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Reference
Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: non-GLP but with QA-statement.
Qualifier:
according to
Guideline:
other: The SCCP's notes of Guidance for the testing of cosmetic ingredients and their safety evaluation (reference: SCCNFP/0321/00 Final).
GLP compliance:
no
Radiolabelling:
yes
Remarks:
Radiolabelled 14C-AHTN (uniformly labelled in the aromatic ring – radiochemical purity 99.6%)
Species:
human
Strain:
other: Full-thickness human female breast skin and abdominal skin.
Sex:
female
Details on test animals and environmental conditions:
Full-thickness human female breast skin and abdominal skin.
Type of coverage:
open
Vehicle:
ethanol
Duration of exposure:
24 hours
Doses:
AHTN was prepared as a 1% (w/v) solution in ethanol (96% v/v). Target dose: 20 µl/cm2
No. of animals per group:
N/A
Control animals:
no
Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Total recovery:
Overall recovery was 92,5%
Dose:
200 ug AHTN/cm2
Parameter:
percentage
Absorption:
ca. 4.1 %
Remarks on result:
other: 24 hours
Remarks:
4.06% of the applied dose in 24 hours
Conversion factor human vs. animal skin:
N/A

Following 24 hours exposure, 0.760 ug test compound/cm2 (corresponding to 0.379% of the applied dose) had permeated through epidermal membranes. The majority of applied test compound (84.7% of the applied dose) was found in the 24 hour surface wipe and donor chamber wash plus wipe. The stratum corneum tape strips contained 3.80% of the applied dose and the epidermis, plus any remaining stratum corneum after tape strpping, 3.49% of the applied dose. The levels of test compound in the epidermis, plus any remaining stratum corneum after tape stripping, filter paper and receptor fluid were combined to produce a total absorbed dose value of 4.06% of the applied dose.

Conclusions:
This in vitro dermal absorption study with 14C-ring labelled AHTN using human epidermal membranes indicated that 4.1% of the applied dose is absorbed over 24 hours.
Executive summary:

The dermal absorption (non-GLP, but with QA-statement) of AHTN was determined over a 24-hr period according to the methodology of the SCCNFP. Radiolabeled 14C-AHTN (uniformly labelled in the aromatic ring – radiochemical purity 99.6%) was applied in 1% (w/v) solution in ethanol (96% v/v) to human epidermal membranes (prepared from full thickness female breast or abdominal skin and assayed for integrity with tritiated water) supported on a piece of filter paper (for strength) in glass diffusion cells (n=12). There were two control cells. The area of the membrane available for absorption was approximately 1 cm2 and the applied dose was a mean of (n=12) 20±0.4 μL/cm2 (hence, 200 μg AHTN/cm2). Receptor fluid (6% Volpo N20 (to enhance solubility) in pH 7.4 phosphate buffered saline) was sampled at 1, 2, 6, 12 and 24 hrs. After 24 hrs the epidermal membranes were wiped and stripped. The amount of material absorbed into the receptor phase after 24 hr was 0.38±0.06% of the applied dose. The majority of applied AHTN (85±2% of the applied dose) was found in the 24-hr surface wipe and donor chamber wash plus wipe. The stratum corneum tape strips contained 3.8±0.3% of the applied dose and the remaining stratum corneum plus epidermis 3.5±0.3% of the applied dose. As per SCCNFP guidelines, levels of AHTN in the remaining stratum corneum plus epidermis, filter paper (on which the epidermis samples rested; 0.19±0.03% of the applied dose) and permeated AHTN were combined to produce a total absorbed dose value of 4.1±0.4% of the applied dose. The evaporative loss (assessed in a separate experiment under the same conditions) over 24 hrs was 2.9% of the applied dose. Overall-recovery of radioactivity at 24 hrs was 92.5±0.7%.

Description of key information

Short description of key information on bioaccumulation potential result:

The presence of AHTN has been demonstrated in breast milk and human tissue. In case of oral absorption or intravenous injection, almost all AHTN is excreted through urine and faeces.

