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Endpoint:
basic toxicokinetics
Type of information:
other: expert statement
Adequacy of study:
supporting study
Study period:
2014-02-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: expert statement
Objective of study:
toxicokinetics
GLP compliance:
no
Conclusions:
Interpretation of results (migrated information): no data
Since no toxicokinetic studies are available for MPA the assessment of the toxicokinetic behavior is based on the physico-chemical properties of the substance.

a) Absorption

Oral absorption:

The very water soluble substance MPA will readily dissolve in the gastrointestinal fluids.
Its relatively low molecular weight of 153 g/Mol, the relative high water solubility and the moderate log Pow of 0.76 favors its absorption in the gastrointestinal tract by passive diffusion. However, since the molecular weight is below 200 g/Mol the substance may also be taken up by passing through aqueous pores of the epithelial barrier.

Therefore, for evaluation of MPA in the chemical safety assessment, the oral absorption of MPA is set at 100 %.

The results of the toxicity studies with MPA do not provide any reason to deviate from this proposed degree of oral absorption.

Dermal absorption:

Making use of the data for molecular weight and the log Pow the skin permeability can be calculated according to the model of Potts and Guy (Potts, R. O., and Guy, R. H.: “Predicting skin permeability” Pharm. Res. 9, 663-669 (1992)).

The model estimates the figure for the skin permeation coefficient kp, which is a measure of the conductance of skin to a particular chemical substance from a particular vehicle. The kp was calculated to be 7.33E-04 cm/hour, therefore a relevant skin penetration may be possible for the substance MPA.

According to the criteria given in the ECHA guidance document “Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance”, November 2012 (version 1.1) 10 % dermal absorption will be considered in case MW > 500 g/Mol and log Pow <-1 and >4, otherwise 100 % dermal absorption is assumed.

In consequence, 100 % dermal absorption is proposed for MPA.

Respiratory absorption:

Deposition pattern for dusts depends on the particle sizes of the substance to be inhaled. In general, particles having an aerodynamic diameter of greater than 10 µm are deposited in the nasopharyngeal region. Particles smaller than 10 µm are capable for reaching the alveolar region of the respiratory tract.

Since the measurement of the particle size distribution of MPA indicates no particles smaller than 10 µm, deposition of MPA after inhalation predominately takes place in the nasopharyngeal region.

After deposition water soluble particles of MPA may readily diffuse/dissolve into the mucus lining of the nasopharyngeal region and may then be dermally absorbed.

b) Distribution

After absorption MPA is expected to distribute easily throughout the body based on its low molecular weight, the log Pow > 0 (some lipophilicity) and the high water solubility.

Since the log Pow is only slightly above zero, MPA can also distribute into cells to a minor extent so that the intracellular concentration may be higher than the extracellular concentration, particularly in fatty tissues.

Since the log Pow of MPA is < 4 the substance is not expected to accumulate within the body.

c) Metabolism

Once absorbed, the mammalian organism may metabolize the substance MPA by hydroxylation of appropriate carbon and nitrogen atoms which may be followed by conjugation.

Executive summary:

Since no toxicokinetic studies are available for MPA the assessment of the toxicokinetic behavior is based on the physico-chemical properties of the substance.

 

a)    Absorption

 

Oral absorption:

 

The very water soluble substance MPA will readily dissolve in the gastrointestinal fluids.

Its relatively low molecular weight of 153 g/Mol, the relative high water solubility and the moderate log Pow of 0.76 favors its absorption in the gastrointestinal tract by passive diffusion. However, since the molecular weight is below 200 g/Mol the substance may also be taken up by passing through aqueous pores of the epithelial barrier.

 

Therefore, for evaluation of MPA in the chemical safety assessment, the oral absorption of MPA is set at 100 %.

 

The results of the toxicity studies with MPA do not provide any reason to deviate from this proposed degree of oral absorption.

 

Dermal absorption:

The maximum absorbed quantity into the skin was determined as 2.44 µg.cm-2for MPA after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA.

Based on these results, it can be stated that there will be a negligible transport of MPA into or through human skin.

 

 

Respiratory absorption:

 

Deposition pattern for dusts depends on the particle sizes of the substance to be inhaled. In general, particles having an aerodynamic diameter of greater than 10 µm are deposited in the nasopharyngeal region. Particles smaller than 10 µm are capable for reaching the alveolar region of the respiratory tract.

