N-[2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyphenyl]acetamide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-01-1991 to 08-02-1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

distribution

excretion

metabolism

Principles of method if other than guideline:

The test substance was administered to Sprague-Dawley rats via oral gavage in corn oil in order to determine the extent of absorption, distribution,into different tissues and elimination through urine. Urine samples were analysed to determine the levels of the test substance and its primary metabolite.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

radiochemical purity 99%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: 7 weeks
- Housing: stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 h light:12 h dark

IN-LIFE DATES: From: 21-01-1991 To: 08-02-1991

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The unlabelled test substance was first dissolved in small volume of acetone and then the radio-labelled test substance was added by volume to it. Corn oil was then weighed in the vial containing both the forms of the test substance. A small stirring bar was placed in the vial and a stream of nitrogen was bubbled into the suspension overnight to evaporate the acetone.

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose / concentration:

four per sex per dose group

Control animals:

no

Details on study design:

The test substance was administered to Sprague-Dawley rats via oral gavage in corn oil in order to determine the extent of absorption, disposition into different tissues and the extent of elimination through urine. In addition, the urine samples were analysed to determine the levels of the test substance and its primary metabolite.

Details on dosing and sampling:

Four rats per sex per dose group, kept individually in Roth-type metabolism cages for acclimation and fasting for 15 h, were administered the test substance dissolved in corn oil at the target concentrations of 50 and 500 mg/kg/day at a dose volume of 4 mL/kg and a target radioactivity of 10-15 µCi.

Urine samples were collected at 6, 12, 24, 48, 72 and 96 h post-dosing under dry ice. Fecal samples were collected at room temperature at same intervals. Room air was trapped through the metabolism cages at approximately 500 mL/min and the expired 14CO2 was trapped at 12, 24, 48, 72 and 96 h post-dosing and stored cold until analysis.Volatile organic metabolites were not collected.

At 96 h post-dosing, the animals were sacrificed by overdose of methoxyflurane and exsanguination. Blood collected at termination was separated into plasma and RBCs for analysis. The metabolism cages were washed with 1% trisodium phosphate and an acetone/water rinse. These wash and rinse samples were analysed for inclusion in the material balance.

The pelt was removed from the carcass, prior to removal of the organs. The liver, kidney, bone marrow from femur, brain, peritoneal fat, heart, lung, urinary bladder, spleen, upper GI tract and its contents, testes, ovaries and uterus were isolated and collected for analysis. The remaining carcass was weighed and stored cold until analysis.

Type:

other: Material balance

Results:

The overall recovery of the administered dose was 98+/-2.1% for males and 92.5+/-2.6% for female animals.

Type:

absorption

Results:

The test substance was not absorbed substantially through oral gavage administration.

Type:

distribution

Results:

The test substance was not found to be distributed to the tissues to a major extent following oral administration.

Type:

metabolism

Results:

The test substance was substantially cleared from the GI tract and does not appear to be extensively metabolised following oral administration.

Type:

excretion

Results:

The majority (greater than 85%) of the oral dose of the test substance was excreted in the faeces and less than 6% was recovered in the urine during the 96 h post dose period.

Details on absorption:

By 24 h post dosing, approximately 86% (males) and 74% (females) of the administered dose was excreted in the faeces. Additionally, approximately 3% of the dose was recovered in urine in the initial 24 h for either sex. Therefore, it is evident that the test substance was not substantially absorbed when orally administered to rats.

Details on distribution in tissues:

Less than 0.15% (males) and 0.04% (females) of the test substance was detected in the tissues 96 h post administration. Liver was the only tissue that had greater than 0.02% of the administered dose for either sex. Less than 0.01% of the administered dose was recovered from the femur bone marrow. From these results, it is evident that, the test substance was not distributed significantly into different tissues.

Details on excretion:

By 24 h post dosing, approximately 86% (males) and 74% (females) of the administered dose was excreted in the faeces. Additionally, approximately 3% of the dose was recovered in the urine in the initial 24 h for either sex. The majority of the elimination of the radioactivity was virtually complete by 48 h post-dosing. Expired CO2 was less than 0.1% for males and 0.1% for females.

Metabolites identified:

no

Details on metabolites:

Analysis of pooled samples from animals of each sex in each dose group showed an unidentified metabolite which effectively accounted for all (>90%) of the detectable radioactivity in the urine samples. The suspected metabolite BDNA did not appear in any of the urine samples.

Conclusions:

Under the conditions of the study, the test substance was not substantially absorbed through oral gavage. Majority of the administered dose was excreted through feces and only a minor proportion of the dose was excreted through urine. The test substance was not distributed to different tissues and most of the test substance was eliminated unchanged.

