Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro-

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, 40 CFR, Part 158, Subdivision F, Section 85-1, October 1982

Qualifier:

according to guideline

Guideline:

other: Directive 87/302 EEC, Part B, No. L133/51 "Toxicokinetik", May 1988.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-labelled

Species:

rat

Strain:

other: BRL-HAN, Wistar, outbred

Sex:

male

Details on test animals or test system 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

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % CMC in 0.9 % NaCl

Duration and frequency of treatment / exposure:

single treatment

Remarks:

Doses / Concentrations:
100 mg/kg and 1000 mg/kg

No. of animals per sex per dose / concentration:

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):

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

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.

Metabolites identified:

no

- 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.

Conclusions:

In GLP-study according to guideline 88/302/EEC Part B the absorption, distribution and excretion of the radiolabelled substance after single application to rat was investigated. The substance was not bioavailable.

Executive summary:

In a GLP-study according to guideline 88/302/EEC Part B the absorption, distribution and excretion of the radiolabelled substance was investigated. Therefore, the substance was applied in two doses: 100 mg/kg bodyweight and 1000 mg/kg bodyweight were given to groups of five male rats.

The animals were killed 168 h p.a.. During the 168 h 0.3% and 0.6% of the applied radioactivity was found in the urine; after 24 h 0.3 % and 0.4% were detected in the urine. These small amounts are interpreted as technic-related faecalic impurities.

Radiolabelled substance in the faeces was on average 119.7 % in the low dose group and 96.0% in the high dose group. The corresponding fractions after 24 h were 101.4% and 79.7 %, respectively.

In both dose groups only neglictible amounts of the radioactive substance were detected in the organs, in the intestinal tract, in the blood and in the carcasse. The maximal concentrations

were 0.139 Ag/g (low dose group) and 1.152 Ag/g (hih dose group)

in the blood, and 0.140 and 1.048 Ag/g in the plasma. The maxima were reached at 1 to 2 h after application. As the concentrations were close to the

detection limit, the kinetics of the elimination and the area under the curve could not be calculated.

Hence, the substance was not bioavailable when applied once orally under the conditions mentioned above.

Reference 2

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.

3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu and under ambient conditions, read-across of toxicological studies from the source to the target form is scientifically justified. In addition, the zeta-potential of both forms indicated that agglomeration increases with lower pH, which further supports the read-across for studies with oral exposure. The low gastric pH increases agglomeration, which supports the conclusion that both forms are not bioavailable upon oral exposure. As the inhalation route is of particular concern for nanoforms, read-across is supported by a 5-day in vivo inhalation study (Hofmann et al., 2016). This short-term inhalation toxicity study compared, among others, a coarse and a fine form of PR254, which are similar to the two forms considered in this analogue approach. The same minor effects were observed at the identical concentration (of 30 mg/m3).

4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’.

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA, 40 CFR, Part 158, Subdivision F, Section 85-1, October 1982

Qualifier:

according to guideline

Guideline:

other: Directive 87/302 EEC, Part B, No. L133/51 "Toxicokinetik", May 1988.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-labelled

Species:

rat

Strain:

other: BRL-HAN, Wistar, outbred

Sex:

male

Details on test animals or test system 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

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % CMC in 0.9 % NaCl

Duration and frequency of treatment / exposure:

single treatment

Remarks:

Doses / Concentrations:
100 mg/kg and 1000 mg/kg

No. of animals per sex per dose / concentration:

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):

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

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.

Metabolites identified:

no

- 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.

Conclusions:

In GLP-study according to guideline 88/302/EEC Part B the absorption, distribution and excretion of the radiolabelled substance after single application to rat was investigated. The substance was not bioavailable.

Executive summary:

In a GLP-study according to guideline 88/302/EEC Part B the absorption, distribution and excretion of the radiolabelled substance was investigated. Therefore, the substance was applied in two doses: 100 mg/kg bodyweight and 1000 mg/kg bodyweight were given to groups of five male rats.

The animals were killed 168 h p.a.. During the 168 h 0.3% and 0.6% of the applied radioactivity was found in the urine; after 24 h 0.3 % and 0.4% were detected in the urine. These small amounts are interpreted as technic-related faecalic impurities.

Radiolabelled substance in the faeces was on average 119.7 % in the low dose group and 96.0% in the high dose group. The corresponding fractions after 24 h were 101.4% and 79.7 %, respectively.

In both dose groups only neglictible amounts of the radioactive substance were detected in the organs, in the intestinal tract, in the blood and in the carcasse. The maximal concentrations

were 0.139 Ag/g (low dose group) and 1.152 Ag/g (hih dose group)

in the blood, and 0.140 and 1.048 Ag/g in the plasma. The maxima were reached at 1 to 2 h after application. As the concentrations were close to the

detection limit, the kinetics of the elimination and the area under the curve could not be calculated.

Hence, the substance was not bioavailable when applied once orally under the conditions mentioned above.

Description of key information

In GLP-study according to guideline 88/302/EEC Part B the absorption, distribution and excretion of radiolabelled substance after single application to rat was investigated. The substance was not bioavailable and has therefore no bioaccumulation potential.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential