Registration Dossier

Administrative data

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2012 - July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, according to OECD 427

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
[U-Naphtalene-14C]-MCP 2484
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
The animals were 8-10 weeks old at the time of dosing. At the
commencement of the study, the weight variation of the animals did not
exceed ± 20% of the average weight.

The animals were housed under conventional conditions in one room. No
other test system was housed in the same room during the study. The room
was ventilated with about 10 air changes per hour and was maintained at a
temperature of 22 ± 2°C and a relative humidity of at least 45% and not
exceeding 65% other than during one occasion of 10 minutes (65.7-68.4%)
and during room cleaning (highest value recorded; 99.9%). Lighting was
artificial with a sequence of 12 hours light and 12 hours dark.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
6 hours
Doses:
approx. 103 mg/kg bw
No. of animals per group:
12
Control animals:
no
Details on study design:
Group 1 (exposure 6 hours, sacrifice at 24 hours post-dose):
The animal of group 1 was used for blood kinetics, excretion of radioactivity in urine and faeces
and assessment of dermal absorption, tissue distribution and mass balance.
The following samples were collected:
Blood Blood samples (tail blood, 200 μl) were collected from the tail vein into
microvettes (Sarstedt) for EDTA-plasma at the following time-points: 30
min, 1h, 2h, 4h, 6h, 8h and 12h. For the 24 h sample, blood at sacrifice
from the abdominal aorta was used.
Excreta Urine and faeces was collected at 0-4h, 4-6h and 6-24 hours.
Air/Volatiles Collection of [14C]-CO2 and volatile [14C]-labeled compounds in the
exhaled air in special glass trap metabolism cages was not needed.
Previous oral ADME TK radioactivity studies indicate no evidence that
[14C]-MCP 2484 form those materials.
Cage wash Cage wash was collected at the end of the collection period.
Skin washing 6 hours after application. See section 4.4.6. for details.
Sacrifice 24 h At sacrifice, the following samples were collected:
- ‘O’-ring + protective device + covers + tapes
- Skin wash at sacrifice
- Surface tape strips (stratum corneum), individually sampled
- Application site (after skin stripping)
- Skin surrounding application site
- Skin (non-treated area)

Organs/tissues At sacrifice, the following organs and tissues were collected:
Abdominal Fat
Adrenal glands
Blood/plasma
Bone
Bone marrow
Brain
Epididymis
GI-tract + contents
Heart
Kidneys
Liver
Lungs
Muscle
Pituitary gland
Residual carcass
Spleen
Testes
Thyroid


Group 2T1 (exposure 6 hours, sacrifice at 6 hours post-dose)
The following samples were collected:
Excreta Urine and faeces were collected at 0-6 hours.
Cage wash Cage wash was collected at the end of the collection period in each
cage.
Skin washing 6 hours after application. See section 4.4.6. for details.
Sacrifice 6 h At sacrifice, the following samples were collected:
- ‘O’-ring + protective device + covers + tapes
- Skin wash at sacrifice
- Surface tape strips (stratum corneum), individually sampled
- Application site (after skin stripping)
- Skin surrounding application site
- Skin (non-treated area)
Organs/tissues At sacrifice, the following organs and tissues were collected
(amendment 1):
Abdominal Fat
Adrenal glands
Blood/plasma
Bone
Bone marrow
Brain
Epididymis
GI-tract + contents
Heart
Kidneys
Liver
Lungs
Muscle
Pituitary gland
Residual carcass
Spleen
Testes
Thyroid


