2-methyl-2H-isothiazol-3-one

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January - 20 April 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline #28

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

other: colorless liquid

Details on test material:

Aqueous solution containing 2-methyl-4-isothiazolin-3-one (MI) was used. The concentration was 300 - 313 ug/ml as [14C]-MI in two formulations as supplied to CTL. Aliquots were diluted at CTL to provide diluted samples for additional studies.

CTEA shampoo formulation consisted of a colorless viscous liquid with an MI content of 100 ug/ml (nominal) as [14C]-MI.

Body lotion formulation consisted of a white viscous liquid with an MI content of 100 ug/ml (nominal) as [14C]-MI.

Faceial cream formulation consisted of a white cream with an MI content of 100 ug/ml (nominal) as [14C]-MI.

All of the above test materials contain [14C] with a radioactive purity of 96.9%; specific activity of 48.5 mCi/g.

Radiolabelling:

yes

Test animals

Species:

human

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

Extraneous tissue was removed from human whole skin samples obtained from surgery or post mortem. The skin samples were immersed in water at 60C for 40-45 seconds and the epidermis teased away from the dermis. Each epidermal membrane was given an identifying number and stored frozen on aluminum foil until required for use.

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: See test material for the various formulations examined.

Duration of exposure:

24 hour

Doses:

Aqueous solutions of MI (313 ug, 104.3 ug and 52.2 ug MI/ml) and three undiluted formulations were examined

No. of animals per group:

six skin membranes obtained from at least 3 subjects were used for each dose group.

Control animals:

no

Details on in vitro test system (if applicable):

Assembly of Diffusion Cells
The type of glass diffusion cell used in this study has an exposed membrane area of 2.54 cm(2). Discs of approximately 3.3 cm diameter of prepared skin membrane were mounted, dermal side down, in diffusion cells held together with individually numbered clamps and placed in a water bath maintained at 32 +/- 1C.

Measurement of Membrane Integrity
Membrane integrity was determined by measurement of the electrical resistance across the skin membrane. membranes with a measured resistance of <10kOhmes (Davies et al., 2004). were regarded as having a lower integrity than normal and not used for exposure to the test materials.

Measurement of Test Substance Absorption
Cells were selected such that each application was represented by six intact membranes from at least three different subjects.

The receptor chambers of the cells, containing small magnetic stirrer bars, were filled with a recorded volume of water as the receptor fluid. MI is soluble in water and this choice of receptor fluid ensured that the test substance could freely partition into the receptor fluid from the skin membrane and never reached a concentration that limited its diffusion.

A pre-treatment sample (0.5 ml) was taken from each receptor chamber for analysis by LSC. An equal volume of fresh receptor fluid was added to each receptor chamber to replace the volume removed. The receptor chambers were stirred continuously throughout the exposure period.

Prior to dosing the aqueous solutions, the cells were placed in a water bath maintained at normal skin temperature of 32 +/-1 C to allow the receptor fluid temperature to equilibriate. For the formulations, which were applied by weight, this was not possible and the cells were not placed in the waterbath until after dosing.

The three aqueous solutions of MI (313 ug, 104.3 ug and 52.2 ug MI/ml) and the undiluted formulations were applied evenly to the skin membranes at rates of 20 ul/cm(2) and 20 mg/cm(2), respectively, and occluded for the duration of the exposure period (24 hr). These applications were designed to simulate as close as possible the potential human dermal exposure to the formulations during normal use. These occluded conditions however, may over-predict true MI absorption. Initial experiments, dosed as above but without occlusion, showed that MI was too volatile to obtain meaningful data.

At recorded intervals (1, 2, 3, 6, 8, 12 and 24 hr), 0.5 ml samples of the receptor fluid were taken for analysis. The volume of fluid in the receptor chamber was maintained by the addition of 0.5 ml fresh receptor fluid to the chamber immediately after the removal of each sample.

