Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10th May 2002 - 19th May 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP, performed to the draft version of the current OECD guideline. Good level of reporting throughout.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
Performed prior to finalisation of the guideline
Deviations:
no
Qualifier:
according to
Guideline:
other: ECETOC (Monograph No. 20, Percutaneous Adsorption, August 1993)
Deviations:
no
Qualifier:
according to
Guideline:
other: SCCNFP (European Commission Directorate General DG XXIV Health and Consumer Protection (2000), Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation, Octer 24, 2000)
Deviations:
no
Principles of method if other than guideline:
The absorption of dibutyltin bis(2-ethylhexylmercaptoacetate), containing 18.5 % w/w tin, was measured in vitro through human and rat epidermis. The first phase of the study was to identify the highest dose that could practically be applied, which was also regarded as likely to be non-damaging to human epiderms, using rat epidermis as a model. During the second phase of the study, the absorption of tin was determined through human and rat epidermis from both occluded and unoccluded applications of this non-damaging dose (100 µL/cm^2 eq. 21120 µg tin/cm^2).
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material : Dibutyltin bis(2-ethylhexylmercaptoacetate)
- Substance type: Colourless liquid
- Physical state: Liquid
- Analytical purity: 99.82 % w/w
- Purity test date: 12th April 2002
- Lot/batch No.: (Batch reference) R&D/S-vH 2002/38/9
- Expiration date of the lot/batch: 1st April 2003
- Storage condition of test material: Deep frozen in the dark
Radiolabelling:
no

Test animals

Species:
other: human and rat epidermis
Strain:
other: Human: not applicable. Rat: Wistar-derived strain
Sex:
not specified
Details on test animals and environmental conditions:
Not applicable.

Administration / exposure

Type of coverage:
other: both occluded and non-occluded applications
Vehicle:
other: 50 % ethanol in water
Duration of exposure:
24 hour contact period
Doses:
- Nominal doses: 21120 µg/cm^2 based upon a tin content of 18.5 % w/w in the dibutyltin bis(2-ethylhexylmercaptoacetate) mixture and corrected for purity and specific gravity.
- Dose volume: 100 µL/cm^2
- Rationale for dose selection: Though slight impairment of the rat epidermis was noted at this dose in the assessment of skin barrier damage, this dose level was presumed not to cause the same level of damage during contact with the more robust human epidermis.
No. of animals per group:
Not applicable.
Details on study design:
APPLICATION OF DOSE:
To both the rat and human epidermis, the test material was added to both undiluted at a rate of 100 µL/cm^2. One group of six treated cells and two control cells for each species were occluded for the entire 24 hour contact period, with the remaining cells were not occluded.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test material is poorly soluble in water alone.

SAMPLE COLLECTION
At 1, 2, 4, 8, 12, 16, 20 and 24 hours, 0.5 mL samples of the receptor fluid were taken from each receptor chamber for analysis. A pre-treatment sample of 0.5 mL was taken from each receptor chamber for analysis. When a sample was taken, an equal volume of fresh receptor fluid was added to each chamber to replace the sample removed. After the final sample of receptor fluid had been taken, at the end on the eposure period, the remaining fluid in the receptor chamber was discarded and the chamber rinsed with fresh receptor fluid (5 mL) which was also discarded. The donor chambers were carefully removed and washed with ethanol (10 mL) and the washings retained for analysis. The surface epidermis was also rinsed with 50 % ethanol in water (5 x 5 mL) and the rinsings were combined and diluted to 100 mL with ethanol prior to storage while awaiting analysis. The epidermis was carefully removed from the receptor chamber and placed in glass scintillation vial.

SAMPLE PREPARATION
- Storage procedure: All samples were stored by deep freeze while awaiting analysis.

