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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Oct. 10, 1989 to Oct. 17, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Well documented study, followed guideline with deviations, GLP (3 animal/sex/group were used against the guideline recommendation of a minimum of 4 animals of one sex/group; exposure period of 30 min is less when compared to guideline recommendation of 6 or 24 hour exposure period)

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:
yes
Remarks:
(3 animal/sex/group were used against the guideline recommendation of a minimum of 4 animals of one sex/group; exposure period of 30 min is less when compared to guideline recommendation of 6 or 24 hour exposure period)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
RADIOLABELED TEST MATERIAL
- Name of test material: 6-chloro-4-nitro-2-aminophenol-UL-14C; Chlororange
- Substance type: Pure active substance
- Physical state: Yellow powder
- Specific activity: 13.7 mCi/mmol
- Locations of the label: Ring
- Storage: Store in the refrigerator at about 5°C.

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Wella AG ; n°Batch O99F9213
- Expiration date of the lot/batch: No data
- Purity test date: No data
- Purity : >98%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: >98%
- Specific activity: 13.7 mCi/mmol
- Locations of the label: Ring-14C
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the refrigerator at about 5°C (radioactive substance) - room temperature (inactive test substance)
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No
Radiolabelling:
yes
Remarks:
Ring-14C

Test animals

Species:
rat
Strain:
other: Sprague-Dawley, Him:OFA, SPF
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Forschungsinstitut für Versuchstierzucht, A-2325 Himberg, Austria.
- Age at study initiation: Not reported
- Weight at study initiation: Approximately 200 g
- Fasting period before study: None
- Housing: Individually in metabolism cages
- Individual metabolism cages: yes
- Diet: Altromin 1311ff diet for rats, ad libitum
- Water: Tap water in Makrolon bottles with stainless steel canules, ad libitum
- Acclimation period: 1 week
Random samples of the feed were analysed for contaminants by Altromin, D-4937 Lage.

ENVIRONMENTAL CONDITIONS
- Temperature: Average of 21°C
- Humidity: Average of 45%
- Air changes (per hr): 10/hour
- Photoperiod: Artificial light from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: Oct. 10, 1989 To: Oct. 17, 1989

Administration / exposure

Route of administration:
dermal
Vehicle:
other: Hair Dye Formulations I and II (Formulation II with and without Welloxon)
Details on exposure:
PREPARATION OF TEST SUBSTANCE FORMUALTIONS/SOLUTION: Two studies (Study A and B) were conducted using the radiolabeled test substance. In Study A and B, formulation 1 and 2 were used, respectively. In Study C, solution of test substance in water/DMSO was used. The details of test formulation or solution were as follows:
Formulation 1: Formulation without H2O2 containing 3% radiolabeled test substance.
Formulation 2: Formulation with H2O2 containing 3% radiolabeled test substance.
Solution of test substance: 9.99% solution of the radiolabeled test substance in water/DMSO (1:1).

TEST SITE
- Preparation of test site: Animals were shaved with electric clippers the day before treatment. Animals were anesthetized with Thiopental prior to treatment.
- Area of exposure: 9 sq cm
- Application of the solution: Formulations were applied by spreading evenly (until the skin was wetted) with a spatula and the solution was applied using a plastic syringe.

REMOVAL OF TEST SUBSTANCE
- Washing: The formulation or the solution was left for 30 min and was then scraped off using a spatula, followed by a rinse-off using first about 100 mL of a 3 % solution of a proprietary shampoo and then water. Rinsing was continued until the rinsing water and the absorbent cellulose tissue which was used to dab the skin dry were free of colour. After rinsing, the area was covered with gauze fixed by adhesive tape and an additional air permeable plastic cone to further prevent licking of the treated area during the 72 h in the metabolism cages.
- Time after start of exposure: 30 min

TEST MATERIAL
- Amount(s) applied: 30.0 mg/animal (study A); 29.3 mg/animal (study B); 30.3 mg/animal (study C)

