Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study. The validity score is reduced to 2 as indicated by ECHA guidance for read-across studies.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Objective of study:
absorption
distribution
excretion
Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, 40 CFR, Part 158, Subdivision F, Section 85-1, October 1982
Qualifier:
according to
Guideline:
other: Directive 87/302 EEC, Part B, No. L133/51 "Toxicokinetik", May 1988.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Substance type: pigment
- Physical state: solid, red powder
- Analytical purity: about 99.7 %
- Lot/batch No.: MS 70037.62
- Expiration date of the lot/batch: December, 1995
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Radiochemical purity (if radiolabelling): About 99.8 %; could only be indirectly determined due to the insolubility of the compound. After extraction of the pigment with water/methanol (1:1) at room temperature for 1h, a colorless liquid containing 0,197 % of the radioactive dose was obtained.

- Specific activity (if radiolabelling): 64 mCi/mmol / 2.37 GBq/mmol / 178.3 µCi/mg
- Storage condition of radiochemical substance (if radiolabelling): At -20 °C, in the dark
- Storage condition of test material: At 4 °C, 20 °C allowed;
- Stability under storage conditions: Stable for at least 5 years
- Stability in Vehicle: Stable for at least 48 hours in water, polyethyleneglycol and carboxymethylcellulose
Radiolabelling:
yes
Remarks:
14C-labelled

Test animals

Species:
rat
Strain:
other: BRL-HAN, Wistar, outbred
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Biological Research Laboratories Ltd., Fuellinsdorf/Switzerland
- Age at study initiation: 7 weeks
- Weight at study initiation: One day prior to the treatment with 14C-IRGAZIN DPP RED 80, body weights ranged from 181-191 g for groups 1 and 2 and from 156-185 g for groups 3 and 4
- Fasting period before study: Prior to dosing with 14C-IRGAZIN DPP RED 80, rats were fasted overnight.
- Housing: Groups of 3 rats
- Individual metabolism cages: yes/no
- Diet (e.g. ad libitum): Pelleted 343-Kliba rat maintenance diet, ad libitum (KLIBA Klingentalmuehle AG, Kaiseraugst/Switzerland).
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: Seven days to laboratory environment, including 1 day to metabolism cages.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 25.0 °C
- Humidity (%): 36.9 - 70.0 %
- Air changes (per hr): 10-15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5 % CMC in 0.9 % NaCl
Details on exposure:
PRE-TESTS
- Solubility: Based on the high target dose level of 1000 mg/kg, about 100 mg unlabelled test article were tested for solubility. After ultrasonic treatment, the amount was not soluble in 300 µl polyethylene glycol (PEG 400), but could be suspended in 1000 µl acetone in the presence of 200 mg CREMOPHOR EL (BASF, Ludwigshafen/Germany).