Group 2T2 (exposure 6 hours, sacrifice at 24 hours post-dose)
Excreta Urine and faeces was collected at 0-6h and 6-24 hours.
Cage wash Cage wash was collected at the end of the collection period in each
cage.
Skin washing 6 hours after application. See section 4.4.6. for details.
Sacrifice 24 h At sacrifice, the following samples were collected:
- ‘O’-ring + protective device + covers + tapes
- Skin wash at sacrifice
- Surface tape strips (stratum corneum), individually sampled
Application site (after skin stripping)
- Skin surrounding application site
- Skin (non-treated area)
Organs/tissues At sacrifice, the following organs and tissues were collected
(amendment 1):
Abdominal Fat
Adrenal glands
Blood/plasma
Bone
Bone marrow
Brain
Epididymis
GI-tract + contents
Heart
Kidneys
Liver
Lungs
Muscle
Pituitary gland
Residual carcass
Spleen
Testes
Thyroid


Group 2T3 (exposure 6 hours, sacrifice at 120 hours post-dose)
The following samples were collected:
Excreta Urine and faeces were collected at 0-6h, 6-24h, 24-48h, 48-72h, 72-
96h and 96-120 hours.
Cage wash Cage wash was collected at the end of the collection period in each
cage.
Skin washing 6 hours after application. See section 4.4.6. for details.
Sacrifice 120 h At sacrifice, the following samples were collected:
- ‘O’-ring + protective device + covers + tapes
- Skin wash at sacrifice
- Surface tape strips (stratum corneum), individually sampled
- Application site (after skin stripping)
- Skin surrounding application site
- Skin (non-treated area)
Organs/tissues At sacrifice, the following organs and tissues were collected
(amendment 1):
Abdominal Fat
Adrenal glands
Blood/plasma
Bone
Bone marrow
Brain
Epididymis
GI-tract + contents
Heart
Kidneys
Liver
Lungs
Muscle
Pituitary gland
Residual carcass
Spleen
Testes
Thyroid
Blood Blood samples (tail blood, 200 μl) will be collected from the tail vein into
microvettes (Sarstedt) for EDTA-plasma at the following time-points: 30
min, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 48h, 72h and 96h post-dose. For the
120 h sample, blood at sacrifice from the abdominal aorta was used.

Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects
Absorption in different matrices:
After dermal application of [14C] MCP2484, 0.040% of the applied radioactivity was absorbed
within 6 hours. The absorption was 0.126% and 0.159% at 24h and 120h post-dose. The
majority of this radioactivity was present in the residual carcass.
Total recovery:
At the application site 12.03%, 10.83% and 8.98% of the applied dose was recovered for group
2T1 (6h), 2T2 (24h) and 2T3 (120h). The major part of the radioactivity (9.05%, 7.93% and
7.32% of the applied dose) was present in the skin strips (3-20) and 2.98%, 2.90% and 1.66%
of group 2T1, 2T2 and 2T3, respectively, was retained in the stripped skin.

Total recovered radioactivity, expressed as % of dose is as follows:
2T1 (6h): 97.03%
2T2 (24h): 96.67%
2T3 (120h): 94.58%

Total unabsorbed in was and remaining on skin:
2T1 (6h): 96.99%
2T2 (24h): 96.54%
2T3 (120h): 94.42%
Percutaneous absorptionopen allclose all
Dose:
3.5ul/cm2
Parameter:
percentage
Absorption:
ca. 0.04 %
Remarks on result:
other: 6h
Dose:
3.5ul/cm2
Parameter:
percentage
Absorption:
ca. 0.126 %
Remarks on result:
other: 24h
Dose:
3.5ul/cm2
Parameter:
percentage
Absorption:
ca. 0.159 %
Remarks on result:
other: 120h

Applicant's summary and conclusion

Executive summary:

As part of an overall effort to determine the complete toxicokinetics of MCP 2484 in the rat, the

present OECD 427 study investigated specifically the in vivo skin absorption, distribution,

metabolism and excretion (ADME) or toxicokinetics following a single dermal administration of

radiolabelled [14C]-MCP 2484. In a previous study, the comprehensive toxicokinetics and

ADME of radiolabeled [14C]-MCP 2484 were determined for the oral route of exposure (see

TNO Study V20218). It was not practical to carry out a toxicokinetic ADME study of MCP 2484

by the inhalation or the respiratory route due to its very low vapour pressure and non-volatility.

