Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 1982 to May 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP study, oxidized form of the test item, acceptable, well-documented study report which meets basic scientific principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
Principles of method if other than guideline:
Method as requested by the National Institute of Environmental Health Sciences, USA
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): CI Vat Blue #1: Indigo
- Substance type: active substance
- Physical state: blue solid
- Analytical purity: essentially pure
- Lot/batch No. unlabeled: #A1A Eastman Kodak
- Purity test date: 19. October 1981
- Lot/batch No. labeled: 1555-105 New England Nuclear
- Radiochemical purity (if radiolabelling): ca. 70%
- Specific activity (if radiolabelling): 13.4 mCi/mmol
- Radioactivity: 5.88 mCi
- Locations of the label (if radiolabelling): [phenyl-14C(U)]-
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston, NY
- Age at study initiation: adult
- Housing: singly
- Individual metabolism cages: yes
- Diet: Purina Rat Chow ad libitum
- Water: furnished water ad libitum
- Acclimation period: at least 2 weeks

Administration / exposure

Route of administration:
other: intravenous, oral gavage, dermal
Vehicle:
other: IV: DMSO; PO: corn oil; dermal: ointment from PEG 1540+PEG 400+propylene glycol
Details on exposure:
Intravenous:
Solvent: DMSO
Concentration: 1 mg/mL
Preparation: 1.45 mL of solution mixed with 9 mL rat serum (7.7 µCi/g dosing solution)

Oral:
Activity adjusted 5-10 µCi per dose
Indigo dissolved in chloroform , chloroform evaporated, resulting solid homogeneously suspended in corn oil.
In addition, labelled indigo was cocrystalized with unlabelled indigo to increase purity to 91%; doses administered: 3.4 mg/kg (2.98 x 10E7 DPM/kg).

Dermal 1:
Vehicle: ointment
Composition: 9 g PEG 1540, 6 g PEG 400, 3 g propylene glycol
Dose level: 28 mg ointment + 2 mg labeled indigo (2.53 x 10E8 DPM)

Dermal 2:
applied as dry powder: 0.5 mg (6.3 x 10E7 DPM)
Duration and frequency of treatment / exposure:
single doses
Doses / concentrations
Remarks:
Doses / Concentrations:
Intravenous: 0.21 to 0.25 mg/kg bw in 0.5 mL
Oral: 3, 31, 305 mg/kg bw
3.4 mg/kg bw purified indigo
Dermal 1: 1 cm² ointment: 3.7 mg/kg bw
Dermal 2: 1 cm² dry powder: 1.5 mg/kg bw
No. of animals per sex per dose:
Inravenous: 3
Oral: 3
Dermal 1: 3
Dermal 2: 3
Control animals:
no
Positive control:
no
Details on study design:
Dose selection as requested by NIEHS
Details on dosing and sampling:
- Tissues and body fluids sampled: urine, faeces, CO2 in breath, blood (plasma and red blood cells), bile, cage washes
- Time and frequency of sampling: see Attachment
- Time of sacrifice:
- Intravenous: 0.25, 0.75, 2, 6, 24, 72, 240 hours
- Oral: 72 hours (low dose only) and 96 hours
- Dermal 1: 192 hours
- Dermal 2: 1 rat - 168 hours; 2 rats - 192 hours

Statistics:
Mean and standard deviation

Results and discussion

Preliminary studies:
NA

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Oral: 3 mg/kg: 58%
31 mg/kg: 34%
305 mg/kg: 33%
of 70% radiochemically pure indigo as urinary excretion decreased dramatically with increased purity, the majority might be absorption of impurities

Dermal: 1 - 2%
2 - 0.7%

Calculations were based on urinary excretion
Details on distribution in tissues:
Intravenous: Highest concentrations in liver, lungs and spleen at 0.25 hours. After 72 and 240 hours significant values only in liver and lungs
Oral: 3 mg - liver, kidney, mesenteric adipose, adrenals, large intestines
31 and 305 mg - liver, kidney, large intestines
no tissue contained >1% of the dose administered
no potential for accumulation of indigo or its metabolites observed

Dermal: skin from the back, kidneys, large intestines, fat and skin beneath administration site
except the later skin all tissue levels were below 0.01% of the dose administered
no signs for bioaccumulation were observed
Details on excretion:
See attached Tables 2 to 8
Major route of excretion: feces for IV and PO administration

Oral: 3 mg/kg bw: 88% feces, 11% urine in 4 days
87% feces, 11% urine in 72 hours
3.4 mg/kg purified: 78% feces, 4% urine in 72 hours
31 mg/kg bw:
305 mg/kg bw:

Dermal: 1 - 0.2% feces, 0.6% urine in 8 days
2 - 0.1% feces, 0.2% urine in 8 days

IV: only 0.08% of total urine excretion within 24 hours was parent indigo
44% feces, 26% urine, 0.5% exhalate in 3 days
43% feces, 35% urine in 10 days
25% bile in 7 hours

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
The majority of the radioactivity excreted were metabolites of indigo, however, no identification of metabolites was done

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Low to very low oral and dermal absorption of indigo, respectively. Excretion mainly via feces after intravenous and oral administration. Only about 2% of indigo was excreted unchanged..
No potential for bioaccumulation of indigo or its metabolites was observed.
Executive summary:

The toxicokinetic properties of Indigo has been investigated in male rats (Research Triangle Institute: Absorption,
Distribution, Metabolism and Excretion of CI Vat Blue#I; Indigo; RTI/2227/00-04P, September 1983). For this purpose, radioactive labeled (14C) Indigo (radiochemical purity ca. 70%) was administered as a single intravenous injection (0.23 mg/kg bw), a single oral gavage (3.0; 31; 305 mg/kg bw) and a single dermal application (1.5 and 3.7 mg/kg bw).

Intravenous injection: 25% of the administered radioactivity were detected in the bile within 7 hours after injection. 35% and 43% of the administered radioactivity were found in the urine and feces of the rats, respectively, within 240 hours after intravenous injection. Excretes via urine, bile and feces contained only 0.5% to 2% unchanged Indigo; 98% of the excreted radioactivity were metabolites.

Oral administration: 9% to 11% and 77% to 87% of the administered radioactivity were excreted with urine and feces, respectively, within 72 hours after gavage. Less than 1% were detected in the exhaled fraction.Comparison with IV data led to an oral bioavailability of approximately 33% to 58%.


After oral administration of the purified indigo 4% and 78% of the radioactivity were found in the urine and feces, respectively, after 72 hours. Taking into account the results with purified indigo, as well as after standardization of the IV data would lead to an oral absorption of about 20%.

The highest tissue levels after oral administration were found in liver and kidneys

Dermal administration: After dermal application of the ointment, 0.8% of the administered radioactivity were found in urine, feces and cage washes; after application of the dry powder, only 0.3% were found, leading to a very limited dermal absorption of indigo.

There were no hints for a possible bioaccumulation of indigo