Executive summary:

A study was conducted to determine the toxicokinetic behaviour of the test substance in rats according to an internal protocol of the test facility. The test substance (dissolved in corn oil) was administered to groups of fasted rats at 50 and 500 mg/kg/day through oral gavage at a dose volume of 4 mL/kg and a target radioactivity of 10-15 µCi. Urine and faecal samples were collected at regular intervals until 96 h post dosing. At 96 h post-dosing, the animals were sacrificed by overdose of methoxyflurane and exsanguination. Blood collected at termination was separated into plasma and RBCs for analysis. The liver, kidney, bone marrow from femur, brain, peritoneal fat, heart, lung, urinary bladder, spleen, upper GI tract and its contents, testes, ovaries and uterus were isolated and collected for analysis. The remaining carcass was weighed and stored cold until analysis. By 24 h post dosing, approximately 86% (males) and 74% (females) of the administered dose was excreted in the faeces. Additionally, approximately 3% of the dose was recovered in the urine in the initial 24 h for either sex. The majority of the elimination of the radioactivity was virtually complete by 48 h post-dosing. Expired CO2 was less than 0.1% for males and 0.1% for females. Less than 0.15% (males) and 0.04% (females) were detected in the tissues 96 h post administration. Liver was the only tissue that had greater than 0.02% of the administered dose for either sex. Less than 0.01% of the administered dose was recovered from the femur bone marrow. Analysis of pooled samples from animals of each sex in each dose group showed an unidentified metabolite which effectively accounted for all (>90%) of the detectable radioactivity in the urine samples. The suspected metabolite BDNA did not appear in any of the urine samples. Under the conditions of the study, the test substance was not substantially absorbed through oral gavage. The majority of the administered dose was excreted through feces and only a minor proportion of the dose was excreted through urine. The test substance was not distributed to different tissues and most of the test substance was eliminated unchanged (Frantz, 1991).

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Principles of method if other than guideline:

The cutaneous penetration kinetics of Disperse Blue 79:1 in human abdominal skin samples were measured using in vitro techniques for a 6 h period along with parallel testing of other substances. The amount of test substance penetrating skin samples and collected in the effluent were measured by liquid scintillation spectrometry or high-performance liquid chromatography (HPLC).

Radiolabelling:

yes

Total recovery:

No information available.

Skin penetration of the test substance, applied to human abdominal skin, could not be detected by HPLC above the minimum detection limit of 1 µg/mL. The results thus indicate that human dermal exposure to the test substance would probably not result in a significant absorbed dose.

Conclusions:

Skin penetration of Disperse Blue 79:1 (Br), applied to human abdominal skin, could not be detected by HPLC above the minimum detection limit of 1 µg/mL. The results thus indicate that human dermal exposure to the test substance would probably not result in a significant absorbed dose.

Executive summary:

The cutaneous penetration kinetics of ³H-water (550 µl) or ¹⁴C-mannitol (550 µl; 1 mg/ml) through excised pig ear and human abdominal skin samples were measured using in vitro techniques for a 6-hr period. In addition, the cutaneous penetration kinetics of ¹⁴C-Disperse Blue 79:1 (Br) (110 µl; 5 mg/ml), Reactive Blue 19 (550 µl; 1 mg/ml) and Direct Blue 218 (550 µl; 1 mg/ml) were evaluated by the same in-vitro techniques using human abdominal skin samples. The CAS numbers were 2580-78-1 (Reactive Blue 19), 10401-50-0 (Direct Blue 218), 3618-72-2 (Dispersion Blue 79:1 (Br)), and 69-65-8 (D-Mannitol). Test substances penetrating skin samples and collected in the effluent were measured by liquid scintillation spectrometry or high-performance liquid chromatography (HPLC).

For pig ear skin, the lag time before skin penetration of ¹⁴C-mannitol or ³H-water reached a steady-state rate was 2.77 and 1.75 hr, respectively. The steady-state rate of penetration of mannitol or water through pig ear skin was calculated to be 0.2 µg/cm²/hr and 6.12 mg/cm²/hr, respectively, while the Kp value was calculated to be 0.21 x 10^-3cm/hr and 6.12 x 10^-3cm/hr, respectively. These results were similar to those reported by Scott and Dick (1990). For human abdominal skin, the lag time before skin penetration of ¹⁴C-mannitol or ³H-water reached a steady-state rate was 3.49 and 2.17 hr, respectively. The steady-state rate of penetration of mannitol or water through human skin was calculated to be 0.006 µg/cm²/hr and 2.43 mg/cm²/hr, respectively, while the Kp value was calculated to be 0.006 x 10^-3cm/hr and 2.43 x 10^-3cm/hr, respectively.