USE OF RESTRAINERS FOR PREVENTING INGESTION: During the exposure time, animals were fixed to avoid licking.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Six samples of each of the formulations and of the solution of the test substance were drawn to check the homogeneity and also to determine the specific radioactivity. The latter was used to calculate the actual applied 14C-dose. Samples of the formulations and the solution were dissolved and diluted with Insta-Gel scintillator, Packard Instrument Co.
Duration and frequency of treatment / exposure:
Once, 30 min application
Doses / concentrationsopen allclose all
Dose / conc.:
2.28 other: mg/cm²
Remarks:
Study A
Doses / Concentrations:
- Mean mass of formulation or solution applied per animal: 1.0 g (Study A)
- Mean mass of the test substance applied per animal: 30.0 mg (Study A)
- Mean mass of the test substance applied per cm2: 2.28 mg (Study A)
- Mean specific activity of the formulations or the solution: 7.544 MBq/g (Study A)

Dose / conc.:
3.26 other: mg/cm²
Remarks:
Doses / Concentrations:
- Mean mass of formulation or solution applied per animal: 0.99 g (Study B)
- Mean mass of the test substance applied per animal: 29.3 mg (Study B)
- Mean mass of the test substance applied per cm2: 3.26 mg (Study B)
- Mean specific activity of the formulations or the solution: 5.285 MBq/g (Study B)
Dose / conc.:
3.37 other: mg/cm²
Remarks:
Doses / Concentrations:
- Mean mass of formulation or solution applied per animal: 0.303 g (Study C)
- Mean mass of the test substance applied per animal: 30.3 mg (Study C)
- Mean mass of the test substance applied per cm2:3.37 mg (Study C)
- Mean specific activity of the formulations or the solution: 13.673 MBq/g (Study C)
No. of animals per sex per dose:
3
Control animals:
no
Positive control:
None
Details on study design:
- Dose selection rationale: The doses were chosen considering practical conditions of hair dyeing in humans.
- Rationale for animal assignment: Animal numbers were allocated using a table of random numbers.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (absorption, distribution, excretion)
- Tissues and body fluids sampled: Rinsing water, treated skin, urine, faeces, organs, carcass.
- Time and frequency of sampling: Urine and faeces were collected daily (0-24, 24-48 and 48-72 h p.a.) from the metabolism cages. Water from the daily rinsing of the cages was combined with urine. Urine and faeces that were passed during the exposure period were recovered. Faeces were mixed with water and then homogenized.

SACRIFICE: The animals were killed 72 hours after the application by exsanguination in anesthesia (Thiopental).
RINSINGS: Application site washings (rinsings), consisted of scraped off formulations dissolved in methanol/water 1:1, solution of shampoo, rinsing water, methanol/water 1:1 extracts of the absorbent tissue used for drying the skin. These rinsing were filtered and 14C-content of filter and filtrate were determined separately and were then combined by calculation.

COLLECTION AND PROCESSING OF SAMPLES: The samples collected were processed prior to radioanalysis as follows:
- Application site: The dyed area plus some surrounding skin was dissolved in Soluene-350, Packard Instrument Co.
- Organs taken: adrenals, blood, brain, fat, femur, heart, kidneys, liver, lungs, muscle, ovaries, skin (untreated), spleen, testes and thyroids.
- Carcass: The skin of the animal was completely removed, to avoid false 14C-concentrations in the carcass due to skin. The residual carcass was homogenized together with the rest of the organs in an electric meat mincer by forcing them several times through the device.

DETERMINATION OF RADIOACTIVITY:
- Filters and weighed aliquots of faeces, organs and carcass were air dried and then combusted in a Sample Oxidizer 306, Packard Instrument Co.
- 14C-hexadecane standards of known radioactivity were combusted after each series of 15 - 30 samples to determine the recovery and to check the carry-over. This recovery was used to correct the sample activity.
- Liquid samples (urine, filtrate of rinsing, dissolved test substance) were incorporated into the liquid scintillator Insta-Gel (Packard Instrument Co.), dissolved skin from the injection site in Permafluor V (Packard Instrument Co.).
- Double determinations were performed of faeces, carcass, adrenals, ovaries and thyroids.
- Double or multiple determinations of other samples were performed if the result of the first determination required verification.