 

Since the measurement of the particle size distribution of MPA indicates no particles smaller than 10 µm, deposition of MPA after inhalation predominately takes place in the nasopharyngeal region.

 

After deposition water soluble particles of MPA may readily diffuse/dissolve into the mucus lining of the nasopharyngeal region and may then be dermally absorbed.

 

b)   Distribution

 

After absorption MPA is expected to distribute easily throughout the body based on its low molecular weight, the log Pow > 0 (some lipophilicity) and the high water solubility.

 

Since the log Pow is only slightly above zero, MPA can also distribute into cells to a minor extent so that the intracellular concentration may be higher than the extracellular concentration, particularly in fatty tissues.

 

Since the log Pow of MPA is < 4 the substance is not expected to accumulate within the body.

 

c)    Metabolism

 

Once absorbed, the mammalian organism may metabolize the substance MPA by hydroxylation of appropriate carbon and nitrogen atoms which may be followed by conjugation.

 

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-07 - 2014-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Species:
other: not applicable. in vitro
Duration of exposure:
24 hours
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human skin from surgical operations
- Ethical approval if human skin: yes
- Type of skin: abdomen
- Preparative technique: Dermatomization, tape stripping and cryo-sectioning
- Thickness of skin (in mm): mean thickness of around 500 ± 100 µm
- Membrane integrity check: yes, with caffeine
- Storage conditions: cooled to 4 °C
- Justification of species, anatomical site and preparative technique:

PRINCIPLES OF ASSAY
- Diffusion cell: Franz cell
- Receptor fluid: PBS pH 7.4
- Solubility od test substance in receptor fluid: yes as determined
- Static system: yes
- Flow-through system: no
- Test temperature: 32 °C ± 1 °C

Total recovery:
The recovery of MPA after extraction development were 105.66 %. if the applied amount is set to 100 %. These value is within the set specifications (recovery of 90 % to 110 %). The development of an extraction method for skin was successful.
Dose:
0.2%
Parameter:
percentage
Absorption:
0.21 %
Remarks on result:
other: 24 hours

Solubility

To analyze the solubility of MPA and to ensure sink conditions during the resorption experiments, one concentration (ca. 1072 µg·mL-1for MPA) in the preselected acceptor medium was prepared and diluted to the linear range for measurement.The solutions were stored for 4 hours at room temperature on a stirrer. Samples were taken in triplicate at time points 0 h, 1 h and 4 h.The following table shows the results of the solubility measurements of MPA in the selected acceptor medium PBS pH 7.4.

Solubility of MPA in PBS pH 7.4

Storage time [h]

0

1

4
Storage temperature

RT

RT

RT
n1 [µg∙mL-1]

1076.60

1062.18

1142.36
n2 [µg∙mL-1]

1080.44

1081.32

1154.58
n3 [µg∙mL-1]

1079.90

1068.38

1177.16
Mean [µg∙mL-1]

1079.0

1070.6

1158.0
SD [µg∙mL-1]

1.7

8.0

14.4
RSD [%]

0.2

0.7

1.2
Theoretical c for t=0 [µg∙mL-1]

1072.44
Appearance

Clear colorless solution with few particles

Clear colorless solution with few particles

Clear colorless solution with few particles
Dissolved [µg∙mL-1]

1079.0 ± 1.7

1070.6 ± 8.0

1158.0 ± 14.4

The measured solubilities ensure sink conditions in the resorption experiments with PBS pH 7.4 as acceptor medium.

Skin quality control experiment (MEA)

In an earlier study the permeability of caffeine from an aqueous solution across full-thickness skin, dermatomized skin, heat-separated epidermis and isolated SC had been studied at Across Barriers to establish quality assurance benchmarks for skin integrity [Bock et al., 2002]. Subsequent quality control studies have been performed on a range of human skin samples.

The following table provides an overview of caffeine permeability through dermatomized skin specimens measured at Across Barriers and includes the Pappvalues determined in the present study.

Comparison of apparent permeability coefficients for caffeine through different dermatomized skin specimens with intact SC. Skin No.617-01-0714 was used in the present study.