Measurement of Mass Balance
After the final receptor fluid sample had been taken, the remaining fluid in the receptor chamber was discarded and the chamber rinsed with fresh receptor fluid which was also discarded. The donor chamber was carefully removed, washed with water and the sample analyzed for MI by LSC.

The epidermal surface of the skin was decontaminated by gently swabbing the application site with natural sponges pre-wetted with 3% Teepol, and with further sponges pre-wetted with water. Decontamination was shown to be complete following assessment of residual radioactivity levels on the skin surface with a Geiger counter. The sponges were digested in Soluene 350 and made up to a recorded volume. A sample was taken for analysis.

To assess penetration through the stratum corneum, successive layers of the stratum corneum were removed by the repeated application of adhesive tape (eg Scotch 3M Magic Tape, 1.25 cm wide), to a maximum of 5 strips. In some cases, it was not possible to take the full 5 tape strips, as the epidermis began to tear during the process described below. in such cases, tape stripping was discontinued at first tearing and the last tape strip taken was digested with the remaining epidermis, so as not to underestimate residues in the remaining epidermis compartment.

The surface of the skin was allowed to dry naturally. A strip of adhesive tape was pressed onto the skin surface and then carefully peeled off to remove the stratum corneum. the adhesive strips were soaked in Soluene 350 to extract any test material. The strips were extracted individually and sequentially numbered prior to analysis by LSC.

The remaining epidermis was carefully removed from the receptor chamber and digested in Soluene 350 and the whole digest analysed.

Reference:
Davies, D.J., Ward, R.J. and Heylings, J.R. (2004). Multi-species Assessment of Electrical Resistance as a Skin Integrity Marker for In vitro Percutaneous Absorption Studies. Toxicology In Vitro 18:351-358.

Results and discussion

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

Aqueous solutions
The fastest rate of MI absorption from all aqueous applications was between 8-12 hr after dosing. Little significant absorption occurred before 6 hr. After 12 hr absorption rapidly slowed, most probably due to depletion of the applied MI reservoir, giving 3 phase of sigmoidal profiles.

313 ug/ml solution
During the first 8 hr of exposure the mean absorption rate for MI was 0.017 ug/cm(2)/hr, with 2.53% (0.158 ug/cm(2)) of the dose being absorbed within that time. Over the following 4 hours, the mean rate had increased to 0.577 ug/cm(2)/hr, with 39.4% (2.47 ug/cm(2)) being absorbed by 12 hr. By the end of the exposure period at 24 hr, 54.7% (3.42 ug/cm(2)) of the dose had been absorbed.

The residual MI in the epidermis after tape stripping at the end of the exposure period was 10.8%, thus the total potentially systemically available proportion of the dose (remaining epidermis + absorbed) was 65.5%.

By the end of the exposure period, only 7.03% of the dose was washed from the surface of the epidermis and 1.55% was recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 86.9%.

104.3 ug/ml solution
Between 0-8 hr after dosing, the mean MI absorption rate was 0.007 ug/cm(2)/h, with 3.32% (0.069 ug/cm(2)) of the dose being absorbed by 8 hr. During the 8-12 hr period, the rate was 0.132 ug/cm(2)/hr, with 28.6% (0.598 ug/cm(2)) having been absorbed by 12 hr after application. By the end of the exposure period at 24hr, 38.0% (0.793 ug/cm(2)) of the dose had been absorbed.

The MI remaining in the epidermis after tape stripping was 22.1%, giving a total potentially systemically available proportion of the dose was 60.1% after 24 hr exposure.

By the end of the exposure period, only 15.0% of the dose was washed from the surface of the epidermis and 4.27% was recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 89.9%.

52.2 ug/ml solution
During the 0-8 hr period after dosing, the mean MI absorption rate was 0.005 ug/cm(2)/hr, with 4.68% (0.049 ug/cm(2)) of the dose being absorbed by 8 hr. Over the 8-12 hr period, the rate was 0.045 ug/cm(2)/hr, with 21.8% (0.228 ug/cm(2)) having been absorbed by 12 hr after application. By the end of the exposure period at 24 hr, 29.8% (0.312 ug/cm(2)) of the dose had been absorbed.

The MI remaining in the epidermis after tape stripping after 24 hr exposure was 35.5%, thus the total potentially systemically available proportion of the dose was 65.3%.

By the end of the exposure period, 14.1% of the dose was washed from the surface of the epidermis, with 4.48% being recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 94.7%.

Formulations
The absorption profiles from the formulations was noticeably different from the aqueous solution, in that the profiles over the 24 hr exposure period were only 2 phase, with a period of slowly increasing absorption of between 6-12 hr, depending on the formulation, followed by a steady state period of absorption up to the end of the exposure period.

100 ug/ml shampoo
During the first 6 hr of exposure, the mean rate of MI absorption was 0.007 ug/cm(2))/hr, with 2.32% (0.046 ug/cm(2)) of the dose being absorbed by 6 hr. A maximum, steady state rate of 0.030 ug/cm(2)/hr was maintained until the end of the exposure period at 24 hr, by which time 29.5% of the dose had been absorbed.

The residual MI in the epidermis after tape stripping at the end of the exposure period was 20.2%, thus the total potentially systemically available proportion of the dose (remaining epidermis + absorbed) was 49.7%.

By the end of the exposure period, 29.7% of the dose was washed from the surface of the epidermis, with 4.06% being recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 91.4%.

100 ug/ml body lotion
Between 0-12 hr after dosing, the mean MI absorption rate was 0.004 ug/cm(2)/h, with 2.25% (0.045 ug/cm(2)) of the dose being absorbed by 12 hr. During the 12-24 hr steady state period, the rate was 0.011 ug/cm(2)/hr, with 8.98% (0.180 ug/cm(2)) having been absorbed by 24 hr.

The residual MI in the epidermis after tape stripping at the end of the exposure period was 16.9%, thus the total potentially systemically available proportion of the dose was 25.9%.

By the end of the exposure period, 69.4% of the dose was washed from the surface of the epidermis, with 3.86% being recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 103%.

100 ug/ml facial cream
During the 0-8 hr period after dosing, the mean MI absorption rate was 0.005 ug/cm(2)/hr, with 1.99% (0.040 ug/cm(2)) of the dose being absorbed by 8 hr. Over the 8-24 hr period to the end of the exposure period, the rate was 0.022 ug/cm(2)/hr, with a total 19.6% (0.391 ug/cm(2)) having been absorbed.

The MI remaining in the epidermis after tape stripping after 24 hr exposure was 16.5%, thus the total potentially systemically available proportion of the dose was 36.1%.

By the end of the exposure period, 49.1% of the dose was washed form the surface of the epidermis, with 2.11% being recovered from the stratum corneum.

The total recovery of dosed [14C]-MI equivalents was 96.0%.

Total recovery:

In the aqueous solutions, total recovery ranged from 86.9 - 94.7%.

Conversion factor human vs. animal skin:

Study was conducted on human skin

Any other information on results incl. tables

Table 1 Summary of MI Absorption from Aqueous Solutions through Human Epidermis

	Mean Absorption Rates
Details of Application of Test Materials	Time Period (hr)	Absorption rate (ug/cm^2/hr + SEM)
313 ug/ml solution	0 -8	0.017 + 0.006
	8 -12	0.577 + 0.103
	0 - 24	0.160 + 0.014
104.3 ug/ml solution	0 -8	0.007 + 0.004
	8 - 12	0.132 + 0.005
	0 - 24	0.037 + 0.005
52.2 ug./ml solution	0 - 8	0.005 + 0.002
	8 - 12	0.045 + 0.010
	0 - 34	0.015 + 0.002