ANALYSIS
- Receptor fluid samples: Samples generated were analysed for tin content by Harwell Scientifics Ltd, Harwell Didcot, Oxon, OX11 0TD UK. Samples were diluted by weight using an inductively coupled plasma mass spectrometer (ICP-MS). The limit of quantitation using the above procedure was set at 0.003 µg tin/mL.
- Donor extracts and wash samples: The entire sample was transferred to an acid washed beaker and the weight recorded. The samples were taken down to dryness before being digested in high purity nitric acid and made up to a known volume in 10% v/v nitric acid. The samples were analysed by ICP AES. Further dilutions were made with 5 % nitric acid as necessary.
- Epidermis samples: The samples were transferred to acid cleaned beakers and the original containers rinsed with 5% v/v nitric acid, which was added to the samples. The sampels were digested in high purity nitric acid and made up to a known volume in 25 % v/v nitric acid. The samples were analysed by ICP-AES. Further dilutions were made with 5 % v/v nitric acid as necessary.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Extraneous tissue was removed from human whole skin samples obtained post mortem
- Preparative technique: The skin samples were immersed in water at 60 °C for 40-45 seconds and the epidermis teased way from the dermis.
- Membrane integrity check: The integrity of the membranes was determined by measurement of their electrical resistance across the skin membrane. Membranes with a measured resistance of < 10kΩ (human) were regarded as having a lower integrity than normal and not used for exposure to the test material.
- Storage conditions: Each epidermal membrane was given an identifying number and stored frozen on aluminium foil until required for use.

SKIN PREPARATION
- Source of skin: Skin was used from male rats of the Wistar-derived stain (Charles River UK Ltd) age 28 ± 2 days. Fur from the dorsal and flank region was carefully shaved using animal clippers, ensuring that the skin was not damaged. The clipped area was excised and any subcataneous fat removed.
- Preparative technique: The skins were soaked for approximately 20 hours in 1.5 M sodium bromide then rinsed in distilled water. The epidermis was carefully peeled from the dermis.
- Membrane integrity check: The integrity of the membranes was determined by measurement of their electrical resistance across the skin membrane. Membranes with a measured resistance of < 2.5 kΩ (rat) were regarded as having a lower integrity than normal and not used for exposure to the test material.
- Storage conditions: Each epidermal membrane was given an identifying number and stored frozen on aluminium foil until required for use.

PRINCIPLES OF ASSAY
- Diffusion cell: Glass diffusion cell with an exposed membrane area of 2.54 cm^2. Discs of approximately 3.3 cm diameter of prepared skin membrane from at least three subjects were mounted, dermal side down, in diffusion cells held together with individually numbered clamps and placed in a water bath maintained at 32 ± 1 °C.
- Receptor fluid: water
- Solubility of test substance in receptor fluid: 1.5 x 10^-8 mg/L
- Test temperature: 32 ± 1°C
- Occlusion: One group of six treated cells and two control cells for each species were occluded for the entire 24 hour contact period, with the remaining cells were not occluded.

Results and discussion

Absorption in different matrices:
Absorption through human epidermis
From the occluded application to human epidermis, the rate of tin absorption gradually increased to reach a maximum of 0.005 µg/cm^2/h during 16-24 h period after application. The average rate over the whole 24 hour exposure was 0.002 µg/cm^2/h. From the unoccluded applivation, the mean 16-24 h absorption rate for tin was 0.013 µg/cm^2/h. In terms of mean percent of applied tin, 0.0004 % was absorbed from the occluded dose, while 0.0010 % was absorbed from the unoccluded dose after 24 h exposure.

Absorption through rat epidermis
Absorption of tin through rat epidermis was much faster. As with human epidermis, the rate of tin absorption gradually increased to reach a maximum of 3.88 µg/cm^2/h (occluded and 2.73 µg/cm^2/h (unoccluded) during the 16-24 h period after application. The average rates over the whole 24 hour exposure period were 2.17 µg/cm2/h and 1.58 µg/cm^2/h. In terms of mean percent of applied tin, 0.261% was absorbed from the occluded dose, while 0.189% was absorbed from the unoccluded dose after 24 hours of exposure.

