Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed methods similar to guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Objective of study:
absorption
distribution
excretion
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
UNLABELED TEST MATERIAL
- Name of test material: 2,2'-[(4-aminophenyl)imino]bisethanol sulfate (APE) (A050)
- TSIN: CFQ 5209
- Substance type: Pure active substance
- Solubility: Test substance is soluble in water

RADIOLABELED TEST MATERIAL
- Name of test material: 14C-2,2'-[(4-aminophenyl)imino]bisethanol sulfate (APE)
- TSIN: CFQ 5209
- Substance type: Pure active substance
- Specific activity: 435 MBq/mmol (11.8 mCi/mmol)
- Locations of the label: Ring

No other information on details of test material (unlabelled and radiolabelled) was provided in the study report.
Radiolabelling:
yes
Remarks:
(14C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: From Charles River, Japan
- Age at study initiation: Six week old
- Weight at study initiation: The body weight of animals was 196±11.3 g
- Fasting period before study: Not reported
- Housing: Not reported
- Individual metabolism cages: Yes
- Diet: Chow (from Crea Japan, CE-2), ad libitum
- Water: Water, ad libitum
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS: No details on environmental conditions were provided in the study report.

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:
dermal
Vehicle:
other: Water, 0.56% ammonia solution and distilled water for formulations 1, 2 and 3, respectively
Details on exposure:
PREPARATION OF TEST SUBSTANCE FORMUALTIONS:
Formulation 1: Unlabelled test substance was added to the 14C-test substance. The 4.8% of 14C test substance was added to the water.
Formulation 2: Unlabelled test substance was added to the 14C-test substance. The 4.8% of 14C test substance was added to the 0.56% ammonia solution.
Formulation 3: Formulation was prepared by mixing dyes 1 and 2 in equal amount. Hair dye 1 was prepared by mixing 4.8% of 14C-test substance, 25.2% distilled water and 70.0% excipients. The dye 2 included Carencia cream oxide C (oxidant). Formulation was prepared just before application.

TEST SITE
- Preparation of test site: Animals were shaved with electric clippers the day before treatment. Animals were anesthetized with Pentobarbital prior to treatment.
- Area of exposure: 2.5 sq cm
- % coverage: 2%
- Type of wrap if used: Treated sites were covered with polyethylene sheets (3 x 3.5 cm) secured with adhesive elastic bandages (Tokyo Eizai Research Center, Silk Tex)
- Time intervals for shavings or clipplings: Animals were shaved on the day before treatment, with electric clippers.

REMOVAL OF TEST SUBSTANCE
- Washing: In case of formulation 3, test site was wiped clean with water-soaked cotton swabs; the cleaning process was repeated five times.
- Time after start of exposure: 30 min

TEST MATERIAL
- Amount applied: 0.2 g/rat (0.74 MBq (20 µCi)/rat)
- Concentration: 4.8% in formulations 1, 2 and 2.4% in formulation 3

VEHICLE: The vehicle used for different formulations was as follows:
Formulation 1: Water
Formulation 2: 0.56% ammonia solution
Formulation 3: Distilled water

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not reported
Duration and frequency of treatment / exposure:
Once, removal of formulation 1 and 2 was not reported. However, formulation 3 was removed after 30 min of application
Doses / concentrations
Remarks:
Doses / Concentrations:
Group 1 and 2: 4.8%
Group 3: 2.4%
No. of animals per sex per dose:
4 animals/group
Control animals:
no
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment: Not reported

TREATMENT: The treatment schedule was as follow:
Group I (formulation 1): Treated with formulation containing 4.8% test substance in aqueous solution
Group II (formulation 2): Treated with formulation containing 4.8% of test substance in 0.56% ammonia solution
Group III (formulation 3): Treated with formulation containing 2.4% test substance (Mixture of dye 1 and 2). Removed after 30 min of treatment.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, feces, blood, expired air, cage washes, treated skin site ( skin area 2-3 cm in circumference) and tissues
- Time and frequency of sampling: Urine, faeces and expired air were collected at 24, 48, 72 and 96 h after treatment. The blood samples were collected at 0.5, 1, 2, 3, 4, 5, 6, 24 and 48 h after treatment. The rinse water for funnel of metabolic cages was collected together with urine. Tissues were collected after sacrifice of animals (96 h after treatment). The details on collection of samples are provided in the study report.

