2,2'-[6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol}

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July 2002 to 27 Sept 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Version / remarks:

Draft Guideline (Dec. 2000)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Radiolabelling:

yes

Remarks:

14C

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Alpk:APfSD

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Biological Services Section, Alderley Park, Macclesfield, Cheshire
- Age at study initiation: no data
- Weight at study initiation: 203 - 238 grams (mean 217 g)
- Fasting period before study: no
- Housing: individual; stainless steel metabolism cages (for excretion / distribution study) & stock cages for blood sampling study
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): Rat and Mouse #1 maintenance diet; Special Diet Services, Stepfield, Whitham; ad libitum
- Water (e.g. ad libitum): tap, ad libitum
- Acclimation period: 4 days (stock cages), 1 day (metabolism cage)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: commercial formulation

Duration of exposure:

6 hours

Doses:

Dose preparation
Two doses were prepared in the commercial formulation selected. The formulation comprised unlabelled and [14C]- radiolabelled CGF-C 1607 homogeneously dispersed in the formulation such that a dose of set volumes of 54 µl/rat or 22 µl/rat were equivalent to the nominal dose levels of 2 mg/rat or 0.8 mg/rat respectively.
Formulations were each prepared 1 day prior to dosing. This was acceptable because the stability of the CGF-C 1607 in the formulations was confirmed by HPLC analysis.

High dose level:
Formulation concentration: 4% CGF-C1607
Nominal dose: 2 mg a.i./rat -> 0.2 mg a.i./cm2
Mean achieved dose: 1.82 mg a.i./rat
Dose volume per 10 cm2 per rat: 54 µl
Specific activity: 257.4 kBq/mg a.i.
Nominal 14C-dose: 500 kBq per rat

Low dose level:
Formulation concentration: 4% CGF-C1607
Nominal dose: 0.8 mg a.i./rat -> 0.08 mg a.i./cm2
Mean achieved dose: 0.788 mg a.i./rat
Dose volume per 10 cm2 per rat: 22 µl
Specific activity: 573.8 kBq/mg a.i.
Nominal 14C-dose: 400 kBq per rat

No. of animals per group:

4 per time point and dose level

Control animals:

no

Details on study design:

Preparation of skin
On the day before dosing, the fur from behind both shoulders of each rat was shaved and the exposed skin was swabbed with acetone to remove sebum. Using cyanoacrylate glue, an Acetal plastic 'O'-ring (25.25 mm internal diameter) with a screw thread cap was attached to the shaved skin behind each shoulder, to define each application site. The screw thread cap held in place a nylon gauze cover. Care was taken to avoid the inclusion of any damaged skin within each defined area. A strip of non-occlusive elasticated bandage was then wrapped around the rat and over the application devices. The total area of skin defined for dose application to each rat was 10 cm2 per rat, comprising two sites each of nominally 5 cm2.

Dose administration
Following acclimatisation in metabolism cages, each rat was weighed. The appropriate volume of dose preparation was applied to the defined skin surface using a positive displacement pipette. Following application, the edge of each pipette tip was used to spread the dispensed dose as evenly as practicable over the defined area, taking care to avoid direct contact of the pipette tip or dose preparation with the plastic O-rings. Separate pipette tips were used for each application site on each rat and the pair of tips were then retained in the same container for subsequent extraction of residual radioactivity with ethyl acetate to enable the actual dose applied to each rat to be calculated.Each application site was protected by a nylon gauze cover. A strip of non-occlusive elasticated bandage was then placed around the rat and over the O-rings.