Distribution of dose and mass balance.
The overall mean recoveries of tin from the test system after 24 hours exposure for the occluded applications were 54.6% and 72.0% of applied tin (human and rat respectively), while 78.6% and 82.1 % was recovered from the unoccluded applications. The lower recovery for the occluded applications was attributed to the reduced amount washed from the donor chamber and epidermis, therefore, as these were not systemic compartments, the absorbed amounts of epidermal residues were probably not affected.
The vast majority of the recovered tin was washed off the epidermis for both human and rat during decontamination at 24 hours. For the occluded applications this gave 49.0% and 66.3% of the dose (human and rat respectively) and 75.4% and 72.2 % of that applied to unoccluded experiments. The mean amounts absorbed by 24 hours were determined to be 0.0004 % and 0.0010 % from human experiments (occluded and unoccluded, respectively) and 0.261% and 0.189% from rat experiments. The mean amounts extracted from the epidermis were 0.615% and 1.03% (human occluded and unoccluded respectively) and 4.78 % and 8.11% (rat occluded and unoccluded respectively).
Total recovery:
- Total recovery: Please refer to tables 3 and 4 under section "Any other information of results incl. tables" for details on total recovery for both human and rat epidermis in both occluded and unoccluded applications.
- Recovery of applied dose acceptable:The OECD guideline 428 states “In all studies adequate recovery should be achieved (the aim should be a mean of 100 ± 10% of the radioactivity and any deviation should be justified). The amount of test substance in the receptor fluid, skin preparation, skin surface washings and apparatus rinse should be analysed, using a suitable technique.”. The recovered doses were found not to fall in this criteria.
- Possible confounding factors: The results indicate that at a dose level of 100µl/cm^2, approximately up to 18-45% of the tin dose is unaccounted for, possibly due to adherence of the test material to the glass apparatus used in the study, especially during the decontamination process.
Percutaneous absorptionopen allclose all
Dose:
100 µL/cm2
Parameter:
percentage
Absorption:
0.005 %
Remarks on result:
other: 16-24 hours
Remarks:
(Standard deviation ± 0.002) in human epidermis, occluded
Dose:
100 µL/cm2
Parameter:
percentage
Absorption:
0.002 %
Remarks on result:
other: 0-24 hours
Remarks:
(Standard deviation ± 0.001) in human epidermis, occluded
Dose:
100 µL/cm2
Parameter:
percentage
Absorption:
3.88 %
Remarks on result:
other: 16-24 hours
Remarks:
(Standard deviation ± 0.484) in rat epidermis, occluded
Dose:
100 µL/cm2
Parameter:
percentage
Absorption:
2.17 %
Remarks on result:
other: 0-24 hours
Remarks:
(Standard deviation ± 0.299) in rat epidermis, occluded
Dose:
100 µL/cm2
Parameter:
percentage
Absorption:
0.013 %
Remarks on result:
other: 16-24 hours
Remarks:
(Standard deviation ± 0.005) in human epidermis, unoccluded
Dose:
100 µL/cm2
Parameter:
percentage
Absorption:
0.007 %
Remarks on result:
other: 0-24 hours
Remarks:
(Standard deviation ± 0.003) in human epidermis, unoccluded
Dose:
100 µL/cm2
Parameter:
percentage
Absorption:
2.73 %
Remarks on result:
other: 16-24 hours
Remarks:
(Standard deviation ± 0.424) in rat epidermis, unoccluded
Dose:
100 µL/cm2
Parameter:
percentage
Absorption:
1.58 %
Remarks on result:
other: 0-24 hours
Remarks:
(Standard deviation ± 0.265) in rat epidermis, unoccluded
Conversion factor human vs. animal skin:
Not applicable. Only human skin results used for results.