SACRIFICE: 96 h after application, the pentobarbital-anesthetized rats were killed by bleeding from the posterior veins.

COLLECTION AND STORAGE OF TISSUES: The various tissues were collected and the respective weights determined. Part or all of the samples were collected and dried at room temperature, and the samples for radioactivity determination were prepared with an automatic sample combustion device.

PREPARATION OF SAMPLES FOR DETERMINATION OF RADIOACTIVITY:
- Urine: 10 mL of Aquasol-2 was added to 1-2 mL of urine and used as the sample for radioactivity determination.
- Feces: Water was added to the total amount, which was homogenized. Part of the homogenate was weighed out and dried at room temperature to prepare the sample for radioactivity determination using an automatic sample combustion device (Aroka ASC-113).
- Expired air (CO2) collection liquid: Approx. 1 g of the liquid was weighed out, and 5 mL of methyl alcohol and 10 mL of Aquasol-2 were added to prepare the sample for radioactivity determination.
- Treated (painted) skin areas and the carcasses: 50-100 mL of a solution of 10% sodium hydroxide and 60% ethyl alcohol was added, and the samples were dissolved by refluxing until the solids disappeared. Part of the solution was collected, dried at room temperature and treated in an automatic sample combustion device to prepare a sample for radioactivity determination.
- The stomach, small intestine and large intestine were collected together with their contents, and a 10% sodium hydroxide solution was added to the whole amount, which was then homogenized. The samples for radioactivity determination were prepared in the same way as the feces. For Formulation 3, water was added to the rinse water of the painted regions to adjust the volume, and the sample for radioactivity determination was prepared in the same way as the urine.

ANALYSIS OF SAMPLES: The radioactivity of the samples was determined with a whole-body scintillation system (Aroka, LSC-3500). Counting efficiency was corrected by a channel comparison method using an external standard radiation source.

Results and discussion

Main ADME resultsopen allclose all
Type:
absorption
Results:
The absorption of test substance was very low in Group 2. No radioactivity was determined in blood of Group 1 and 3 animals.
Type:
distribution
Results:
After absorption, test substance was deposited mainly in bladder, kidneys and liver.
Type:
excretion
Results:
Test substance excreted through both urine and feces.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
- The blood radioactivity levels after application of test substance in formulations 1 and 3 were below the detection limits at all determination times.
- In Group II animals, the blood levels after application increased, reaching the highest values (0.28 Eq µg/mL) 2 h after application. The blood serum radioactivity levels were over 70% of the maximum level 1 h after application and reached the maximum 2 h after application. 6 h after treatment, almost no radioactivity was detected in the blood.The half-life was 2.2±0.7 h (mean ± standard deviation).
- The radioactivity determined in blood of Group II animals at 1, 2, 3, 4 and 5 h after treatment was 0.20±0.09, 0.28±0.13, 0.23±0.09, 0.15±0.06 and 0.12±0.04 µg Eq/mL of test substance, respectively.
Details on distribution in tissues:
- Group I (Formulation 1): After treatment, the tissue radioactivity reached the maximum value 4 h after application, then decreased. The tissues with relatively high radioactivity levels 2 and 4 h after application were the bladder, kidneys and liver. 24 h after treatment, no radioactivity was detected in any tissues other than the kidneys and liver. The brain, hypophysis, thyroid and fat showed relatively low radioactivity levels at all times as compared to the other tissues. In terms of radioactivity distribution, the sites of highest concentration 2 and 4 h after application were the muscle, small intestine and skin, but the radioactivity decreased in these tissues 24 h after application, and almost none was detected in tissues other than the large intestine and liver.
- Group II (Formulation 2): The tissue radioactivity levels were low on the whole, but as observed over time, remained their maximum values 2 h after application, diminishing thereafter. 2 h after treatment, the tissues with the highest radioactivity levels as compared to the other tissues were the bladder, kidneys and liver, but all of the tissues were seen to decrease in radioactivity level 4 h after application. 24 h after Treatment, the levels dropped still further, and at 96 h, almost no radioactivity was observed outside the kidney and liver. No radioactivity was detected at any of the test times in the brain, hypophysis, thyroid, adrenals or fat. The radioactivity levels were relatively high in the muscle and small intestine 4 h after application and in the large intestine 24 h after application, but except for small amounts of radioactivity detected in the large intestine and liver, no radioactivity was found to persist in any of the tissues 96 h after application.
- Group III (Formulation 3):The tissue radioactivity levels after application of formulation 3 were lower than for formulations 1 and2. However, 2 h after application, radioactivity was detected in the liver, kidneys and bladder. 4 h after application, the radioactivity levels were extremely reduced even in the tissues in which radioactivity was detected 2 h after application. Except for low levels detected in the small and large intestines, the radioactivity distributions revealed almost no radioactivity detected in the various tissues at any of the test times.

