Dimethyltin dichloride

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8th June - 13th August 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

Draft dated 1996

Deviations:

not specified

Principles of method if other than guideline:

Purity: 89 % dimethyltin dichloride, 11 % monomethyltin dichloride

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyltin dichloride

EC Number:

212-039-2

EC Name:

Dimethyltin dichloride

Cas Number:

753-73-1

Molecular formula:

C2H6Cl2Sn

IUPAC Name:

dimethyltin dichloride

Test material form:

solid: crystalline

Details on test material:

Methyltin chloride mixture containing 11% monomethyltin trichloride and 89% dimethyltin dichloride (DMTC) - tin content: 53.5% w/w

Radiolabelling:

no

Test animals

Species:

other: human skin and rat skin

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Human skin:
Extraneous tissue was removed from human whole skin samples. The skin samples were immersed in water at 60°C for 40-45 seconds and the epidermis teased off the dermis. Each epidermal membrane was given an identifying number and stored frozen on aluminium foil until required for use.

Rat skin:
Skin was from male rats of the Wistar-derived strain (supplied by Charles River UK Ltd, Margate, Kent, UK.), aged 28 days ± 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 subcutaneous fat removed. The skins were soaked for approximately 20 hours in 1.5M sodium bromide then rinsed in distilled water. The epidermis was carefully peeled from the dermis. Each epidermal membrane was given an identifying number and stored frozen on aluminium foil until required for use.

Administration / exposure

Type of coverage:

other: dermal absorption was determined under both occluded and unoccluded conditions

Vehicle:

ethanol

Duration of exposure:

24 hrs

Doses:

Based on results of three experiments to determine a non-damaging dosage, the methyltin chloride mixture was applied to both human and rat epidermal membranes at a rate of 100 µg/cm² as a 10 µl/cm² dose of a 10000 µg/ml solution in ethanol. This application was equivalent to a nominal dose rate for tin of 53.5µg/cm², based upon a tin content of 53.5% w/w in the methyltin chloride mixture, or 89 µg/cm² based on DMTC content.

No. of animals per group:

6 cells per species

Details on in vitro test system (if applicable):

MEASUREMENT OF MEMBRANE INTEGRITY
Samples of epidermis were mounted in glass diffusion cells with an exposed area of 2.54 cm². The cells were placed in a water bath maintained at 32 ± 1 °C.

The integrity of the membranes was determined by measurement of their electrical resistance across the skin membrane. Membranes with a measured resistance <10 kΩ (human) or <2.5 kΩ (rat) were regarded as having a lower integrity than normal and not used for exposure to the test material.


ASSESSMENT OF SKIN BARRIER DAMAGE
This assessment was only performed for human epidermis.

The receptor chambers of 3 cells containing intact membranes were filled with a recorded volume of receptor fluid (water) and the cells placed in a waterbath maintained at 32 ± 1°C. The methyltin chloride mixture was applied undiluted to these cells at a rate of 0.1g/cm² and the cells occluded for the entire exposure period (24h). Concurrently with these experiments, 2 untreated control cells were assessed under identical conditions as the treated cells.

After the 24h contact period, the donor chamber were emptied and the surface of the membrane decontaminated by flushing with water (5 x 5 ml volumes), followed by physiological saline (5ml). The receptor chambers were emptied and rinsed with physiological saline (5ml).

The procedure used for the assessment of skin integrity was repeated and a damage ratio calculated by dividing the pre-treatment resistance measurement by the post treatment resistance measurement. If the membrane has been damaged by the test material, the electrical resistance falls, compared to the initial measurement. When the change to the barrier properties of the membrane is more than 3-fold, this is regarded as significant impairment. Damage ratios <3 are regarded as insignificant. In this experiment with the methyltin chloride mixture, the mean damage ratio for the treated cells was 24 (versus a control value of 1.4), which clearly indicated significant damage to the barrier function of the epidermis.

The experiment was repeated using the minimum amount that could practically weighed onto the cells (0.002 g/cm²). This dose level gave a mean damage ratio of 6.8 (versus a control value of 1), which again was regarded as have significantly damaged the epidermal barrier.