Moreover, respiratory exposure was not considered to be a relevant route in humans compared

to the oral or dermal exposure routes for this material.

Therefore, the primary purpose of the present study was to evaluate the in vivo skin absorption,

distribution, metabolism and excretion after a single dermal dose with 3.5 μl/cm2 (equivalent to

approximately 100 mg/kg) [14C]-MCP 2484 in male Sprague-Dawley rats. Following OECD 427

guidelines, dermal exposure was carried out for 6 hours; then the skin application site was

wiped and washed with a soap solution to remove the radiolabeled material. Groups of rats

(n=4) were sacrificed at 6h, 24h and 120h post-dose to determine the extent of skin absorption

of [14C]-MCP 2484 over these three time intervals, its retention at the skin application site and

its distribution to the tissues, and its metabolism and excretion.

Major findings from the in vivo skin absorption study are summarized below:

% of Radioactive Dose Absorbed Dermally - 0.040% in 6 hrs to 0.159% in 120 hrs

Systemic absorption and excretion of absorbed radioactivity

Based on mass balance, very little or 0.040% of the applied radioactive dose of MCP 2484 was

absorbed into systemic circulation after 6 hours as evidenced by the radiometric recoveries in

blood, urine, cage wash, faeces and internal tissues including residual carcass. At 6h, the

radiolabeled dose was removed from the skin application site by wiping and the washing with

soap solution and absorption monitored further. Skin absorption was found to be 0.126% after

24 hours and 0.159% after 120 hours. The absorbed radioactivity appears to be mainly

excreted in the faeces (<0.001%, 0.003% and 0.038% in 6, 24 and 120h group animals,

respectively) with smaller percentage excreted in the urine (<0.001%, 0.001% and 0.009% in 6,

24 and 120h group animals, respectively). Based on radioactivity recovered in blood, internal

tissues, urine and faeces, there was very little indication that radiolabeled MCP 2484 is

absorbed systemically into the blood stream through the skin. In fact, less than 0.001% of the

applied radioactive dose was found in blood at 6, 24 or 120h. As discussed below, the

recoveries findings at the skin application site and the recoveries findings in the wipes and

soap washes indicated that the radioactive dose is predominantly unabsorbed through the skin.

Hence, most if not all the radioactivity is recovered in the wipes, soap washes or retained at the

skin application site.

Retention at skin application site (skin stripping) and radioactivity recoveries findings

While a majority of the radioactivity (84.75 to 85.23%) was recovered as unabsorbed material

in the wiping procedure (wipes, soap washes, O-ring protective device, etc.), the remainder of

the radioactivity was recovered at the skin application site, mainly in the stratum corneum

portion of the skin. At the application site, 12.03%, 10.83% and 8.98% of the applied dose was

recovered for group 2T1 (6h), 2T2 (24h) and 2T3 (120h). The major part of the radioactivity

(9.05%, 7.93% and 7.32% of the applied dose) was present in the skin strips (3-20). Skin

stripping revealed that most radioactivity was retained or localized in the upper stratum

corneum layer and/or hair at 120 hours post-application. After 6 hours, following removal of the

applied dose at t=6h, 2.98% of the administered dose remained in the stripped skin. The

amount remaining in the stripped skin after this time point decreased to 1.66% after 120 hours.

Mass Balance Recovery of Radioactivity

Mean recovery of radioactivity in the dose groups ranged from 94.6% to 97.0%.

Distribution to tissues

After dermal absorption, very little radioactivity (ca. 0.003 to 0.012% of radioactive dose) was

found distributed to other tissues and between <0.001 and 0.001 % was present in individual

organs. Tissues showing the highest radioactivity levels at 120 hours included organs like the

liver, kidney, lung, abdominal fat and spleen, With such low skin absorption, bioaccumulation in

the internal tissues appears unlikely.