PREPARATION OF DOSING SOLUTIONS:
- Formulation: The acetone used to suspend the test article was evaporated at room temperature under a gentle stream of nitrogen. The residue
suspended in CREMOPHOR was mixed with 300 µl PEG 400 and 500 µl 0.5 % CMC (carboxymethyl-cellulose) in 0.9 % NaCl. A homogeneous suspension was obtained.
In conclusion, 100 mg test article per 100 g rat body weight, first suspended in 1000 µl acetone in the presence of 200 mg CREMOPHOR, could be efficiently formulated in 300 µl PEG 400 and 500 µl 0,5 % CMC in 0.9 % NaCl after evaporation of the acetone under a gentle stream of nitrogen.
Consequently, the low dose level (10 mg test article/100 g rat body weight) was formulated similarly.
- Due to lack of solubility of the test article, actual amounts of 14C-IRGAZIN DPP RED 80 present in the various stock suspertsions and suspensions for application were only indirectly determined by means of weighing.
Group 1:
A weighed amount of 8.035 g stock suspension I corresponding to 150.27 mg 14C-IRGAZIN DPP RED BO at a specific radioactivity of 4.13 yCi/mg was quantitatively transferred into a glass-beaker, previously weighed. After addition of 7.5 ml 0.5 % CMC in 0.9 % NaCl, the gross weight of the resulting
suspension for application (suspension A) was determined. By subtraction of the glass-beaker weight, the net weight (15.527 g) of suspension A was determined. Based on the intended dose level of 100 mg/kg, an average rat body weight of 185 g, an excess of about 5 % and a target application volume of 1.0 ml/100 g rat, weighed aliquots of 2.0 ml (Specific weight of all suspensions for application assumed to be 1.0 g/ml) suspension A were separated and thereafter directly administered.
Group 3:
In a similar way, a weighed amount of 7.697 g stock suspension I corresponding to 143.95 mg 14C-IRGAZIN DPP RED BO was transferred into a weighed glass-beaker and mixed with 7.2 ml 0.5 % CMC in 0.9 % NaCl. The net weight of the resulting suspension for application (suspension B) was 14.726 g.
Based on the intended dose level of 100 mg/kg, an average rat body weight of 160 g for the animals, an excess of about 10 % and a target
application volume of 1.0 ml per 100 g rat body weight, weighed aliquots of 1,8 ml * suspension 8 were separated and thereafter directly administered. A weighed aliquot of 2.0 ml * suspension B was administered to one animal with 185 g body weight.
Group 2:
A weighed amount of 11.884 g stock suspension II corresponding to 1619.67 mg 14C- IRGAZIN DPP RED BO at a specific radioactivity of 0.41 yCi/mg was transferred into a previously weighed glass-beaker and mixed with 8.1 ml 0.5 % CMC in 0.9 % NaCl. The net weight of the resulting suspension
for application (suspension C) was calculated to be 20.367 g. Based on the intended dose level of 1000 mg/kg, an average rat body weight of
185 g, an excess of about 7 % and a target application volume of 1.0 ml/100 g rat, weighed aliquots of 2.5 ml suspension C were separated and thereafter directly administered.
Group 4:
A weighed amount of 11.485 g stock suspension II (i.e. 1565.29 mg 14C-IRGAZIN DPP RED BO at a specific radioactivity of 0.41 yCi/mg) was transferred into a previously weighed glass-beaker and 7.8 ml 0.5 % CMC in 0.9 % NaCl added. The net weight of the resulting suspension for application (suspension 0) was 19.174 g.
Based on the intended dose level of 1000 mg/kg, an average rat body weight of 169 g, an excess of about 6 % and a target application volume of 1.0 ml/100 g rat, weighed aliquots of 2.2 ml suspension D were separated and thereafter directly administered.

TREATMENT:
- Administration volume: 1.0 ml/100 g of body weight
- Accommodation: All-glass metabolism cages, 1 day prior to the treatment and during the experiment.
The metabolism cages were ventilated with about 600 ml air per minute.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
- Stability of the 14C-Labelled Test Article in the Formulation: 14C-IRGAZIN DPP RED 80 was proved to be sufficiently stable in the formulation.
Duration and frequency of treatment / exposure:
single treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg/kg and 1000 mg/kg
No. of animals per sex per dose:
20 total
Details on study design:
- Dose selection rationale:
Low dose: 100 mg/kg of body weight (spec, radioactivity: 4.0 µCi/mg or 0.15 MBq/mg)
High dose: 1000 mg/kg of body weight (spec, radioactivity: 0.4 µCi/mg or 0.015 MBq/mg)

- Rationale for animal assignment (if not random): Since the study was performed in various separate experiments, animal numbers were given randomly at each batch.

- Duration of recovery: 168 hours
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum or other tissues, cage washes, bile
- Time and frequency of sampling: followed for 168 hours
- Other: No volatiles (expired air) were collected.
- The levels of radioactivity appearing in urine, feces, blood/plasma and organs/tissues were determined.