Any other information on results incl. tables

Table 1: Summary of tin absorption through human epidermis

1. Details of Application of Test Materials

2. Mean Absorption Rates

3. Mean Amount and Percent of Dose Absorbed

Time Period (h)

Absorption Rate (µg/cm2/h ± SEM)

Time (h)

Amount (µg/cm2)

Percent Absorbed

Dibutyltin bis(2-ethylhexyl mercaptoacetate)

100 µL/cm2(21120 µg tin/cm2)

Occluded

Duration of exposure 24 hours n = 5

 

 

 

16-24

 

0-24

 

 

 

0.005 ± 0.002

 

0.002 ± 0.001

 

1

2

4

8

12

16

20

24

 

0.029

0.031

0.032

0.036

0.038

0.047

0.062

0.088

 

0.0001

0.0001

0.0001

0.0002

0.0002

0.0002

0.0003

0.0004

Dibutyltin bis(2-ethylhexyl mercaptoacetate)

100 µL/cm2(21120 µg tin/cm2)

Unoccluded

Duration of exposure 24 hours n = 5

 

 

 

16-24

 

0-24

 

 

 

0.013 ± 0.005

 

0.007 ± 0.003

 

1

2

4

8

12

16

20

24

 

0.027

0.038

0.0046

0.057

0.074

0.105

0.154

0.205

 

0.0001

0.0002

0.0002

0.0003

0.0004

0.0005

0.0007

0.0010

 

Table 2: Summary of tin absorption through rat epidermis

1. Details of Application of Test Materials

2. Mean Absorption Rates

3. Mean Amount and Percent of Dose Absorbed

Time Period (h)

Absorption Rate (µg/cm2/h ± SEM)

Time (h)

Amount (µg/cm2)

Percent Absorbed

Dibutyltin bis(2-ethylhexyl mercaptoacetate)

100 µL/cm2(21120 µg tin/cm2)

Occluded

Duration of exposure 24 hours n = 6

 

 

 

16-24

 

0-24

 

 

 

3.88 ± 0.484

 

2.17 ± 0.299

 

1

2

4

8

12

16

20

24

 

0.196

0.390

1.39

5.76

13.3

24.0

38.1

55.1

 

0.001

0.002

0.007

0.027

0.063

0.114

0.181

0.261

Dibutyltin bis(2-ethylhexyl mercaptoacetate)

100 µL/cm2(21120 µg tin/cm2)

Unoccluded

Duration of exposure 24 hours n = 6

 

 

 

16-24

 

0-24

 

 

 

2.73 ± 0.424

 

1.58 ± 0.265

 

1

2

4

8

12

16

20

24

 

0.109

0.296

1.18

4.63

10.1

18.1

28.1

39.9

 

0.001

0.001

0.006

0.022

0.048

0.086

0.133

0.189

 

Table 3: Summary of Tin Distribution in the Test System at 24 Hours – Human Epidermis

A.      Occluded application

Test Compartment

Percent of Dose Recovered

(%)

Mean % Recovered

SD

Cell 50

Cell 51

Cell 56

Cell 93

Cell 96

Donor chamber

Skin wash

Epidermis

Receptor fluid

10.3

43.1

0.969

< 0.001

3.53

55.9

0.373

< 0.001

6.77

50.1

0.373

0.001

0.751

31.0

0.802

< 0.001

3.53

64.8

0.559

< 0.001

4.99

49.0

0.615

<0.001

3.67

12.8

0.265

<0.001

Total

54.4

59.8

57.2

32.6

68.9

54.6

13.5

 

B.      Unoccluded application

Test Compartment

Percent of Dose Recovered

(%)

Mean % Recovered

 

Cell 57

Cell 62

Cell 64

Cell 67

Cell 99

Donor chamber

Skin wash

Epidermis

Receptor fluid

2.07

53.4

0.261

< 0.001

0.224

107

0.298

0.002

0.090

35.3

0.839

0.002

2.50

98.2

2.05

<0.001

6.19

83.2

1.70

0.001

2.21

75.4

1.03

0.001

2.47

30.2

0.814

0.001

Total

55.7

107

36.3

103

91.0

78.6

31.1

 