ANALYSIS OF SAMPLES: 14C was determined in a liquid scintillation counter (Tri-Carb 300CD, Packard Instrument Co.) with automatic external standardization and facilities to subtract background counting rates and to compute quench corrected decay rates.


Statistics:
T-test was used for analysis of differences between the sexes and the studies (p = 0.05).

Results and discussion

Main ADME resultsopen allclose all
Type:
absorption
Results:
Absorption through dermal route was low.
Type:
distribution
Results:
The test substance concentration was near or below the detection limits in all analyzed organs.
Type:
excretion
Results:
The test substance was mainly excreted by urine.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
- The mean absorption was low in studies A (0.248% or 5.6 μg/cm²) and B (0.189% or 3.52 μg/cm²), it was statistically significant higher in study C (1.213% or 40.0 μg/cm²) where the test substance solution was applied. The absorption was also transformed into absolute terms (i.e. microgram equivalents of the test substance per square cm).
- The percutaneous absorption at the site of application (mean 14C-content) was 0.38, 0.82 and 0.59% of the administered 14C-amount 3 days post-administration in studies A, B and C, respectively. The mean 14C-content of the skin in study B was statistically significantly higher than that found in study A.
- The majority of the applied 14C was removed from the skin by rinsing 30 min after the beginning of the cutaneous treatment (means of 95.4 to 98.7%).
Details on distribution in tissues:
- Organs: After cutaneous application all mean concentrations of 14C in the organs 72 hours post-dose were near or below the detection limits. In studies A to C relatively highest concentrations were noted in thyroids (below detection limits) and kidneys. Lowest concentrations were detected in lungs, brain and heart in study A and in testes, brain and muscle in studies B and C.
- Carcass: The remaining mean amounts of 14C in the carcass 3 days after cutaneous application were very low and below or near the detection limit in studiesA and B. In study C they were slightly above the detection limit. The mean percentage of the administered 14C-amounts were 0.0015, 0.0026 and 0.0059% for studies A, B and C, respectively, which translates to mean percent recoveries of the absorbed 14C-amounts of 0.6, 1.4 and 0.5%.
Details on excretion:
- After cutaneous application means of 0.247, 0.187 and 1.207% of the applied 14C were recovered in the urine and feces within 72 hours in studies A, B and C, respectively. 14C was excreted mainly via urine (means of 89-92% of the eliminated amounts; 88-92% of the absorbed amounts); to a lesser extent it was eliminated via feces (means of 8-11% of the eliminated amounts). The mean excretion was fast: 91, 89 and 93% of the totally eliminated amounts were excreted in the first 24 hours after application in studies A, B and C, respectively.

Any other information on results incl. tables

Total recovery: Means of 96.0 – 99.7% of the applied 14C-doses were recovered in the various types of samples in studies A, B and C. Sex differences: In study A the mean 14C-amount in the application site sum of urine, sum of feces and cutaneous absorption were all higher in males but without gaining significance statistically. The 14C-concentrations found in the organs were in most cases slightly higher in males than in females. In study C, the mean 14C-amount found in the application site, sum of urine, sum of feces, carcass and cutaneous absorption were higher in males gaining significance only in urine 48 - 72 h post dose. The mean 14C-concentrations found in thyroids and carcass were significantly higher in males than in females.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: no bioaccumulation potential based on study results
Dermal absorption of 6-chloro-4-nitro-2-aminophenol (Chlororange) after 30 min of exposure was low. When absorbed; excretion took place mainly via the urine. Radioactivity in all analysed organs was near or below the detection limit 72 hours post-application.