The details on result of distribution of test substance are reported in the study report.
Details on excretion:
- 96 h after application of test substance in formulation 1, 5.6% of the radioactivity dose was excreted with the urine and 0.9% with the feces. The total clearance 96 h after treatment was 6.5%, of which over 85% was secreted 24 h after application. 96 h after application, 0.14% of the radioactivity dose persisted in the carcass and 91.4% in the painted skin areas.
- After treatment with formulation 2, the main clearance route of the radioactivity was the urine; 4.0% of the radioactivity dose was excreted with the urine 96 h after application. 1.0% was excreted in the feces and 0.2% in the expired air 96 h after application. 0.14% of the radioactivity dose persisted in the carcass and 92.2 % in the painted skin area 96 h after application.
- After treatment with formulation 3, only 0.03% of the radioactivity dose was excreted with the urine 24 h after application, and beyond 24 h, no radioactivity was detected in the urine or feces. 96 h after application, no radioactivity was detected even in the carcasses, while the painted skin areas and rinse water accounted for 4.4% and 88.4% of the radioactivity dose.
- The % cumulative radioactivity (urine/feces) excreted for Group I animals at 24, 48, 72 and 96 h after treatment was 5.57±3.02 (4.95±2.74/0.62±0.29), 5.91±3.14 (5.13±2.79/0.77±0.37), 6.16±3.20 (5.32±2.83/0.84±0.38) and 6.50±3.26 (5.56±2.86/0.94±0.41) % of test substance applied, respectively.
- The % cumulative radioactivity (urine/feces/expired air) excreted for Group II animals at 24, 48, 72 and 96 h after treatment was 4.53±1.50 (3.75±1.23/0.66±0.26/0.12±0.02), 4.89±1.52 (3.84±1.23/0.88±0.28/0.17±0.02), 5.08±1.49 (3.91±1.21/0.97±0.27/0.20±0.02) and 5.23±1.48 (3.96±1.21/1.04±0.26/0.23±0.02) % of test substance applied, respectively.
- The amount of total radioactivity (urine) excreted for Group III animals at 24 h after treatment was 0.03±0.01. No radioactivity was observed thereafter.

Any other information on results incl. tables

Total Recovery: The % total recovery of test substance in Group I, II and Group III (formulation 1, 2 and 3) was 98.00±3.6%, 97.35±3.74% and 92.84±4.67%, respectively.

Autoradiography: 2 h after application of formulation 2, the high concentration of radioactivity in the painted skin region was evident. The distribution of the radioactivity in the areas outside the painted skin regions was extremely low. Some distribution of the radioactivity in the contents of the small intestine and in the kidneys was observed. In the case of formulation 3 as well, intensive radioactivity was concentrated in the painted skin region. Extremely little radioactivity was distributed outside the painted region but some was detected in the bladder.

Applicant's summary and conclusion

Conclusions:
Interpretation of results : no bioaccumulation potential based on study results
Absorption of 2,2'-[(4-aminophenyl)imino]bisethanol sulfate (A050) in Groups I, II and III to rats was very low when administered once dermally. 2,2'-[(4-aminophenyl)imino]bisethanol sulfate (A050) (in Groups I, II and III) was distributed into liver, kidney and bladder and excreted via the urine and feces after absorption.
Executive summary:

The absorption, distribution and excretion of 2,2'-[(4-aminophenyl)imino]bisethanol sulfate (A050) after dermal administration was determined by following the methods similar to the OECD guideline 417 (Toxicokinetics).