Table 4: Summary of Tin Distribution in the Test System at 24 Hours – Rat Epidermis

A.      Occluded application

Test Compartment

Percent of Dose Recovered

(%)

Mean % Recovered

SD

Cell 2A

Cell 3A

Cell 6A

Cell 7A

Cell 13A

Cell 18A

Donor chamber

Skin wash

Epidermis

Receptor fluid

0.141

37.9

3.91

0.169

0.250

48.6

3.17

0.176

0.431

46.5

4.47

0.408

0.029

70.7

2.98

0.264

2.93

98.2

8.57

0.324

0.221

95.7

5.59

0.223

0.667

66.3

4.78

0.261

1.12

26.1

2.08

0.092

Total

42.1

52.5

51.8

74.0

110

102

72.0

28.4

 

B.      Unoccluded application

Test Compartment

Percent of Dose Recovered

(%)

Mean % Recovered

SD

Cell 4A

Cell 5A

Cell 9A

Cell 12A

Cell 14A

Cell 19A

Donor chamber

Skin wash

Epidermis

Receptor fluid

0.076

68.9

2.61

0.102

5.15

50.1

7.46

0.152

0.551

96.5

13.0

0.099

0.810

88.4

5.59

0.224

0.22

88.4

8.02

0.283

2.80

41.2

11.9

0.275

1.60

72.2

8.11

0.189

2.00

22.7

3.90

0.083

Total

71.7

62.8

110

95.0

96.9

56.2

82.1

21.5

 

 

 

Applicant's summary and conclusion

Conclusions:
1. Following 24 hours dermal contact, the amount of dibutyltin bis(2-ethylhexlymercaptoacetate) required to alter the barrier function of rat epidermis was approximately 100 µL/cm^2 (= 21120 µg tin/cm^2).
2. The results indicate that at a dose level of 100 µL/cm^2, approximately up to 18-45 % of the tin dose was unaccounted for, possibly due to adherence of the test material to the glass apparatus used during the study, especially during the decontamination process.
3. At 100 µL/cm^2, the absorption of tin through human epiderims was very slow, when compared with the absorption rates of other penetrants measured using the same in vitro technique. (Dugard et al 1984; Dugard and Scott, 1984).
4. The proportions of dibutyltin bis(2-ethylhexylmercaptoacetate) absorbed through human epidermis were 0.0004% and 0.0010% (occluded and unoccluded respectively) of dose after 24 hours exposure, compared to 0.261% and 0.189% through rat epidermis.
5. The absorption of tin from dibutyltin bis(2-ethylhexylmercaptoacetate) through rat epidermis significantly overestimated absorption through human epidermis.
6. The vast majority of the applied tin dose was washed from the surface of the epidermis during the decontamination process, with only relatively small proportions of the dose (human up to 1%; rat up to 10%) remaining associated with the epidermis and therefore not regarded as systemically available.
Executive summary:

In the 2003 Dermal Absorption study by Ward, 00 µL/cm2 (= 21120 µg tin/cm2) was found to alter the barrier function of the rat epidermis. At 100 µL/cm2, approximately up to 18-45 % of the tin dose was unaccounted for, possibly due to adherence of the test material to the glass apparatus. The absorption of tin through human epiderims was very slow, when compared with the absorption rates of other penetrants. The proportions of dibutyltin bis(2-ethylhexylmercaptoacetate) absorbed through human epidermis were 0.0004% and 0.0010% (occluded and unoccluded respectively) after 24 hours exposure, compared to 0.261% and 0.189% through rat epidermis. The majority of the applied tin dose was washed from the surface of the epidermis during decontamination, only a relatively small proportion of the dose (human up to 1%; rat up to 10%) remained associated with the epidermis and therefore was not regarded as systemically available.