Interim wash
Six hours after dosing, the application sites of all rats were washed. Each un-anaesthetised rat was held above a tray to enable the collection of any excreta and its bandage removed and retained for analysis. In turn, the O-ring caps with nylon gauze covers attached were removed. Each application site was washed using 6 pieces of natural sponge prewetted
with a 3% aqueous solution of a liquid soap solution and 6 with water, and then dried using 2 dry sponges. Care was taken to avoid the transfer of any test substance
from the skin surface and onto the O-rings during washing. All of the sponges used for each rat were retained in a single container for 14C]- analysis. The O-ring caps with nylon gauze
covers fitted were re-attached, a new elasticated bandage was fitted to all rats. Urine and faeces were collected from each cage immediately after each skin wash was completed. Any urine and faeces collected on the tray during washing was added to the corresponding excreta collections. Each cage was then rinsed with water and a cage wash collected.

Collection of excreta
For the duration of each experiment, urine and faeces were collected from each cage at approximately 6, 24,48, 72, 96 and 120 hours. With the exception of the terminal collection, each cage was washed with water immediately after the collection of urine. Excreta were stored at approximately -20°C and cage washes were refrigerated prior to analysis.

Termination of study and collection of samples
Sub-groups of 4 rats were terminated immediately after the 6 hour exposure and 24, 72 and 120 hours after dosing. Each un-anaesthetised rat was held above a tray to enable the collection of any excreta and the bandage removed and added to the same container as previous bandages for each rat. The nylon gauze covers and O-ring caps were removed, leaving the remaining O-rings in place. The nylon gauze covers were retained in the same container as the O-ring caps for each rat. Residual dose preparation was collected from each application site by washing each 5 cm2 skin site with soap solution (3% aqueous solution of a liquid soap solution) and water, applied using pieces of pre-wetted natural sponge. Typically 6 sponges of soap solution and 6 of water were used to wash each site, including the inner surface of the acetal O-rings, although contact of the first few sponges with these devices was avoided to prevent the transfer of test substance residues from the skin surface. Each application site was finally dried, using two pieces of dry sponge. For each rat, all sponges and washes were collected into a single container.
Following skin washing, each rat was killed by exsanguination under terminal anaesthesia and blood sample were taken heparinised tubes to separate plasma. The skin beneath each application site together with an annular ring of skin around each application site was excised and the O-rings detached and transferred into the same container as used for the O-ring caps for each rat. The skin beneath the O-rings was washed and dried with additional sponges to remove any residues and the sponges added to the other sponges for each rat.
Each application site was tape-stripped with successive pieces of adhesive tape to remove the stratum comeum. Tape stripping was continued until the stratum comeum was completely removed, as shown by non-adhesion of further tape strips to the viable epidermis underneath the stratum comeum. Tape strips from both application sites were collected in a single container for each rat.
The residual skin was retained in a single container for each rat.
Any urine present in the bladder was collected and added to the corresponding excreted sample.
The following whole tissues were removed:
- brain
- spleen
- liver
- kidneys
- pituitary
- thymus
- adrenal glands
- testes
- epididymis
- perirenal adipose tissue (fat)
Each residual carcass (with removed gastrointestinal tract including contents) was similarly retained for radioactivity analysis.

Following removed of rats and the collection of excreta, each metabolism cage was washed with circa 200 ml of ethanol:water (1:1 v/v) for further radioactive analysis

Results and discussion

Signs and symptoms of toxicity:

no effects

Absorption in different matrices:

High dose animals (2 mg [14C]-CGF-C1607/ animal)
Less than 0.01% of the applied dose was excreted in urine and 0.01% or less of the applied dose was found in any tissue at any time point.
Up to 24 hours, less than 0.05% of the dose was excreted in faeces, however at later time points small residues were noted in faeces due to animals chewing their site definition devices.
The concentrations of radioactivity in blood and plasma were below the limit of detection at all time points investigated.
The mean amount of dose absorbed (in urine, faeces, cage wash, tissues, G.I. tract with contents, residual carcass) remained approximately constant during the different timepoints (0.15-0.21% of the applied dose) when excluding animals showing oral ingestion of the test substance or incomplete skin wash.
Radioactivity present in the stratum corneum beneath the application sites amounted to means of 1.32%, 1.61%, 1.2% and 1.34% of the applied dose 6, 24, 72 and 120 hours, respectively.
In the remaining skin at the application site means of 0.68% 0.47%, 0.36%, 0.22% of the applied dose were found 6, 24, 72 and 120 hours, respectively.