SAMPLING
- Urine: Urine was collected into dry-ice/ethanol cooled tubes. It was taken 8, 24, 48, 72, 96, 120, 144 and 168 hours after the administration. Additionally, at the sampling intervals of 8 and 24 hours, metabolism cages were rinsed with a small amount of bidistilled water (about 2-5 ml) to remove remaining droplets of urine which were added to the initially sampled urine. Final volumes were noted.
- Feces: Feces was collected at room temperature 24, 48, 72, 96, 120, 144 and 168 hours after the administration. Fresh weights were noted.
- Blood: After 168 hours, animals were killed by carbon dioxide. Blood was collected in the thoracic cavity after heart incision and sampled into heparinized tubes. Aliquots (0.5 - 1.0 ml) of blood were separated and thereafter remaining blood centrifuged and the plasma decanted.
- Organs/Tissues/Intestinal Tract/Carcass: The following organs and tissues were taken after 168 hours: Heart, lung, liver, stomach (contents were separately determined), spleen, adrenal glands, kidneys, testicles, epididymes, muscle, bones (femur), brain, skin (back region), skin of hind paws, fat, thyroid gland and pancreas.
In addition, residual carcass and intestinal tract with contents were taken.
Weights of rats and all specimens mentioned were noted.
- Cage Wash: At the end of the study, the cages were washed with water/acetone (1:1, v/v). The washing solutions were then used for determination of remaining radioactivity.
- Blood: Blood (100-200 microlitres) was withdrawn retroorbitally from the individual rats at 0 (prior to dosing), 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144 and 168 hours after the administration. Blood was collected into heparinized tubes. An aliquot was immediately used for analysis. The rest of the blood was centrifuged, the plasma decanted and stored until analysis at -20 °C.

ANALYSES and SAMPLE PREPARATION
- Urine: The radioactivity in urine was determined by liquid scintillation counting of subsamples (duplicates).
- Feces: Feces was lyophilized, homogenized and subsamples combusted for determination of radioactivity.
- Intestinal Tract/Carcass/Bones: The intestinal tract (with contents), carcass and contents of the stomach were homogenized after limited storage at -20 °C. Thereafter, subsamples were combusted for determination of radioactivity. Bones were directly combusted.
- Organs/Tissues/Blood/Plasma: The radioactivity in organs/tissues and blood was determined after digestion of subsamples with tissue solubilizer. Plasma samples were directly measured.
- Cage Wash: Solid materials (feed, feces, etc.) were homogenized. The radioactivity in the cage wash was determined by combustion of subsamples.
- Blood/Plasma: The radioactivity in blood was determined by digestion of subsamples followed by liquid scintillation counting. Plasma samples were directly measured.

ANALYTICAL METHODS
- Measurement of Radioactivity: liquid scintillation counting. Except for organs/tissues, blood and plasma, all measurements were corrected for the respective scintillation background.

CALCULATIONS
- Levels of Radioactivity in Urine, Feces, Organs/Tissues, Blood, Intestinal Tract, Residual Carcass and Cage Wash:
- All levels were expressed in percentage of the radioactivity administered to the rats after scintillation background correction (urine, feces and cage wash) and sample background correction obtained from untreated rats (organs/tissues, blood/plasma, intestinal tract and residual carcass).
- Additionally, radioactivity in blood/plasma, organs/tissues, intestinal tract and residual carcass was expressed in microgram (yg) parent equivalents per g of sample weight without instrumental background correction.
- Correlation to Background Levels: To be able to compare these values to the limits of quantitation obtained from untreated rats (RCC Project 287627), all dpm-values per sample were normalized to 100 mg. Only for those organs which had total weights below 100 mg (adrenal glands and thyroid gland), the dpm-value per sample was assumed to be identical to a 100 mg sample in order to avoid artificially high background values.
- The limit of quantitation (LOQ) was defined as double background levels of blood, plasma, organs/tissues, intestinal tract and residual carcass determined in untreated control animals. The LOQ's were expressed in µg parent equivalents per g tissue on the basis of the various specific radioactivities used.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, blood/plasma and organs/tissues, cage washes, bile
- Time and frequency of sampling: followed for 168 hours
- From how many animals: (samples pooled or not)
- Method type(s) for identification:
Liquid scintillation counting, NMR, TLC)
- Limits of detection and quantification:
- Other:

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):

Results and discussion

Main ADME resultsopen allclose all
Type:
absorption
Results:
14C-pigment in organs and serum were below the limit of quantitation (< 0.05%). For the high dose, 0.5% of the dose was detected in urine which is considered to originate from feces contamination.
Type:
distribution
Results:
Radioactivity in organs were below the limit of quantitation (< 0.05%).
Type:
excretion
Results:
Excretion via the feces within 24h

Toxicokinetic / pharmacokinetic studies

Details on absorption:
For both dose groups, absorption is neglible. Plasma concentrations are below the limit of quantification with a few exceptions around the 2h time point for which values are at less than 1.5 fold of the limit of quanitation. Considering that the test material contained 0.2% of an extractable, non coloured impurity, these plasma values are not indicative of uptake of the pigment.

