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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Although no guideline is followed, the study design is clearly described, adequate and reliable. Study conducted at reputable lab with study director with extensive experience.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
no guideline followed
Deviations:
not applicable
Principles of method if other than guideline:
Because the majority (55%) of orally administered DPPD (N,N'-diphenyl-p-phenylenediamine) is exreted in feces of rats with <0.1% excreted in urine, this study was designed to focus on fecal excretion with a thorough assessment of biliary elimination of the test chemical N,N'-di-o-tolyl-p-phenylenediamine (R-898). This chemical is typically ~20% of the registration substance DAPD.
The R-898 molecule was synthesized with radioactivity (14C) positioned in the center ring. This radiotracer was added to the submission substance to allow quantitation of excreted material. The specific activity of the radiochemical was 11.66 mCi/mmol with 98.7% radiochemical purity.
One oral gavage dose administered to each rat. Three sampling sites were included in the study - urine, feces, and bile. Urine & fecal samples were collected at 6, 12, 24, 36, 48 & 72 hrs following dosing in the Phase 1 (intact) group. Sampling was terminated at 48 hrs in the Phase 2 group.
Samples were analyzed for radioactivity using liquid scintillation counting and HPLC with both UV and flow-thru scintillation counting.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
The test material was R-898 (N,N-di-o-tolyl-p-phenylenediamine), a major component of the submission substance DAPD. The molecular formula is C20H20N2 with molecular weight of 288.4. SMILES notation: Cc1ccccc1Nc2ccc(Nc3c(C)cccc3)cc2. The lot # was 960718A.

Test solutions were prepared by dissolving DAPD + [14C]R-898 in corn oil prior to oral gavage dosing. Two trials were performed - the dosing soln for the first trial (intact bile ducts) delivered a dose of 8.4 mg R-898/kg, 30.3 mg DAPD/kg, and ~21 uCi per rat. Prior to addition of [14C]R-898 to theDAPD in the dosing corn oil dosing solution, the submission substance was estimated to contain 23% of this component; after addition of [14C] material, 28%. Levels of R-898 in the submission substance was estimated using HPLC/UV quantitation.

The 2nd trial (bile ducts cannulated) delivered a dose of 7.4 mg R-898/kg, 26.7 mg DAPD/kg, and ~15 uCi per rat.

The lot numbers were 137170393 for the DAPD, and 91004140 for the labeled R-898.

Due to the very long shelf life of the submission substance (5 yr+), the stability of the radiolabel and chemicals in the dosing solution were considered to be assured under these conditions.
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
Male rats were obtained from Charles River Labs, North Carolina. They were provided drinking water & Purina Rat chow ad libitum. Rats were housed 4 per cage during minimum of one-week acclimation period and until study initation. The environment was 73 +/- F degrees, 40-70% relative humidity, and with 12/12 hr photocycle. Each rat in group 2 (6 rats) was subjected to cannulations of the bile duct to provide delivery of bile. Following duct cannulations, the rats were allowed to recover for 48 hr before oral gavage dosing. Group 1 rats (4 rats) remained intact without cannulations.

At dosing, rats were individually housed in glass metabolism cages that provided separation of urine & feces. Body weights of rats were in the range of 216 to 251 grams at dosing. Rats were not fasted prior to dosing.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test solution was dosed in volume of 5 ml/kg. The submission substance was at a level of ~4 mg/ml in the dosing solution. Corn oil was used as it is common vehicle for animal oral gavage testing, and was shown to solubilize the test compound.
Duration and frequency of treatment / exposure:
One oral gavage dose administered to each rat.
Doses / concentrations
Remarks:
Doses / Concentrations:
Dose for Phase 1 was ~30 mg DAPD/kg plus ~8 mg R-898/kg, and for Phase 2, 27 mg DAPD/kg, 7 mg R-898/kg. The radioactivity doses were ~21 uCi rat in Phase 1, and ~15 uCi/rat in Phase 2.
No. of animals per sex per dose:
Males only were used in study. A total of 6 rats were cannulated for use in Phase 2. Due to poor health, only 4 rats were retained for collection of bile samples.
Control animals:
no
Positive control:
No
Details on study design:
The dose employed was below that known to be toxic, but provided sufficient amount of chemical to allow measurements of excreted material following oral dosing. Animals were assigned in random manner.
Details on dosing and sampling:
Three sampling sites were included in the study - urine, feces, and bile. Urine & fecal samples were collected at 6, 12, 24, 36, 48 & 72 hrs following dosing in the Phase 1 (intact) group. Sampling was terminated at 48 hrs in the Phase 2 group.

Samples were analyzed for radioactivity using liquid scintillation counting and HPLC with both UV and flow-thru scintillation counting.
Statistics:
Standard deviations were calculated for radioactivities collected.

Results and discussion

Preliminary studies:
None

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
Identification of metabolites was not conducted. However, HPLC profiles demonstrate (a) the parent compound R-898 in the bile is a minor fraction (<2.5%) of that collected in the bile, and (b) >90% of the bile radiolabel elutes in the HPL chromatographic region associated with metabolites exhibiting higher polarity than the parent cmpd. 40% of that radioactivity was located in one peak (B4).

Any other information on results incl. tables

Radioactivity Excreted Following Oral

Dosing of [14C] R-898 to Intact Rats

14C [% of dose admin.] (S. Dev.)
Urine  Fecal Total
Ave. Amnts (n=4)   1.1 (0.3) 74.5 (6.1) 75.5 (6.2)

Radioactivity Excreted Following Oral

Dosing of [14C] R-898 to Cannulated Rats

14C [% of dose admin.] (S. Dev.)
Bile collected (g) Bile  Urine  Fecal Total
Ave. Amnts (n=4) 30.2 (3.6) 30.1 (7.3) 0.6 (0.2) 43.8 (4.3) 74.4 (3.5)

Bile Metabolites of [14C] R-898 Following Oral

Administration to Cannulated Rats (% of Eluted 14C)

HPLC Peak Retention Time (min) Mean  SD
B1 7.2 - 8.5 9.7 5.4
B2 8.5 - 10.7 16.8 6.2
B3 11.9 - 14.0 50.0 10.7
B4 (R-898) 18.6 - 20.3 1.5 0.3
TOTAL 78.0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Oral administration of radioactive R-898, a major component of the submission substance, has shown the radioactivity is excreted primarily in the feces (75%). The biliary excretion accounted for ~40% of that amount. Urinary excretion was minor (<2.5%). Biliary metabolites were shown to be polar substances, accounting for >95% of the radioactivity excreted by this route.
Executive summary:

The study was designed to assess routes of excretion in rats of R-898, a major component of the submission substance. Urinary & fecal samples were collected over period up to 72 hrs following oral dosing in intact rats, up to 48 hrs in bile duct-cannulated animals. Results showed that ~75% of dosed radioactivity is excreted in feces with 30% of the dosed amount entering the GI tract via the biliary route. <2% was excreted in the urine. HPLC analysis of bile demonstrated that 95+ % of the metabolites excreted by this route exhibited greater polarity than the parent compound, suggesting metabolic formation of oxidation and conjugation products of this component.