The authors considered the radioactivity present in the skin beneath each application site as unabsorbed, although it was recognized, that some of this material may be absorbed beyond the duration of exposure investigated. While the fraction found in the stratum corneum would not be regarded as bioavailable, the content in the remaining skin was added as potentially bioavailable to the absorbed doses as a worst case approach. Accordingly, the mean percentages in the remaining application site skin was added to the means of the absorbed doses per timpoint and the bioavailable fraction of [14C]-CGF-C1607 is 0.8%, 0.7%, 0.5% and 0.4% of the applied dose 6, 24, 72 and 120 hours, respectively.

The mean amount of [14C]-CGF-C1607 in the skin wash after 6 hours ranged from 86 to 93% of the applied dose. A much lower additional mean fraction (0.8-2.5% of the applied dose) was removed by the terminal skin wash.
Residues on the O-rings and covers (mean 0.9 - 1.2% of the applied dose) were attributed to deposits made during the skin washing procedure. Bandage residues amounted to means of 0.03-0.5% of the dose.

Exclusion of data
Some results for 1 rat (termination time 72 hours after dosing) were substituted for calculation of means, due to oral ingestion by chewing its O-rings and grooming the exposed skin sites, apparent both a low recovery in the terminal skin wash (0.49% of the applied dose) and the high carcass residue (1.54% of the applied dose) due to distribution of radioactivity over the pelt. Consequently, increase in radioactivity in the 48-72 hour faecal collection, corresponding terminal cage wash and that remaining in the gastrointestinal tract was attributable to ingested and unabsorbed dose. Thus, the anomalous data for 48-72 hour faeces, cage wash, gastrointestinal tract plus contents and residual carcass have been substituted by the corresponding mean data of the 6, 24 hour and the remaining 72 hour group rats. This gives an estimated absorbed dose of 0.20%.
The results for 1 rat (termination time 120 hours after dosing) was excluded for calculation of means, because the 6 hour skin wash was shown to be incomplete (47.35% of the applied dose). However, this exclusion is considered not to affect the integrity of this study as all other rats produced consistent results.
For the remaining 3 rats of this group anomalous data for the 96-120 hour faecal collection, corresponding terminal cage wash and G.I. tract contents due to O-ring removal have been substituted as described above to give estimated dermally absorbed doses of 0.19%, 0.19% and 0.20% respectively.


Low dose animals (0.8 mg [14C]-CGF-C1607/ animal)
Less than 0.01% of the applied dose was excreted in urine and was found in any tissue at any time point.
Until 72 hours after application, up to 0.03% of the dose was excreted in faeces, however, at 120 hours small residues were noted in faeces due to animals chewing their site definition devices.
The concentrations of radioactivity in blood and plasma were below the limit of detection at all time points investigated.
The mean amount of dose absorbed (in urine, faeces, cage wash, tissues, G.I. tract with contents, residual carcass) remained approximately constant during the different timepoints (0.12-0.31% of the applied dose) when excluding animals showing oral ingestion or leakage of the test substance.
Radioactivity present in the stratum corneum beneath the application sites amounted to means of 1.62%, 1.57%, 1.11% and 0.87% of the applied dose 6, 24, 72 and 120 hours, respectively.
In the remaining skin at the application site means of 0.51%,0.48%, 0.32% and 0.52% of the applied dose were found 6, 24, 72 and 120 hours, respectively.

The authors considered the radioactivity present in the skin beneath each application site as unabsorbed, although it was recognized, that some of this material may be absorbed beyond the duration of exposure investigated. While the fraction found in the stratum corneum would not be regarded as bioavailable, the content in the remaining skin was added as potentially bioavailable to the absorbed doses as a worst case approach. Accordingly, the mean percentages in the remaining application site skin was added to the means of the absorbed doses per timpoint and the bioavailable fraction of [14C]-CGF-C1607 is 0.6%, 0.8%, 0.5% and 0.8% of the applied dose 6, 24, 72 and 120 hours, respectively.