Short description of key information on absorption rate:

Dermal exposure is the major exposure route. It has been demonstrated that AHTN absorption was very low. It was lost through reverse diffusion and/or desquamation. A worst-case absorption rate has been selected for the risk assessment.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
4.1
Absorption rate - inhalation (%):
100

Additional information

There are no data available on the toxicokinetics of AHTN after inhalation exposure, but exposure is very low due to the very low vapour pressure of AHTN. For inhalation exposure, an assumption of 100% absorption as a worst-case will be used in the risk characterization. The latter assumption would probably overestimate exposure from dust because absorption in the lung is likely to occur only for dissolved AHTN and AHTN is poorly soluble in water (1.25 mg/L).

For oral exposure, a default of 50% absorption will be used in the risk characterization. As a support, based on urine, cage washing and tissue levels in the study by Wu (2002), see table 1 below, absorption of about average 50% can be concluded.

Table 1 Total radioactivity recovered from urine, cage rinse, faeces and tissues after oral dosing with AHTN in rat expressed as percentage of dose.

Group ID

Sex

% Dose in Urine

% Dose in Cage Rinse

% Dose in Faeces

% Dose in Tissues

% Dose Recovered

1 (15 mg/kg bw/day)

M

F

11.04

14.16

2.92

7.97

52.18

44.76

26.66

28.55

92.80

95.44

2 (100 mg/kg bw/day)

M

F

35.06

28.20

6.27

3.98

35.00

41.74

19.63

18.82

95.95

92.74

In the in vivo human dermal study, a 6 hour unoccluded exposure with 0.24% AHTN in 70% ethanol, intended to simulate a typically high exposure from the use of alcohol-based products such as perfumes or eau de toilette, resulted in absorption into the skin (at least 15%). However, most of the material in this skin reservoir was not absorbed, but was recovered from dressings over the site of exposure over a 120 hr period, presumably from reverse diffusion and/or desquamation. Based on amounts excreted, primarily in the urine, >2% in total was actually absorbed under the conditions of this experiment (Hawkins, Girkin, Brooker - 1996).

An in vivo study in rats supports the assumption that a good indication of the amount absorbed is the amount excreted (Hawkins, Elsom, Girkin - 1997). Although approximately 14% of the applied radioactivity was not recovered, from a separate evaporation study, it appears that under non-occlusive conditions about 24% of an applied dose may evaporate (Ford, hawkins, Schwarzenbach - 1994).

A similar picture (although, as expected, with considerably higher absorption) was seen in vivo with rats where the material was applied for 6 hours under occlusion in 70% alcohol. Here again, a reservoir in the skin of about 13% of the applied dose was formed after the 6-hour application with about 2 of this 13% reservoir being lost presumably from reverse diffusion and/or desquamation to the dressing after dose removal. Based on the amount remaining in the tissues (excluding that at the site of dosing, i.e. 1.5%) at sacrifice (1.7%) and the amount excreted (17.1%), and almost all in the faeces (14.5%), a total absorption under the conditions of this experiment of 18.8% can be concluded (Ford, hawkins, Schwarzenbach - 1999).

The principal differences with the human study is the much larger absorption because of the application under occlusion and the well-known fact that rat skin is more permeable than human.

Recently, an in vitro 24-hour absorption study using 1% AHTN in 96% alcohol with human epidermal membranes was performed according to the recommendations of the SCCNFP. In this study, 0.38% of the applied dose was found in the receptor fluid after 24 hr under non-occlusive conditions, however, 3.5% of the applied dose remained in the epidermis. Adding these amounts to the small amount remaining on the filter paper used to support the membranes leads to the calculation of total absorption of 4.1% of the applied dose (Green, Brain - 2001).

From a separate experiment, it appears that under the same conditions about 29% of an applied dose may evaporate from human skin in vitro (Kirkpatrick, Dean, Mellor 1997).