Six week old male Sprague Dawley rats (from Charles River, Japan) were used in study. The body weight of animals at study initiation was 196±11.3 g. After treatment animals were housed individually in metabolic cages. 4 animals were included in each treatment group. Each animal received a dose of 0.2 g (0.74 MBq (20 µCi) test substance, on the shaved skin. Water, 0.56% ammonia solution and distilled water was used as vehicle for preparation of formulation 1, 2 and 3, respectively. The treatment schedule was as follows:

Group I (formulation 1): Treated with formulation containing 4.8% test substance in aqueous solution

Group II (formulation 2): Treated with formulation containing 4.8% of test substance in 0.56% ammonia solution

Group III (formulation 3): Treated with formulation containing 2.4% test substance (Mixture of dye 1 and 2). Removed after 30 min of application.

Urine, faeces, blood, CO2 (expired air) and cage wash samples were collected at different time points during the study. Animals anesthetized with pentobarbital were sacrificed 96 h after treatment. After sacrifice, treated skin site (skin area 2-3 cm in circumference) and tissues samples were collected.

The blood radioactivity levels after application of test substance in formulations 1 and 3 were below the detection limits at all determination times. In Group II animals, the blood levels after application increased, reaching the highest values (0.28 Eq µg/mL) 2 h after application. The blood serum radioactivity levels were over 70% of the maximum level 1 h after application and reached the maximum 2 h after application. 6 h after treatment, almost no radioactivity was detected in the blood. The half-life was 2.2±0.7 h (mean ± standard deviation).

In Group I animals, the tissue radioactivity reached the maximum value 4 h after application, then decreased. The tissues with relatively high radioactivity levels 2 and 4 h after application were the bladder, kidneys and liver. 24 h after application, no radioactivity was detected in any tissues other than the kidneys and liver.

In Group 2, the tissue radioactivity levels were low overall, but as observed over time, remained their maximum values 2 h after application, diminishing thereafter. 2 h after treatment, the tissues with the highest radioactivity levels as compared to the other tissues were the bladder, kidneys and liver, but all of the tissues were seen to decrease in radioactivity level 4 h after application.

The tissue radioactivity levels after application of formulation 3 were lower than for formulations 1 and 2. However, 2 h after application, radioactivity was detected in the liver, kidneys and bladder. 4 h after application, the radioactivity levels were extremely reduced even in the tissues in which radioactivity was detected 2 h after application.

96 h after application of test substance in formulation 1, 5.6% of the radioactivity dose was excreted with the urine and 0.9% with the feces. The total clearance 96 h after application was 6.5%, of which over 85% was secreted 24 h after application. 96 h after application, 0.14% of the radioactivity dose persisted in the carcass and 91.4% in the painted skin areas.

After application of formulation 2, the main clearance route of the radioactivity was the urine; 4.0% of the radioactivity dose was excreted with the urine 96 h after application. 1.0% was excreted in the feces and 0.2% in the expired air 96 h after application. 0.14% of the radioactivity dose persisted in the carcass and 92.2% in the painted skin area 96 h after application.

When Formulation 3 was administered, only 0.03% of the radioactivity dose was excreted with the urine 24 h after application, and beyond 24 h, no radioactivity was detected in the urine or feces. 96 h after application, no radioactivity was detected even in the carcasses, while the painted skin areas and rinse water accounted for 4.4% and 88.4% of the radioactivity dose.

The % total recovery of test substance in Group I, II and Group III (formulation 1, 2 and 3) was 98.00±3.6%, 97.35±3.74% and 92.84±4.67% after treatment, respectively.

In the autoradiography experiment, 2 h after application of formulation 2, the high concentration of radioactivity in the painted skin region was evident. The distribution of the radioactivity in the areas outside the painted skin regions was extremely low. Some distribution of the radioactivity in the contents of the small intestine and in the kidneys was observed. In the case of formulation 3 as well, radioactivity was concentrated in the painted skin region. Extremely little radioactivity was distributed outside the painted region but some was detected in the bladder.

Based on above, absorption of 2,2'-[(4-aminophenyl)imino]bisethanol sulfate (A050) in Groups I, II and III to rats was very low when administered once dermally. 2,2'-[(4-aminophenyl)imino]bisethanol sulfate (A050) (in Groups I, II and III) was distributed into liver, kidney and bladder and excreted via the urine and feces after absorption.