In a further attempt to find a non-damaging dose to human epidermis, a third series of experiments were designed. Three solutions of the methyltin chloride mixture 10000, 50000 and 100000 µg/ml) were prepared in ethanol and each applied to three replicate epidermal membranes at a rate of 10 µl/cm² (a, 100, 500 or 1000 µg methyltin chloride mixture /cm²). For control purposes, ethanol only was applied to 2 cells at a rate of 10 µl/cm². The applications were not occluded until the ethanol vehicle had evaporated naturally and then remained covered until the end of the 24h contact period, when the cells were decontaminated and post-treatment resistance measurements taken, as previously described. The determined mean damage ratios were 1.33, 5.1 and 9.7 for the 100, 500 or 1000 µg methyltin chloride mixture /cm² applications respectively (versus 0.93 for the control applications), indicating that a non-damaging dose of methyltin chloride mixture would lie between 100 and 500 µg/cm².


MEASUREMENT OF TEST SUBSTANCE ADSORPTION
The receptor chambers of cells containing intact human and rat epidermal membranes (6 cells per species) were filled with a recorded volume of receptor fluid (water) and placed in a water bath maintained at 32 ± 1°C, A pre-treatment sample (0.5 ml) was taken from each receptor chamber for analysis by ICP-MS. An equal volume of fresh receptor fluid was added to each receptor chamber to replace the volume removed.

From information gained from the above experiments to define a non-damaging dose of the test material, the methyltin chloride mixture was applied to both human and rat epidermal membranes at a rate of 100 µg/cm² as a 10 µl/cm² dose of a 10000 µg/ml solution in ethanol. This application was equivalent to a nominal dose rate for tin of 53.5µg/cm², based upon a tin content of 53.5% w/w in the methyltin chloride mixture. For the cells designated to be occluded, as soon as the ethanol vehicle had evaporated naturally, the cells were occluded for the remainder of the 24h contact period.

At recorded intervals, samples (0.5ml) of the receptor fluid were taken for analysis by ICP-MS. The volume of fluid in the receptor chamber was maintained by the addition of (0.5ml) of fresh receptor fluid to the chamber immediately after the removal of each sample.


MASS BALANCE DETERMINATION
After the final sample of receptor fluid had been taken at the end of the exposure 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 water (10 ml) and the washings retained for analysis.

The surface of the epidermis was rinsed with water (5 x 5 ml) and the rinsings were combined prior to analysis.
The epidermis was carefully removed from the receptor chamber and placed in a glass scintillation vial.
All samples were stored refrigerated while awaiting analysis.

Results and discussion

Absorption in different matrices:

Human skin - unoccluded application (Mean of 6 replicates - % of dose recovered)
Donor chamber 2.43
Skin wash 8.02
Epidermis 20.15
Absorbed (receptor fluid) 0.25
TOTAL RECOVERY 30.85

Human skin - occluded application (Mean of 5 replicates - % of dose recovered)
Donor chamber 6.71
Skin wash 3.45
Epidermis 43.26
Absorbed (receptor fluid) 1.39
TOTAL RECOVERY 54.80

Rat skin - unoccluded application (Mean of 5 replicates - % of dose recovered)
Donor chamber 2.19
Skin wash 11.89
Epidermis 24.18
Absorbed (receptor fluid) 9.95
TOTAL RECOVERY 48.21

Rat skin - unoccluded application (Mean of 6 replicates - % of dose recovered)
Donor chamber 2.41
Skin wash 4.01
Epidermis 51.08
Absorbed (receptor fluid) 10.04
TOTAL RECOVERY 67.54

Total recovery:

Due to volatility of the test substance (25.1 Pa at 25 ºC) and the use of ethanol, the overall recovery of tin from the test system was low.

Percutaneous absorption

Percutaneous absorption 1

Parameter:

percentage

Absorption:

20 %

Remarks on result:

other: 24 hours

Remarks:

unoccluded human - amount remaining in epidermis considered potentially absorbable

Percutaneous absorption 2

Parameter:

percentage

Absorption:

34 %

Remarks on result:

other: 24 hours

Remarks:

unoccluded rat - - amount remaining in epidermis considered potentially absorbable

Any other information on results incl. tables

Preliminary Study:

During preliminary assessments, the direct application of a non-damaging occluded dose of the methyltin chloride mixture to the epidermis could not be achieved, however a dose of 100 µg/cm² (applied as a 10000 µg/ml solution in ethanol at a rate of 10 µl/cm² and occluded only after the ethanol had evaporated), was determined not to damage the epidermis (damage ratio = 1.33). Ethanol itself was determined not to affect the barrier function of the epidermis.