Skin number
Mean Papp[cm∙s-1] (n=3)
RSD [%]

061-01-0701

9.38E-08

29

153-01-0104

8.41E-08

28

059-01-0601

8.24E-08

39

060-01-0601

5.63E-08

6

062-01-0701

5.34E-08

18

155-01-0204

5.03E-08

27

209-01-0605

3.82E-08

7

157-01-0304

3.23E-08

27

057-01-0601

3.21E-08

16

150-01-1203

2.54E-08

56

058-01-0601

2.00E-08

7

308-01-1107

1.28E-08

25

239-01-0707

1.04E-08

3

617-01-0714*

6.14E-08

15

*Evaluated as n=2 because one Franz cell was declared as outlier due to a significant higher transport most likely derived from a damaged skin biopsy.

 

The skin tightness is comparable with skins used in the past. The permeation coefficient for caffeine applied as infinite dose is in the same magnitude compared with previous data.

Mass balance of MPA after transport through human skin over a time period of 24 h.

 
MPA
Applied amount [µg·cm-2]

559.61
Amount remaining on skin surface [µg·cm-2]

585.51
Amount in stratum corneum [µg·cm-2]

0.34
Amount in epidermis/dermis [µg·cm-2]

0.00
Amount resorbed [µg·cm-2]

5.53
Sum of absorbed & not absorbed [µg·cm-2]

591.27
Recovery from applied amount [%]

105.66

All calculations and evaluations were performed with the measured amounts of test substance in the test items. 559.61 µg·cm-2MPA was applied (corresponds to 0.1 % (w/v)). The recovery of MPA after extraction development were 105.66 % for MPA if the applied amount is set to 100 %. These value is within the set specifications (recovery of 90 % to 110 %). The development of an extraction method for skin was successful.

Transport of MPA through human skin

The permeation coefficient was calculated from the linear range of the transport curve (time points 4 to 8; 8 h to 24 h). The permeation coefficient was 1.96E-8 cm∙s-1± 5.86E-9 cm∙s-1. After 18 hours MPA could be detected for the first time. For the calculation of the permeation coefficient at least five points are necessary; the fourth time point after 8 h was additionally included. The transported amount after 24 hours was 2.61 µg∙cm-2± 1.03 µg∙cm-².

Mass balance reported as µg·cm-2and recovery reported as % for MPA after transport through human skin over a time period of 24 h.

Parameter
MPA
Amount remaining on skin surface [µg·cm-2]
(Not absorbed: residual test items in donor chamber + first two tape strips)

1219.65 ± 30.06
Amount in stratum corneum [µg·cm-2]
(All tape strips except for the first two strips)

2.44 ± 0.65
Amount in epidermis/dermis [µg·cm-2]
(All slices of the skin layers)

0.00
Absorbed into skin [µg·cm-2]
(Sum of stratum corneum + epidermis/dermis)

2.44 ± 0.65
Amount resorbed [µg·cm-2]
(Found in acceptor medium)

2.61 ± 1.03
Sum [µg·cm-2]
(Absorbed + resorbed + not absorbed)

1224.70 ± 31.74
Applied amount [µg·cm-2]

1156.95
Recovery of sum from applied amount [%]
(Absorbed + resorbed + not absorbed)

106

 Penetration of MPA

see attached figure

The maximum absorbed quantity into the skin was 2.44 µg.cm-2for MPA after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA. The amount of MPA resorbed or transported through the skin was 2.61 µg.cm-2which corresponds to 0.23 % of the applied dose. Therefore, the permeation coefficient for MPA was determined as 1.96E-8 cm∙s-1± 5.86E-9 cm∙s-1. The lag time (first time point where MPA could be detected in the acceptor compartment of the Franz diffusion cell) was eight to ten hours. After the resorption experiment MPA could only be detected in the stratum corneum. It did not penetrate the deeper skin layers in measurable amounts. The sum of absorbed MPA has been calculated by adding the amount MPA found in the stratum corneum to the amount in the epidermis/dermis.