The mean amount of [14C]-CGF-C1607 in the skin wash after 6 hours ranged from 94% to 98% of the applied dose. A much lower additional mean fraction (0.9-3.4% of the applied dose) was removed by the terminal skin wash.
Residues on the O-rings and covers (mean 0.4 – 0.6% of the applied dose) were attributed to deposits made during the skin washing procedure. Bandage residues amounted to means of 0.04-0.45% of the dose.

Exclusion of data
The results for 1 rat (termination time 6 hours after dosing) was excluded for calculation of means, because of incomplete skin wash (high recovery of radioactivity in the stratum corneum (5.8%), remaining skin (1.46%) and carcass (3.72%)) and some of the dose had leaked from the application sites. However, this exclusion is considered not to affect the integrity of this study as all other rats produced consistent results.
Some results for all rats at termination time 120 hours after dosing) were substituted for calculation of means, due to oral ingestion by chewing its O-rings following their detachment and grooming the exposed skin sites. Consequently, anomalous data for 48-72 hour faeces, 72-96 hour faeces, 96-120 hour faeces, terminal cage wash, G.I. tract with contents and residual carcass have been substituted by the corresponding mean data of all other low dose treated and non-excluded rats. This gives estimated absorbed doses of 0.24%, 0.24%, 0.23% and 0.26% for the affected animals, respectively.

Total recovery:

High dose animals (2 mg [14C]-CGF-C1607/ animal)
The overall recovery resulted in means ranging from 89.45% to 98.98% of the applied dose at the different timepoints investigated.

Low dose animals (0.8 mg [14C]-CGF-C1607/ animal)
The overall recovery resulted in means ranging from 96.87% to 103.48% of the applied dose at the different timepoints investigated.

Percutaneous absorption

Percutaneous absorption 1

Time point:

6 h

Dose:

2 mg/animal

Parameter:

percentage

Absorption:

0.8 %

Remarks on result:

other: based on urine + faeces + cage wash + tissues + G.I. tract with contents + residual carcass + residual skin at application site

Percutaneous absorption 2

Time point:

24 h

Dose:

2 mg/animal

Parameter:

percentage

Absorption:

0.7 %

Remarks on result:

other: based on urine + faeces + cage wash + tissues + G.I. tract with contents + residual carcass + residual skin at application site

Percutaneous absorption 3

Time point:

72 h

Dose:

2 mg/animal

Parameter:

percentage

Absorption:

0.5 %

Remarks on result:

other: based on urine + faeces + cage wash + tissues + G.I. tract with contents + residual carcass + residual skin at application site

Percutaneous absorption 4

Time point:

120 h

Dose:

2 mg/animal

Parameter:

percentage

Absorption:

0.4 %

Remarks on result:

other: based on urine + faeces + cage wash + tissues + G.I. tract with contents + residual carcass + residual skin at application site

Percutaneous absorption 5

Time point:

6 h

Dose:

0.8 mg/animal

Parameter:

percentage

Absorption:

0.6 %

Remarks on result:

other: based on urine + faeces + cage wash + tissues + G.I. tract with contents + residual carcass + residual skin at application site

Percutaneous absorption 6

Time point:

24 h

Dose:

0.8 mg/animal

Parameter:

percentage

Absorption:

0.8 %

Remarks on result:

other: based on urine + faeces + cage wash + tissues + G.I. tract with contents + residual carcass + residual skin at application site

Percutaneous absorption 7

Time point:

72 h

Dose:

0.8 mg/animal

Parameter:

percentage

Absorption:

0.5 %

Remarks on result:

other: based on urine + faeces + cage wash + tissues + G.I. tract with contents + residual carcass + residual skin at application site

Percutaneous absorption 8

Time point:

120 h

Dose:

0.8 mg/animal

Parameter:

percentage

Absorption:

0.8 %

Remarks on result:

other: based on urine + faeces + cage wash + tissues + G.I. tract with contents + residual carcass + residual skin at application site

Any other information on results incl. tables

Analytical results

Radiochemical purity was 98.4% (stock solution), 97.6% (high dose formulation) and 98.7% (low dose formulation). These small differences in the radiochemical purity were not considered to be

relevant.