Metabolic profiling

Due to the low dermal absorption, no metabolite profiling was performed, as the amount of

radioactivity in urine, faeces and plasma samples was far below 50000 dpm/ml for the urine

and faeces homogenates and below 10000 dpm/ml for the plasma samples, needed for reliable

radiometric HPLC analysis and chromatographic comparison for presence of parent and polar

conjugated metabolites. However, a previous oral absorption study with radiolabelled MCP

2484 has provided evidence that the parent material, if systemically absorbed, can undergo

metabolism in the liver to produced oxidized and conjugated metabolites (i.e. glucuronide or

sulphate) that ultimately find their way into the blood and urine. The exact chemical identity of

such metabolites was not been fully characterized owing to the lack of available metabolite

reference for comparison as well as the confounding issue that MCP 2484 is a complex mixture

containing many isomeric components. The most likely metabolic pathway to occur with MCP

2484 probably involved oxidation of the “tetradecyl’ side-chain alkyl group in its structure to

form the corresponding alcohol metabolite or subsequent further oxidized carboxylic acid

metabolites (and their conjugates) [See TNO V20218 oral absorption final report for further

discussion, TNO 2013]. As discussed above, the radioactivity that is absorbed through the skin

into blood appears to be mainly excreted in the faeces (<0.001%, 0.003% and 0.038% of

applied radioactivity dose at 6, 24 and 120 hr, respectively) with smaller percentage excreted in

the urine (<0.001%, 0.001% and 0.009% at 6, 24 and 120h, respectively).

Toxicokinetic Analysis

Due to the very low skin absorption into systemic circulation and the low levels of radioactivity

found in the collected blood samples (i.e., close to limit of detection), very limited toxicokinetic

analysis could be carried out in this study. The dermal blood absorption was too low to provide

any definitive compartmental model fit. However, WinNonlin was used to roughly estimate likely

AUC values for the dermal blood kinetic curve so that relative comparison could be made with

the AUC by the oral route from a previous study [see TNO Study V20218, Oral Absorption Final

Report, TNO (2013)]. The estimated dermal blood AUC was 1.23 to 1.42 h*μg/mL for a 100

mg/kg dermal dose. For relative comparison, a 100 mg/kg oral dose gave an AUC of 111

h*μg/mL. Taken together these findings suggest that dermal absorption or relative dermal

bioavailability was probably at least one order to two orders of magnitude less than oral

absorption based on simple AUC comparison (i.e., ratio of the two AUCs for same dose level).

Hence, dermal absorption is expected to be much less than oral absorption at the same

comparable exposure level of 100 mg/kg bw.

Conclusion

In conclusion, the data from the present dermal study clearly indicate that absorption of

radioactivity into systemic circulation (blood) following dermal exposure to [14C]-MCP 2484 for 6

hours is extremely low. The low dermal systemic bioavailability was also evidenced by the low

blood concentrations (close to limit of detection) observed. Definitive toxicokinetic analysis was

very limited because of low blood concentrations observed. Based on comparison with an oral

absorption ADME study carried out separately and comparison of their AUCs, dermal

absorption occurs even to a lesser extent (at least one order to two orders of magnitude less)

than oral absorption at the same 100 mg/kg dose range. Due to the low systemic absorption,

distribution to the individual tissues was very low (<0.001%) and not expected to pose any

bioaccumulation potential via the dermal route. Overall, systemic absorption of MCP 2484 by

the skin is extremely low and these findings would not be expected to cause systemic exposure

concerns since so little material was absorbed via this route. It is believed that the high

molecular weight MW 599, average), low water solubility (<0.01 mg/L) and high log Kow (>10)

physico-chemical characteristics of MCP 2484 may be the main contributing factors limiting its

dermal absorption [see references, Roberts and Walters (2008); U.S. EPA (1992); Bronaugh

and Maibach (1989); Flynn (1990)].