These results show that significantly more ³H-water can penetrate both pig ear skin and human abdominal skin than ¹⁴C-mannitol. However, the total penetration of water and mannitol after 6 hr of exposure was 2.7-times and 55-times less for human skin than for pig ear skin. Similarly, both the steady-state penetration rates and permeability constants for water and mannitol were approximately 2.5-times and 30-times less, respectively, for human skin than for pig ear skin. These results would suggest that pig ear skin is not a good animal model for human cutaneous penetration of chemicals.

For the ¹⁴C-DB-79:1 applied to human abdominal skin, the lag time before skin penetration reached a steady-state rate was 3.38 hr. The steady-state rate of penetration of DB-79:1 through human skin was calculated to be 0.0057 µg/cm²/hr. The Kp value for the skin penetration of this dye, however, could not be calculated. Skin penetration of the Reactive Blue 19 and Direct Blue 218 applied to human abdominal skin could not be detected by HPLC above the minimum detection limit of 1 µg/ml.

Little or no Disperse Blue 79:1 (Br), Reactive Blue 19 or Direct Blue 218 was detected penetrating human skin samples. These results indicate that human dermal exposure to any of these 3 dye compounds would probably not result in a significant absorbed dose.

(Sun, 1995).

Description of key information

In an oral toxicokinetic study in rats, the majority of the administered dose was excreted through faeces and only a minor proportion of the dose was excreted through urine. The test substance was not distributed to different tissues and most of the test substance was eliminated unchanged. An assumption of approximately 16% (in case of males) and 26% (in case of females) of test substance absorption through the oral route could be made from the extent of faecal elimination, for the purpose of safety assessment a conservative estimate of 30% was assumed.

 

In an in vitro dermal penetration study in human abdominal cells, no skin penetration was detected by HPLC above the minimum detection limit of 1 µg/mL. The results thus indicate that human dermal exposure to the test substance would probably not result in a significant absorbed dose. For the purpose of safety assessment, a conservative value of 10% was also chosen for dermal penetration

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

30

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

50

Additional information

A study was conducted to determine the toxicokinetic behaviour of the structural analogue in rats according to an internal protocol of the test facility. The test substance (dissolved in corn oil) was administered to groups of fasted rats at 50 and 500 mg/kg/day through oral gavage at a dose volume of 4 mL/kg and a target radioactivity of 10-15 µCi. Urine and faecal samples were collected at regular intervals until 96 h post dosing. At 96 h post-dosing, the animals were sacrificed by overdose of methoxyflurane and exsanguination. Blood collected at termination was separated into plasma and RBCs for analysis. The liver, kidney, bone marrow from femur, brain, peritoneal fat, heart, lung, urinary bladder, spleen, upper GI tract and its contents, testes, ovaries and uterus were isolated and collected for analysis. The remaining carcass was weighed and stored cold until analysis. By 24 h post dosing, approximately 86% (males) and 74% (females) of the administered dose was excreted in the faeces. Additionally, approximately 3% of the dose was recovered in the urine in the initial 24 h for either sex. The majority of the elimination of the radioactivity was virtually complete by 48 h post-dosing. Expired CO2 was less than 0.1% for males and 0.1% for females. Less than 0.15% (males) and 0.04% (females) were detected in the tissues 96 h post administration. Liver was the only tissue that had greater than 0.02% of the administered dose for either sex. Less than 0.01% of the administered dose was recovered from the femur bone marrow. Analysis of pooled samples from animals of each sex in each dose group showed an unidentified metabolite which effectively accounted for all (>90%) of the detectable radioactivity in the urine samples. The suspected metabolite BDNA did not appear in any of the urine samples. Under the conditions of the study, the test substance was not substantially absorbed through oral gavage. The majority of the administered dose was excreted through faeces and only a minor proportion of the dose was excreted through urine. The test substance was not distributed to different tissues and most of the test substance was eliminated unchanged (Frantz, 1991).

A study was conducted to determine the in vitro dermal penetration of the three dyes in human abdominal cells. No skin penetration was detected by HPLC above the minimum detection limit of 1 µg/mL. The results thus indicate that human dermal exposure to the test substance would probably not result in a significant absorbed dose (Sun, 1995).

Although an assumption of approximately 16% (in case of males) and 26% (in case of females) of test substance absorption through the oral route could be made from the extent of faecal elimination, for the purpose of safety assessment a conservative estimate of 30% was assumed. A conservative value of 10% was also chosen for dermal penetration.