Table 2 Summary of MI Absorption from Formulations through Human Epidermis

	Mean Absorption Rates
Details of Application of Test Materials	Time Period (hr)	Absorption rate (ug/cm^2/hr + SEM)
100 ug/ml Shampoo	0 -6	0.007 + 0.002
	6 - 24	0.030 + 0.005
	0 - 24	0.026 + 0.004
100 ug/ml Body Lotion	0 - 12	0.004 + 0.001
	12 - 24	0.011 + 0.002
	0 - 24	0.007 + 0.001
100 ug/ml Facial Cream	0 - 8	0.005 + 0.001
	8 - 24	0.022 + 0.004
	0 - 24	0.017 + 0.003

Applicant's summary and conclusion

Conclusions:

Given the reactive nature of MI, [14C]-label present in the epidermis or having passed through the epidermis may represent parent compound or ring-opened degradation products/metabolites. It was not possible to determine if the [14C]-material, present in the tissue at the end of the study, was permanently bound or available for further absorption. Expressing the results as [14C]-MI equivalents the folowing conclusions can be drawn:

MI in aqueous solutions was readily absorbed across the epidermis following a 24 hr occluded exposure-29.8, 38.0 and 54.7% of applied dose at MI concentrations of 52.2, 104 and 313 ug/ml, respectively. When including [14C]-label retained in the epidermis, 65.3, 60.1 and 75.5% of the applied dose was 'potentially' systemically available at MI concentrations of 52.2, 104 and 313 ug/ml, respectively.

When MI (100 ug/ml) was formulated in a shampoo, body lotion and facial cream, 29.5, 9.0 and 19.6% of the applied dose was absorbed across the epidermis (24 hr, occluded exposure), respectively. When including [14C]-label retained in the epidermis, 49.7, 25.9 and 36.1% of the applied dose was 'potentially' systemically available from a shampoo, body lotion and facial cream, respectively, at 100 ug/ml MI concentration.

Overall, the rates of absorption of MI (100 ug/ml concentration) across human epidermis were slower when formulated in personal care formulations (0.007 to 0.026 ug/cm(2)/hr during 24 hr exposure) compared to the rate of absorption of MI (104 ug/ml concentration) in aqueous vehicle (0.037 ug/cm(2)/hr during 24 hr exposure). In addition, rates of absorption MI, in either aqueous solution or in formulations, were minimal within the first 6 hours of exposure.

Executive summary:

Study Design

The absorption of 2 -methyl-4 -isothiazolin-3 -one (MI: RH-573) from aqueous solutions (nominally 300, 100 and 50 ug MI/ml) and from three 100 ug MI/ml formulations, (shampoo, body lotion and facial cream) has been measured in vitro through human epidermis. The aqueous solutions were applied to the epidermal membranes at a rate of 20 ul/cm(2), while the formulations were applied at 20 mg/cm². The absorption of MI from these doses was measured under occlusion over an exposure period of 24 hr. The absorption process was followed using [¹⁴C]-labelled MI, which was incorporated into the doses. At the end of the exposure period, the distribution in the test system was measured. The results in this study are reported as [¹⁴C]-MI equivalents, subsequently referred to as MI.

Results

Aqueous Solutions

The fastest rates of MI absorption from all the aqueous applications was between 8 -12 after dosing. Little significant absorption occurred before 6 hr. After 12 hr, absorption rapidly slowed, most probably due to depletion of the applied MI reservoir.

313 ug/ml solution

The fastest rate of MI absorption occurred between 8 -12 hr at 0.577 ug/cm(2)/hr, with 39.4% (2.47 ug/cm2)) being absorbed by 12 hr. By the end of the exposure period at 24 hr, 54.7% (3.42 ug/cm²) of the dose had been absorbed. The residual MI in the epidermis after tape stripping at the end of the exposure period was 10.8%, thus the total potentially systemically available proportion of the dose (remaining epidermis + absorbed) was 65.5%.

The total recovery of dosed [¹⁴C]-MI equivalents was 86.9%.

104.3 ug/ml solution

During the 8-12 hr period, the rate was 0.132 ug/cm²/hr, with 28.6% (0.598 ug/cm²) having been absorbed by 12 hr after application. By the end of the exposure period at 24hr, 38.0% (0.793 ug/cm²) of the dose had been absorbed. The MI remaining in the epidermis after tape stripping was 22.1%, giving a total potentially systemically available proportion of the dose was 60.1% after 24 hr exposure.