Although in selective animals red colored extremities due to contamination with feces were observed the first two days after administration, also negligible radioactivity levels were detected after 168 hours in the skin of hind-paws, indicating lack of skin absorption.
Details on distribution in tissues:
- Plasma
Low Dose Level: 112 mg/kg: In male rats exposed to a single oral dose level of 112 mg/kg, the mean maximum plasma concentration (Cmax) of 0.140 µg/g was reached 2 hours after the administration of 14C-Pigment Red 254. These radioactivity levels corresponded to about 1,5 times the limit of quantitation in plasma. At 12 hours after the administration, radioactivity levels in plasma were below the limit of quantitation.
Therefore, no elimination kinetics and no area under the curve were calculated.
High Dose Level: 1111 mg/kg: In male rats exposed to a single oral dose level of 1111 mg/kg, the highest mean plasma concentration of 1.048 µg/g was reached 1 hour after the administration of 14C-Pigment Red 254 and was slightly above the limit of quantitation. After 12 hours, radioactivity levels in plasma were below the limit of detection.

- Blood
Low Dose Level: 112 mg/kg: The mean maximum blood concentration of 0.139 yg/g (i,e, about 1,3 times the limit of quantitation) was reached 2 hours after administration of 14C-Pigment Red 254 at a dose level of 112 mg/kg. After 8 hours, radioactivity levels in blood were below the limit of quantitation.
High Dose Level: 1111 mg/kg: At the high dose level of 1111 mg/kg, the highest mean blood concentration (Cmax) of 1.152 µg/g was reached 1 hour after the administration of 14C-Pigment Red 254 and corresponded to the limit of quantitation. After 4 hours, radioactivity levels in blood were below the limit of quantitation.

In organs, the levels of radioactivity were below the limit of quantitation (< 0.05%).
Details on excretion:
At 168 hours after low dose level (101 mg/kg) administration, residual radioactivity levels in all organs/tissues, blood/plasma and residual carcass were below the limit of quantitation. Residual radioactivity in intestinal tract with contents (0,036 µg/g) corresponded to two times the limit of quantitation.
At 168 hours after high dose level (1058 mg/kg) administration, residual radioactivity levels in organs/tissues and blood/plasma were below the limit of quantitation. Residual radioactivity in stomach contents (0.376 µg/g), intestinal tract with contents (1.624 µg/g) and residual carcass (0.322 µg/g) corresponded to about 2, 9 and 2 times the limit of quantitation, respectively.

At 168 hours after the administration, only very low amounts (0.3 % and 0.6 %) of radioactivity were found in the urine at the low and high dose level, respectively. Within 24 hours, urinary excretion amounted already to 0.3 % and 0.4 %, respectively. Due to the sampling technique of urine and feces and taking into account the water rinse of metabolism cages performed at 8 and 24 hours after the administration, radioactivity found in the urine may originate for the most part from a contamination via the feces. It is also possible that this represents the extractable 14C-impurity test material.

Excretion of 14C-Pigment Red 254 proceeded via the feces and amounted, on average, to 119.7 % and 96.0 % at the low and high dose level, respectively. Already after 24 hours, fecal excretion accounted for 101.4 % (low dose level) and 79.7 % (high dose level) of the radioactivity administered. Together with the cage wash (1.8 % and 7.7 %), total excreted radioactivity accounted, on average, for 121.5 % and 104.3 % at the low and high dose level, respectively.
At both dose levels, the radioactivity was below the lmit of quantitation for blood and carcass. Values slightly above the limit of quantitation were detected in the content of the gastrointestinal tract.

Recoveries of radioactivity accounted, on average, for 121.5 % and 104.3 % of the radioactivity administered at the low and high dose level, respectively. The high recovery of radioactivity at the low dose level reflected the less accurate determination of the amounts of 14C-labelled test article present in the initial stock solution.

Metabolite characterisation studies

Metabolites identified:
no

Any other information on results incl. tables

- One animal of group 2 showed slight diarrhoea on the second day following high dose level administration. All animals of group 4 showed slight diarrhoea after 8 or 12 hours following high dose level administration.

- The first two days after high dose level administration, all animals showed red coloured front- and hind-paws, tail and abdominal skin, due to contamination with feces.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results