 

Conclusions:
The study was performed as a risk assessment for the test substance MPA. It was the objective to find out, which amounts of the substance are capable of permeating through and penetrating into the human skin. In practice, the maximum possible exposure concentrations are 0.1 % (w/v) for MPA , that can contaminate human skin. These concentrations are based on the finished mixtures of the substances that will be marketed.
During the study the expected concentration in a product were doubled and the duration of incubation set to 24 h to get a measurable transport of the substances through the skin. Even with the doubled concentration (0.2 % for MPA) and a duration of 24 h a very low transport was determined for MPA (1.96E-8 cm∙s-1 ± 5.86E-9 cm∙s-1). The maximum absorbed quantity into the skin was 2.44 µg.cm-2 after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA.
Based on these results, it can be stated that there will be a negligible transport of MPA into or through human skin, especially as the real life exposure levels are half of the used ones. Furthermore it can be assumed that the contact time will be much shorter, because the skin will be cleaned faster.
 
Executive summary:

In the present study, a comparative in vitro examination of the dermal absorption of N-[(3/5-Methyl-1H-pyrazol-1-yl) methyl] acetamide (MPA) was performed according to OECD-Guideline 428. The test item was applied to human skin as follows, as no interferences with analytics were determined:

 

·     Test item A:           1 mL of 0.2 % MPA (w/v) in PBS pH 7.4

The amount of MPA which can be absorbed into the human organism through the skin was determined using human excised skin as an in vitro model for dermal absorption. The resorbed amounts of the test substance were quantified over a time period of 24 hours by analyzing samples from the acceptor compartment of Franz diffusion cells.

After the resorption experiment the amounts of MPA taken up into the skin were determined. For this penetration study the stratum corneum was stripped by the so-called “tape stripping technology”. The strips were collected in two samples. The deeper skin layers were sliced in parallel sections using a cryo microtome. The skin slices were collected in one sample.

 

The used skin was qualified by performing a resorption study with caffeine over a period of 24 hours. Resorbed caffeine was quantified by a method developed and validated at Across Barriers. The resorption data were compared with historical data gained by the testing facility.

 

The analytical method for MPA was obtained from sponsor and adapted. For the quantitative analysis of the test substances the following validation parameters were measured: the system suitability, the linearity and the LLOQ. This was carried out according to EMA Guideline for Bioanalytical method validation (EMEA/CHMP/192217/2009).

The amount of MPA resorbed or transported through the skin was 2.61 µg.cm-2which corresponds to 0.23 % of the applied dose. After the resorption experiment MPA could only be detected in the stratum corneum. It did not penetrate in measurable amounts into the deeper skin layers.

During the study the expected concentrations of 0.1% in a product were doubled and the duration of incubation set to 24 h to get a measurable transport of the substances through the skin. Even with the doubled concentrations (0.2 % for MPA) and a duration of 24 h a very low transport was determined for MPA (1.96E-8 cm∙s-1± 5.86E-9 cm∙s-1). The maximum absorbed quantity into the skin was 2.44 µg.cm-2for MPA after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA.

Based on these results, it can be stated that there will be a negligible transport of MPA into or through human skin, especially as the real life exposure levels are half of the used ones. Furthermore it can be assumed that the contact time will be much shorter, because the skin will be cleaned faster.


Description of key information

Theoretical assessment of the toxicokinetic behaviour of MPA:

 a) Absorption

1)     Oral absorption:

 The very water soluble substance MPA will readily dissolve in the gastrointestinal fluids.

Its relatively low molecular weight of 153 g/Mol, the relative high water solubility and the moderate log Pow of 0.76 favors its absorption in the gastrointestinal tract by passive diffusion. However, since the molecular weight is below 200 g/Mol the substance may also be taken up by passing through aqueous pores of the epithelial barrier.

 Therefore, for evaluation of MPA in the chemical safety assessment, the oral absorption of MPA is set at 100 %.

 The results of the toxicity studies with MPA do not provide any reason to deviate from this proposed degree of oral absorption.

 2) Dermal absorption:

The maximum absorbed quantity into the skin was determined as 2.44 µg.cm-2for MPA after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA.

Based on these results, it can be stated that there will be a negligible transport of MPA into or through human skin.

 3) Respiratory absorption:

 Deposition pattern for dusts depends on the particle sizes of the substance to be inhaled. In general, particles having an aerodynamic diameter of greater than 10 µm are deposited in the nasopharyngeal region. Particles smaller than 10 µm are capable for reaching the alveolar region of the respiratory tract.

