difference in the distribution of dose between human and rat skin membranes, with approximately a 5-fold greater proportion of dose absorbed through rat skin. The proportion of dose that was degraded over the 24-hour exposure period was approximately 80 % for both the rat and human skin groups.
Executive summary:

The aim of the test was the determination of the dermal absorption of the test material.

The test procedure was OECD Guideline 428 (Skin Absorption: In Vitro Method).

The result was as follows:

Maximum proportion of the high dose that is absorbed through human skin: 9.7%

Maximum proportion of the low dose that is absorbed through human skin: 5.1 %