The homogeneity in both dose preparations was shown to be satisfactory throughout the periods of dosing.

Stability analyses of [14C]-CGF-C1607 in both dose preparations demonstrated, that the test substance was stable beyond the periods of use in this study.

Applicant's summary and conclusion

Executive summary:

The dermal absorption of [14C]-CGF-C1607 in a 4% formulation was investigated for different dose volumes in male Wistar rats. A measured amount (54 µl or 22 µl) of each formulated dose was applied under semiocclusive conditions to 10 cm2 of shaved skin per rat corresponding to doses of 2 mg and 0.8 mg CGF-C 1607 per animal for 6 hours. After a 6 hour exposure, groups were either terminated or the application sites of the remaining rats were washed to remove the unabsorbed dose. Urine, faeces and cage wash were collected from each cage after the 6 hour skin wash, and then at daily intervals after dosing for the duration of each experiment. Groups of four rats were terminated at 6, 24, 72 and 120 hours after dosing. The skin was washed before exsanguination to remove any unabsorbed residual dose. The protective covers were removed, the skin under the O-rings washed to remove any unabsorbed residual dose and the application site skin was then tape-stripped to remove the stratum corneum. All samples, including selected tissues and residual carcasses were analysed for radioactivity.

 

Following dermal exposure to the high dose (2 mg [14C]-CGF-C1607), the majority of the applied radioactivity (86-93%) was washed from the skin surface using soap solution and water. Much lower fractions remained associated with the application site (i.e. 1.2%-1.6% in the stratum corneum and 0.2%-0.7% in the remaining skin). The residue associated with the application site declined slightly at later time-points. The concentrations of radioactivity in blood and plasma were below the limit of detection at all time points investigated. The mean amount of dose absorbed (in urine, faeces, cage wash, tissues, G.I. tract with contents, residual carcass) remained approximately constant during the different timepoints (0.15-0.21% of the applied dose) when excluding animals showing oral ingestion of the test substance or incomplete skin wash.

Following dermal exposure to the low dose (0.8 mg [14C]-CGF-C1607), similar results were obtained. The majority of the substance was washed from the skin surface (94% - 98%) and lower fractions remained associated with the stratum corneum (0.9%-1.6%) and the remaining skin (0.3%-0.5%) of the application site. Concentrations in blood and plasma were below the limit of detection and the mean amount of dose absorbed (in urine, faeces, cage wash, tissues, G.I. tract with contents, residual carcass) remained approximately constant (0.12-0.31% of the applied dose) when excluding animals showing oral ingestion or leakage of the test substance.

The authors considered the radioactivity present in the skin beneath each application site as unabsorbed, although it was recognized, that some of this material may be absorbed beyond the duration of exposure investigated. While the fraction found in the stratum corneum would not be regarded as bioavailable, the content in the remaining skin was added as potentially bioavailable to the absorbed doses as a worst case approach. Accordingly, the mean percentages in the remaining application site skin was added to the means of the absorbed doses per timepoint and the bioavailable fraction of [14C]-CGF-C1607 in the high dose is 0.8%, 0.7%, 0.5% and 0.4% of the applied dose 6, 24, 72 and 120 hours, respectively. For the low dose groups, the bioavailable fraction is 0.6%, 0.8%, 0.5% and 0.8% of the applied dose 6, 24, 72 and 120 hours, respectively.

 

In conclusion, following a 6 hour exposure period to 2 mg or 0.8 mg [14C]-CGF-C1607 applied as a 4% formulation, the absorption of the test substance was very low.