 Since the measurement of the particle size distribution of MPA indicates no particles smaller than 10 µm, deposition of MPA after inhalation predominately takes place in the nasopharyngeal region.

 After deposition water soluble particles of MPA may readily diffuse/dissolve into the mucus lining of the nasopharyngeal region and may then be dermally absorbed.

 b)   Distribution

 After absorption MPA is expected to distribute easily throughout the body based on its low molecular weight, the log Pow > 0 (some lipophilicity) and the high water solubility.

 Since the log Pow is only slightly above zero, MPA can also distribute into cells to a minor extent so that the intracellular concentration may be higher than the extracellular concentration, particularly in fatty tissues.

 Since the log Pow of MPA is < 4 the substance is not expected to accumulate within the body.

 c)    Metabolism

 Once absorbed, the mammalian organism may metabolize the substance MPA by hydroxylation of appropriate carbon and nitrogen atoms which may be followed by conjugation

Experimental determination of absorption through the skin:

The study was performed as a risk assessment for the test substance MPA. It was the objective to find out, which amounts of the substance are capable of permeating through and penetrating into the human skin. In practice, the maximum possible exposure concentrations are 0.1 % (w/v) for MPA , that can contaminate human skin. These concentrations are based on the finished mixtures of the substances that will be marketed.

During the study the expected concentration in a product were doubled and the duration of incubation set to 24 h to get a measurable transport of the substances through the skin. Even with the doubled concentration (0.2 % for MPA) and a duration of 24 h a very low transport was determined for MPA (1.96E-8 cm∙s-1 ± 5.86E-9 cm∙s-1).  The maximum absorbed quantity into the skin was 2.44 µg.cm-2  after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA.

Based on these results, it can be stated that there will be a negligible transport of MPA into or through human skin, especially as the real life exposure levels are half of the used ones. Furthermore it can be assumed that the contact time will be much shorter, because the skin will be cleaned faster.

 

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
0.21

Additional information

Since no toxicokinetic studies are available for MPA the assessment of the toxicokinetic behavior is based on the physico-chemical properties of the substance.

 

a)    Absorption

 

Oral absorption:

 

The very water soluble substance MPA will readily dissolve in the gastrointestinal fluids.

Its relatively low molecular weight of 153 g/Mol, the relative high water solubility and the moderate log Pow of 0.76 favors its absorption in the gastrointestinal tract by passive diffusion. However, since the molecular weight is below 200 g/Mol the substance may also be taken up by passing through aqueous pores of the epithelial barrier.

 

Therefore, for evaluation of MPA in the chemical safety assessment, the oral absorption of MPA is set at 100 %.

 

The results of the toxicity studies with MPA do not provide any reason to deviate from this proposed degree of oral absorption.

 

Dermal absorption:

The maximum absorbed quantity into the skin was determined as 2.44 µg.cm-2for MPA after 24 hours contact with the skin. This corresponds to 0.21 % of the applied dose MPA.

Based on these results, it can be stated that there will be a negligible transport of MPA into or through human skin.

 

Respiratory absorption:

 

Deposition pattern for dusts depends on the particle sizes of the substance to be inhaled. In general, particles having an aerodynamic diameter of greater than 10 µm are deposited in the nasopharyngeal region. Particles smaller than 10 µm are capable for reaching the alveolar region of the respiratory tract.

 

Since the measurement of the particle size distribution of MPA indicates no particles smaller than 10 µm, deposition of MPA after inhalation predominately takes place in the nasopharyngeal region.

 

After deposition water soluble particles of MPA may readily diffuse/dissolve into the mucus lining of the nasopharyngeal region and may then be dermally absorbed.

 

b)   Distribution

 

After absorption MPA is expected to distribute easily throughout the body based on its low molecular weight, the log Pow > 0 (some lipophilicity) and the high water solubility.

 

Since the log Pow is only slightly above zero, MPA can also distribute into cells to a minor extent so that the intracellular concentration may be higher than the extracellular concentration, particularly in fatty tissues.

 

Since the log Pow of MPA is < 4 the substance is not expected to accumulate within the body.

 

c)    Metabolism

 

Once absorbed, the mammalian organism may metabolize the substance MPA by hydroxylation of appropriate carbon and nitrogen atoms which may be followed by conjugation.