The total recovery of dosed [¹⁴C]-MI equivalents was 89.9%.

52.2 ug/ml solution

Over the 8-12 hr period, the rate was 0.045 ug/cm²/hr, with 21.8% (0.228 ug/cm²) having been absorbed by 12 hr after application. By the end of the exposure period at 24 hr, 29.8% (0.312 ug/cm²) of the dose had been absorbed. The MI remaining in the epidermis after tape stripping after 24 hr exposure was 35.5%, thus the total potentially systemically available proportion of the dose was 65.3%.

The total recovery of dosed [¹⁴C]-MI equivalents was 94.7%.

Formulations

The absorption profiles from the formulations was noticeably different from the aqueous solution, in that the profiles over the 24 hr exposure period were only 2 phase, with a period of slowly increasing absorption of between 6-12 hr, depending on the formulation, followed by a steady state period of absorption up to the end of the exposure period.

100 ug/ml shampoo

A maximum, steady state rate of 0.030 ug/cm²/hr was maintained until the end of the exposure period at 24 hr, by which time 29.5% of the dose had been absorbed. The residual MI in the epidermis after tape stripping at the end of the exposure period was 20.2%, giving a total potentially systemically available proportion of the dose was 49.7%.

The total recovery of dosed [¹⁴C]-MI equivalents was 91.4%.

100 ug/ml body lotion

During the 12-24 hr steady state period, the rate was 0.011 ug/cm²/hr, with 8.98% (0.180 ug/cm²) having been absorbed by 24 hr. The residual MI in the epidermis after tape stripping at the end of the exposure period was 16.9%, thus the total potentially systemically available proportion of the dose was 25.9%.

The total recovery of dosed [¹⁴C]-MI equivalents was 103%.

100 ug/ml facial cream

Over the 8-24 hr period to the end of the exposure period, the rate was 0.022 ug/cm²/hr, with a total 19.6% (0.391 ug/cm²) having been absorbed. The MI remaining in the epidermis after tape stripping after 24 hr exposure was 16.5%, giving a total systemically available proportion of the dose was 36.1%.

The total recovery of dosed [¹⁴C]-MI equivalents was 96.0%.

Conclusions

Given the reactive nature of MI, [¹⁴C]-label present in the epidermis or having passed through the epidermis may represent parent compound or ring-opened degradation products/metabolites. It was not possible to determine if the [¹⁴C]-material, present in the tissue at the end of the study, was permanently bound or available for further absorption. Expressing the results as [¹⁴C]-MI equivalents the folowing conclusions can be drawn:

MI in aqueous solutions was readily absorbed across the epidermis following a 24 hr occluded exposure-29.8, 38.0 and 54.7% of applied dose at MI concentrations of 52.2, 104 and 313 ug/ml, respectively. When including [¹⁴C]-label retained in the epidermis, 65.3, 60.1 and 75.5% of the applied dose was 'potentially' systemically available at MI concentrations of 52.2, 104 and 313 ug/ml, respectively.

When MI (100 ug/ml) was formulated in a shampoo, body lotion and facial cream, 29.5, 9.0 and 19.6% of the applied dose was absorbed across the epidermis (24 hr, occluded exposure), respectively. When including [¹⁴C]-label retained in the epidermis, 49.7, 25.9 and 36.1% of the applied dose was 'potentially' systemically available from a shampoo, body lotion and facial cream, respectively, at 100 ug/ml MI concentration.

Overall, the rates of absorption of MI (100 ug/ml concentration) across human epidermis were slower when formulated in personal care formulations (0.007 to 0.026 ug/cm²/hr during 24 hr exposure) compared to the rate of absorption of MI (104 ug/ml concentration) in aqueous vehicle (0.037 ug/cm²/hr during 24 hr exposure). In addition, rates of absorption MI, in either aqueous solution or in formulations, were minimal within the first 6 hours of exposure.