This same regime was used in experiments to determine the absorption through human and rat epidermis and the distribution of tin within the test system. Similar experiments were also carried out where the applications were left unoccluded throughout the 24h exposure period.

Absorption through human epidermis:

From the occluded applications to human epidermis, an initial rate of tin absorption of 0.015 µg/cm²/h measured during the first 6h of exposure. Between 6-24h a maximum absorption rate of 0.037 µg/cm²/h was achieved. From the unoccluded application a similar pattern of absorption was seen, except that the tin absorption rates were 5-6 times slower during the 0-6h and 6-24h periods (0.003 µg/cm²/h and 0.006 µg/cm²/h). In terms of percent of applied tin, 1.4% was absorbed from the occluded dose, while only 0.25% was absorbed from the unoccluded dose after 24h exposure.

Absorption through rat epidermis:

Absorption of tin through rat epidermis was much faster than through human epidermis. From the occluded application, tin absorption maintained an essentially constant rate (0.233 µg/cm²/h) throughout the entire 24h exposure. From the unoccluded application, the absorption process was essentially complete within the first 3h of exposure, with a maximum mean absorption rate of 1.07 µg/cm²/h during this period. From both applications, 10% of the applied tin was determined to have been absorbed by 24h after dosing.

Distribution of dose and mass balance:

The overall recovery of tin from the test system after 24h exposure was low with 55% and 67% of applied tin being recovered from the occluded applications to human and rat epidermis respectively, while only 31% and 48% was recovered from the unoccluded applications. Since (a) more tin was recovered from the occluded experiments than from the unoccluded exposures and (b) the methyltin chloride mixture is volatile, the assumption is that the losses of tin from the occluded experiments (essentially a sealed system) occurred during the period that the ethanol vehicle was evaporating prior to occlusion. If this is the case, then recovery would be expected to be lower from the unoccluded experiments compared to those that were occluded. This hypothesis is also supported by the fact that tin absorption is faster through rat epidermis than through human epidermis, thus allowing less time for tin to volatilise from the surface of rat epidermis during this early stage of the absorption process, giving rise to the higher recovery values from the rat experiments.

A high proportion of the recovered tin was present in the epidermis for both human and rat. For the occluded applications this amounted to about half of the dose and about fifth of that applied to unoccluded experiments. The amounts determined to have been absorbed into the receptor fluid were low (0.25% and 1.4% from human experiments and 10% from both rat experiments). Approximately 10% for human and 6.4% - 14% for rat was washed off the surface of the epidermis and the donor chamber.

Applicant's summary and conclusion

Conclusions:

1. Following 24h dermal contact with the methyltin chloride mixture, amounts >100 µg/cm² can alter the barrier function of human epidermis.
2. The results indicate that at a dose level of 100 µg/cm² of the methyltin chloride mixture (89% as DMTC), 70% of the tin dose probably evaporates from the surface of human skin under unoccluded conditions.
3. At this 100 µg/cm² dose level, the absorption of tin from this methyltin chloride mixture through human epidermis is very slow, and a significant amount remains in the epidermis.
4. The absorption of tin from this methyltin chloride mixture through rat epidermis significantly overestimated absorption through human epidermis.
5. Absorption of tin through human epidermis after 24h exposure under unoccluded conditions is estimated to be 20%, considering the remaining in epidermis as potentially absorbable. Through rat epidermis, 34% of the applied tin was absorbed by 24h under similar conditions.

The non-irritating concentration of the methyltin chloride mixture can be quoted as 100 µg/cm², 89 µg/cm² based on DMTC content.