As the other skin absorption studies have limitations such as 6 hour exposure time or a small number of samples, for risk characterization, 4.1% as a value for skin absorption will be used as a conservative estimate.

Intravenous administration of AHTN to rats and the pig results in rapid distribution (kirkpatrick, Elsom, Girkin - 1997). Excretion in the rat is primarily via the faeces as was seen in the dermal study (Hawkins, Kirkpatrick, Aikens, Saxton - 1995), but in the pig the principle route of excretion is via urine (Kirkpatrick, Elsom, Girkin - 1997) similar to what was seen in the human study (Hawkins, Girkin, Brooker - 1996). In neither of these studies was any evidence of accumulation seen. However, clearance from the fat was slower than from other organs. It is noteworthy that in the intravenous studies, no unmetabolised AHTN is present in the urinary radioactivity. This means that all AHTN present in urine is metabolised (for rat 21.5%, and for pig, 86.2%). The faeces (the major excretion route of the rat) were not analysed for metabolites or parent.

An oral study with pregnant and later lactating rats shows that orally dosed AHTN and its metabolites can end up in the mother’s milk (Hawkins, Kirkpatrick, Saxton - 1996). The levels seen in the milk of the lactating dams can aid in the interpretation of the study where neonate rats were exposed to AHTN and its metabolites through nursing.

AHTN is also found in human milk at a maximum level of 267 ng/kg milk fat, a mean level of 16 ng/kg milk fat and a median of 0 ng/kg milk fat.

In summary, for the purpose of risk characterization, 50% absorption for oral exposure and 100% for inhalation will be used. For dermal absorption of AHTN in humans, 4.1% has been taken forward to the risk characterisation.

source: EU Risk Assessment Report AHTN (May 2008)

Discussion on bioaccumulation potential result:

Dermal absorption is the prime route of exposure, hence any summary on basic toxicokinetics should involve skin absorption. For the chemical safety assessment regarding oral absorption, the absorption is set to 50%. It should be stressed that AHTN should not be ingested and is not suitable for oral consumption. Hence, toxicokinetic considerations are less relevant for ingestion. Inhalation is also minor with respect to the dominant dermal route of exposure. Oral and especially dermal exposures involve additional absorption barriers. Studies in which test animals were exposed through intravenous exposure were considered relevant, even though this is not a realistic route of exposure. Excretion in rats and pig after intraveneous injection occurs primarily in the faeces and urine respectively. In these studies and also in the human study, no accumulation was observed. Nevertheless, since AHTN has been measured in the human body, a low bioaccumulation potentials has been selected.

Discussion on absorption rate:

The Scientific Committee on Cosmetic Products and Non-Food Products intended for consumers (SCCNFP) released their evaluation and opinion on AHTN that was adopted during the 14th plenary meeting of 24 October 2000 (SCCNFP/0372/00) (see IUCLID entry 13. Assessment reports “SCCNFP 2000”). The percutaneous absorption had to be assessed. A subsequent in-vitro human skin penetration study was carried out (see IUCLID entry 7.1.2 “Humans (in vitro), RIFM 38099, Green 2001”). Although the study is highly relevant and reliable, the SCCNFP judged that it was not conform to the SCCNFP Notes of Guidance and also argued that the use of ethanol as vehicle strongly affects the tissue and therefore the potential skin penetration of the substance. As an alternative, the SCCNFP considered an in-vivo human study (see IUCLID entry 7.1.2 “Man (in vivo), RIFM 28836, Hawkins 1996”). The SCCNFP concluded that a 2% absorption can be assumed as a conservative value.

The EU Risk Assessment Report AHTN (2008) used the above-mentioned in-vitro test for the determination of skin absorption, which was 4.1%. To avoid any discussion on the most reliable or accurate absorption that should be used in the chemical safety assessment, the worst value of 4.1% has been selected for further use.