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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study was not conducted under GLP and limited information is provided on animals used and environmental conditions.
Justification for type of information:
Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.
Reason / purpose:
read-across source
Details on distribution in tissues:
51.4% in urine, 35.3% in faeces, 3.3% in organs/tissues, and 3.2% in cage rinse
Details on excretion:
Approximately 50% of the dose was excreted in the urine and 35% in the faeces. The standard deviations revealed substantial animal variation in the distribution of excreted 14C activity. The urinary excretion for the first 24 hours represented over 40% of the original dose. Approximately 90% of the original dose was excreted in the urine and feces by 72 hours after dosing. Approximately 3-4% of the dose remained in the animal at the 72-hour sacrifice.
Metabolites identified:
yes
Details on metabolites:
The solvent soluble metabolites were examined using TLC and HPLC. Both systems revealed the presence of 2 metabolites as monitored by 14C-containing zones. The ratio of the major to minor component was approximately 2:1. Non-labeled standards of potential test substance metabolites were also analyzed. In both the TLC and HPLC system, the major metabolite co-chromatographed with the N,N'-diacetyl-PPD standard. The project was halted before the hydrolysis, extractions, and derivatization procedures were satisfactorily refined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Objective of study:
excretion
metabolism
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
One dose level, details on test animals and environmental conditions not complete.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: > 99%
Radiolabelling:
yes
Remarks:
14C-p-phenylenediamine

Test animals

Species:
rat
Strain:
not specified
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Not reported
- Age at study initiation: Not reported
- Weight at study initiation: 250 g
- Fasting period before study: Not reported
- Housing: Not reported
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): Not reported
- Water (e.g. ad libitum): Not reported
- Acclimation period: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Airflow: 600-700 mL/min
- Photoperiod (hrs dark / hrs light): Not reported

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
other: Tween 20: 1.15% saline (20:80)
Details on exposure:
Non-labeled test substance was recrystallized in methanol and stored in a freezer. The 14-C-labelled test substance was purified by HPLC. The dose was formulated in Tween 20:1.15% saline (20:80) immediately prior to dosing. Each 250 g male rat received a single 10 mg/kg ip dose. Each rat was administered approximately 2.42 µCi of 14C radioactivity.
Duration and frequency of treatment / exposure:
72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
10 mg/kg in a 20 mL volume (2.42 µCi 14C radioactivity)
No. of animals per sex per dose:
3/time point
Control animals:
no
Details on study design:
Immediately after dosing, each rat was housed individually in a glass metabolism unit. Urine and feces samples were collected separately at 6-, 24-, 48-, and 72-hour intervals after dosing. Samples were frozen and stored immediately after collection. Preliminary studies revealed no evolution of [14C]-CO2 following [14C]-PPD dosing; therefore, expired CO2 was not trapped. The rats were sacrificed 72 hours after dosing. Upon sacrifice blood was drawn and refrigerated in heparinized tubes. The heart, lungs, liver, spleen, kidney, G.I. tract, testes, brain, samples of fat, skin, and muscle were excised and weighed. The carcass was weighed, frozen, and stored with all excised tissues and organs. The metabolism units were rinsed with dilute detergent, water, and acetone into bottles, and refrigerated. Radioactivity in the urine and cage rinses was analyzed directly by liquid scintillation counting (LSC). Samples of feces, blood, tissues, and carcasses were analyzed for 14C by tissue oxidation using a tissue oxidizer.

Urine samples were examined for the distribution of [14C]-PPD metabolites. The quality of organo-soluble metabolites was determined by extraction with ethyl acetate. Urine was also subjected to enzymatic hydrolysis. Radioactivity levels in the organic phase were compared between non-hydrolyzed and enzymatically hydrolyzed urine. The organo-soluble radioactivity was analyzed by thin layer chromatography (TLC) and autoradiography. The organo-soluble extracts were also analyzed by HPLC using LSC analysis of eluant fractions. Procedures were also examined for preparing extracts that were relatively free of endogenous compounds. Column chromatography was examined. The purpose was to isolate fractions that were enriched with PPD metabolites and relatively free of urinary contaminants.

Metabolite fractions from column clean up procedures and organic solvent extractions were purified by HPLC. Fractions from the HPLC eluant were collected and analyzed for 14C content. The radioactive fractions containing the major metabolite were evaporated to dryness under N2. The purified metabolite was then analyzed by GC/Mass Spectrometry. Authentic standards of PPD, N-acetyl-PPD, and N,N'-diacetyl-PPD were also analyzed and compared to the metabolite.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, heart, lungs, liver, spleen, kidney, GI tract, testes, brain, fat, skin, and muscle.
- Time and frequency of sampling: Urine and faeces: 6, 24, 48, and 72 hours

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: Total (72 h)
- Method type(s) for identification: HPLC, TLC, GC

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
51.4% in urine, 35.3% in faeces, 3.3% in organs/tissues, and 3.2% in cage rinse
Details on excretion:
Approximately 50% of the dose was excreted in the urine and 35% in the faeces. The standard deviations revealed substantial animal variation in the distribution of excreted 14C activity. The urinary excretion for the first 24 hours represented over 40% of the original dose. Approximately 90% of the original dose was excreted in the urine and feces by 72 hours after dosing. Approximately 3-4% of the dose remained in the animal at the 72-hour sacrifice.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
The solvent soluble metabolites were examined using TLC and HPLC. Both systems revealed the presence of 2 metabolites as monitored by 14C-containing zones. The ratio of the major to minor component was approximately 2:1. Non-labeled standards of potential test substance metabolites were also analyzed. In both the TLC and HPLC system, the major metabolite co-chromatographed with the N,N'-diacetyl-PPD standard. The project was halted before the hydrolysis, extractions, and derivatization procedures were satisfactorily refined.

Any other information on results incl. tables

Table 1: Disposition of 14C Activity 72 Hours After

a Single 10 mg/kg Dose of [14C]PPD

Sample

µg±SD

Percent of±SD

Urine

1286 ± 293

51.4 ±11.7

Feces

886 ± 203

35.3 ± 8.1

Organs, Tissues

82 ± 25

3.3 ± 1.0

Cage Rinse

78 ± 31

3.2 ± 1.2

 

Table 2: Cumulative Percent Excretion of 14C Activity

from Rats Dosed with 10 mg/kg [14C]PPD

Sample

Cumulative Percent Urinary Excretion

Cumulative Percent Fecal Excretion

6 h

24.8

-

24 h

45.6

22.9

48 h

50.5

32.8

72 h

51.4

35.4

Applicant's summary and conclusion

Conclusions:
In rats administered a single 10 mg/kg ip dose, approximately 50% of the dose was excreted in the urine and 35% in the afeces. The standard deviations reveal substantial animal variation in the distribution of excreted 14C activity. Approximately 3-4% of the dose remained in the animal at the 72-hour sacrifice. Two metabolites were detected.
Executive summary:

The excretion and distribution of 14C radioactivity in male rats were determined for a 72-hour period after the single 10 mg/kg i.p. dose. Approximately 50% of the dose was excreted in the urine and 35% in the faeces. The standard deviations reveal substantial animal variation in the distribution of excreted 14C activity. Approximately 3-4% of the dose remained in the animal at the 72-hour sacrifice. Sufficient quantities of 14C-labeled metabolites were excreted in the 6- and 24-hour urine samples to consider urinalysis as a plausible method for biological monitoring. The solvent soluble metabolites were examined using the TLC and HPLC. Both systems revealed the presence of 2 metabolites as monitored by 14C-containing zones. The ratio of the major to minor component was approximately 2:1. Non-labelled standards of potential test substance metabolites were also analyzed. In both the TLC and HPLC system, the major metabolite co-chromatographed with the N,N'-diacetyl-PPD standard. The project was halted before the hydrolysis, extractions, and derivatization procedures were satisfactorily refined.