Executive summary:

The absorption, distribution and excretion of 6-chloro-4-nitro-2-aminophenol (Chlororange) after dermal administration was determined by following the OECD guideline 417 (Toxicokinetics).

Male and female Sprague-Dawley (Him:OFA, SPF) rats (from Forschungsinstitut für Versuchstierzucht, A-2325 Himberg, Austria) weighing approximately 200 g were used in study. Animals were housed individually in metabolism cages. Animals were maintained under standard laboratory conditions (temperature: 21°C, humidity: 45%; Air changes: 10 air changes/ h; photoperiod: Artificial light from 6.00 a.m. to 6.00 p.m. / d). 

Three studies (Studies A, B and C) were conducted using the radiolabeled test substance. In Studies A and B, formulations 1 and 2 were utilized, respectively. In Study C, a solution of test substance in water/DMSO was used. The details of test formulation/ solution were as follows:

  • Formulation 1: Formulation without H2O2 containing 3% radiolabeled test substance.
  • Formulation 2: Formulation with H2O2 containing 3% radiolabeled test substance.
  • Solution of test substance: 9.99% solution of the radiolabeled test substance in water/DMSO (1:1).

3 rats/sex/study were allocated using a table of random numbers. The amount of test substance applied per animal was approximately 30.0 mg in each study. The test formulation or solution was applied to the clipped dorsal skin of rats for 30 min.

After treatment, the test substance was scraped and washed off and the skin rinsed with a shampoo. During the exposure time, animals were fixed to avoid licking. After rinsing, the area was covered with gauze fixed by adhesive tape and an additional air permeable plastic cone to further prevent licking of the treated area during the 72 h in the metabolism cages.

Urine and faeces were collected daily (0-24, 24-48 and 48-72 h p.a.) from the metabolism cages. Water from the daily rinsing of the cages was combined with urine. Faeces were mixed with water and then homogenized.

The animals were killed 72 hours after the application by exsanguination in anesthesia (Thiopental).

14C-radioactivity was determined in a liquid scintillation counter (Tri-Carb 300CD, Packard Instrument Co.) with automatic external standardization and protocols to subtract background counting rates and to compute quench corrected decay rates.

Following dermal application, the majority of the dyestuff was removed by rinsing 30 min after application, i.e., 95.4 to 98.7 % of the applied dose was found in the washing water. Seventy two hours after application, the amount of radioactivity remaining at the application site (skin) was less than 1 % for all three studies (A, B and C). The highest value (0.82 % of the applied dose) was noted for the formulation with H2O2 (Study B). For the formulation without H2O2 (Study A) and for the solution (DMSO/water; Study C), the figures were 0.38 and 0.59 %, respectively. The lowest absorption rate was obtained in the formulation containing H2O2

The proportion of the applied dose that was absorbed was calculated by adding the amounts in urine, faeces and carcass:

  • A.Formulation without H2O2: 0.248 % (equal to 5.6 μg/cm²),
  • B. Formulation with H2O2: 0.108 % (equal to 3.52 μg/cm²), and
  • C.Pure dyestuff in water/DMSO: 1.213 % (equal to 40.0 μg/cm²).

After cutaneous application, all mean concentrations of 14C in the organs 72 hours post-dose were near or below the detection limits. In studies A to C the highest relative concentrations were noted in the thyroid (below detection limits) and kidney. The lowest concentrations were detected in lungs, brain and heart in study A and in testes, brain and muscle in studies B and C.

Means of 96.0 – 99.7% of the applied 14C-doses were recovered in studies A, B and C.

Based on above, dermal absorption of 6-chloro-4-nitro-2-aminophenol (Chlororange) after 30 min of exposure was low. When absorbed; excretion took place mainly via the urine. Radioactivity in all analysed organs was near or below the detection limit 72 hours post-application.

This toxicokinetics test is classified as acceptable, and satisfies the guideline requirements of the OECD 417 method.