Executive summary:

Study design:

The absorption of a methyltin chloride mixture containing 53.5% w/w tin as monomethyltin trichloride (11%w/w) and dimethyltin dichloride (89% w/w) has been measured in vitro through human and rat epidermis. The tin species absorbed were not individually identified; absorption determinations were based on measurements of total tin in the samples analysed. The first phase of the study was to identify the highest dose that could practically be applied, which was also determined to be non-damaging to human epidermis. During the second phase of the study the absorption of tin was determined from both occluded and unoccluded applications of this non-damaging dose to human and rat epidermis (10 µl/cm² of a 10000 µg methyltin chloride mixture /ml in ethanol, which was equivalent to a dose of 100 µg methyltin chloride mixture /cm²).

Results:

Determination of a non-damaging dose to human epidermis

A non-damaging occluded dose of the methyltin chloride mixture could not be applied directly to the epidermis, however a dose of 100 µg/cm² (applied as a 10000 mg/ml solution in ethanol at a rate of 10 µl/cm² and occluded only after the ethanol had evaporated), was determined not to damage the epidermis. The non-irritating concentration of the methyltin chloride mixture can therefore be quoted as 100 µg/cm². The dimethyltin dichloride content of this mixture is 89%.

This same regime was used in experiments to determine the absorption of tin through human and rat epidermis and its distribution within the test system, from both occluded and unoccluded applications of the methyltin chloride mixture.

Absorption through human epidermis

From the occluded applications to human epidermis, an initial rate of tin absorption of 0.015 µg/cm²/h was measured during the first 6h of exposure. Between 6-24h a maximum absorption rate of 0.037 µg/cm²/h was achieved. From the unoccluded application, tin

absorption rates were 5-6 times slower during the 0-6h and 6-24h periods (0.003 µg/cm²/h and 0.006 µg/cm²/h). In terms of percent of applied tin, 1.4% was absorbed into the receptor fluid from the occluded dose, while only 0.25% was absorbed from the unoccluded dose after 24h exposure. Percentages remaining in the epidermis accounted to 43.26 and 20.15%, respectively, under occluded and unoclluded conditions.

Absorption through rat epidermis

Absorption of tin through rat epidermis was much faster than through human epidermis. From the occluded application, tin absorption maintained an essentially constant rate (0.233 µg/cm²/h) throughout the entire 24h exposure. From the unoccluded application, the absorption process was essentially complete within the first 3h of exposure, with a maximum mean absorption rate of 1.07 µg/cm²/h during this period. From both applications, 10% of the applied tin was determined to have been absorbed into the receptor fluid by 24h after dosing. Percentages remaining in the epidermis accounted to 51.08 and 24.18%, respectively, under occluded and unoccluded conditions.

Distribution of dose and mass balance

The overall recovery of tin from the test system after 24h exposure was low with 55% and 67% of applied tin being recovered from the occluded applications to human and rat epidermis respectively, while only 31% and 48% was recovered from the unoccluded applications. The data indicate that the unaccounted portion of the dose probably volatilised from the surface of the skin and was not captured by this test system.

A high proportion of the recovered tin was present in the epidermis for both human and rat. For the occluded applications this amounted to about half of the dose and about fifth of that applied to unoccluded experiments. The amounts determined to have been absorbed into the receptor fluid were low (0.25% and 1.4% from human experiments and 10% from both rat experiments). Approximately 10% for human and 6.4% - 14% for rat was washed off the surface of the epidermis and the donor chamber.

Conclusion:

1. Following 24h dermal contact with the methyltin chloride mixture, amounts >100 µg/cm² can alter the barrier function of human epidermis.

2. The results indicate that at a dose level of 100 µg/cm² of the methyltin chloride mixture (89% as DMTC), 70% of the tin dose probably evaporates from the surface of human skin under unoccluded conditions.

3. At this 100 µg/cm² dose level, the absorption of tin from this methyltin chloride mixture through human epidermis is very slow, and a significant amount remains in the epidermis.

4. The absorption of tin from this methyltin chloride mixture through rat epidermis significantly overestimated absorption through human epidermis.

5. Absorption of tin through human epidermis after 24h exposure under unoccluded conditions is estimated to be 20%, considering the remaining in epidermis as potentially absorbable. Through rat epidermis, 34% of the applied tin was absorbed by 24h under similar conditions.