Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Full methodology and analytical results provided, although presumed non-GLP. Detailed discussion of results and analytical data included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was designed to characterize the urinary metabolites of ODA and to determine the relative distribution of these metabolites after oral and dermal exposure to ODA. The laboratory rat was used as the model to examine these parameters.

Male rats were dosed orally by gavage with 0.5 or 200 mg [phenyl-14C(U)]-ODA/kg of body weight or were exposed dermally for six hours to 1 mg [phenyl-14C(U)]-ODA. Urine and faeces were collected for a period of 48 hours following the oral gavage dose and for 24 hours following the dermal exposure. Organ and tissue distribution of radioactivity was determined for the dermal application group.
GLP compliance:
not specified
Remarks:
Although appearing to be conducted to a GLP standard, no mention of GLP nor certification are included within the report.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4,4'-Oxydianiline
- Molecular formula (if other than submission substance): Specified; identical
- Molecular weight (if other than submission substance): Not specified
- Smiles notation (if other than submission substance): Not specified
- InChl (if other than submission substance): Not specified
- Structural formula attached as image file (if other than submission substance): Specified; identical
- Substance type: Organic
- Physical state: Solid
- Analytical purity: 99+%
- Impurities (identity and concentrations): Negligible impurities specified
- Composition of test material, percentage of components: Not applicable
- Isomers composition: Not applicable
- Purity test date: Not specified.
- Lot/batch No.: 19644
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling): 99+%
- Specific activity (if radiolabelling): 14C
- Locations of the label (if radiolabelling): Phenyl-14C(U)3-oxydianiline; located on phenyl rings
- Expiration date of radiochemical substance (if radiolabelling): Not specified
- Stability under test conditions: Not specified
- Storage condition of test material: Not specified
- Other:
Radiolabelling:
yes
Remarks:
14C as Benzenamine, 4,4-oxybis-, [phenyl- 14C(U)]-

Test animals

Species:
rat
Strain:
other: Crl:CD®BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: Not specified.
- Weight at study initiation: Not specified.
- Fasting period before study: Not specified.
- Housing: specified only as" appropriate cages"
- Individual metabolism cages: yes; "Roth"-type glass metabolism unit designed to separate urine from feces
- Diet (e.g. ad libitum): Purina® Certified Rodent Chow® #5002 (PCRC) ad libitum.
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 50±10%.
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle.

IN-LIFE DATES: Not specified.

OTHER:
Haskell Laboratory has an animal health monitoring program to periodically analyze water for contaminants and sample freshly washed cages and cage racks for bacteria. The program is monitored and administered by the laboratory veterinarian; data are maintained separately from study records. All parameters were found to be within acceptable ranges.

Administration / exposure

Route of administration:
other: Oral gavage and dermal
Vehicle:
other: Oral Gavage - polyethylene glycol. Dermal dose: Formulated in methanol: polyethylene glycol [PEG]:water (10:20:70) such that 100 µl contained 1 mg of 14C-ODA.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Oral Gavage Dose

One group of four male rats received a single oral (target) dose of 0.5 mg 14C-ODA/kg body weight formulated in PEG and a second group of four male rats received a single oral (target) dose of 200 mg 14C-ODA /kg body weight formulated in PEG. Doses where calculated as follows:

The 0.5 mg/kg dose consisted of a mixture of
• 0.76 mg of 14C-ODA,
• 0.5 ml Methanol (Fisher Lot #96288), and
• 4.5 ml Polyethylene Glycol 300 (Baker Lot #536621).

One ml delivered a 0.61 mg/kg (actual) dose to a 250 gram rat. Each rat received either 21.8 µCi or 26.1 µCi of 14C radioactivity with a specific radioactivity content of 143.4 or 171.7 µCi /mg.

The 200 mg/kg dose consisted of a mixture of
• 1.13 mg of l4C-ODA,
• 250.72 mg non-labelled ODA,
• 0.5 ml Methanol (Fisher Lot #96288), and
• 4.5 ml Polyethylene Glycol 300 (Baker Lot #536621).

One ml of this mixture delivered a 201 mg/kg (actual) dose to a 250 gram rat. Each rat received 35.07 (µCi of l4C radioactivity with a specific radioactivity content of 0.70 µCi/mg).


Dermal Dose

Four male rats received a cannula in the jugular vein. Rats were allowed to recover from surgery for one day before dosing. Following recovery, each rat received a single dermal application of 1mg I4C-ODA. The dermal dose was formulated in methanol:polyethylene glycol [PEG]:water (10:20:70) such that 100 ul contained 1 mg of 14C-ODA. Non-labeled ODA was mixed with l4C-ODA such that each rat received 18.8 µCi of 14C radioactivity with a specific radioactivity content of 18.85 µCi/mg and an application concentration of 0.167 (µg/cm2.

On the day of exposure, the application site on the back of the animal between the shoulder blades was shaved. A foam rubber protective device was secured in place over the application site with surgical adhesive. The dose mixture was applied in a 2x3 cm area within the shaved area. A metal (perforated) protective cover with non-occlusive gauze was then placed over the foam rubber protective device and secured in place. Each rat was placed into a "Roth"-type glass metabolism unit immediately after receiving the radioactive dose. Rats were maintained ad libitum on PCRC and water. Six hours following application, the metal cover and gauze were removed and the application site was washed. The washing consisted of five cycles of rubbing 5% aqueous Micro® detergent over the application site with a cotton swab and removing the wash with a dry cotton swab. Swabs were placed in scintillation vials for subsequent assay for radioactivity.

Blood samples were drawn through the cannula at 1, 2,4, 6, 12, and 24 hours following the dermal application. Approximately 0.3 mL of whole blood was drawn at each time point. Urine and feces were collected from each rat 12 and 24 hours after the dermal application. At study sacrifice, carcass and organs where further collected.

DIET PREPARATION
Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified; assumed historical usage.
- Concentration in vehicle: See above
- Amount of vehicle (if gavage): See above
- Lot/batch no. (if required): Not specified
- Purity: Not specified

HOMOGENEITY AND STABILITY OF TEST MATERIAL: Not specified
Duration and frequency of treatment / exposure:
Oral: Single dose only, each treatment level.
Dermal: Single dose only.
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 mg/kg bw/day (nominal)
Remarks:
Oral Gavage Dose
One group of four male rats received a single oral (target) dose of 0.5 mg 14C-ODA/kg body weight formulated in PEG and a second group of four male rats received a single oral (target) dose of 200 mg 14C-ODA /kg body weight formulated in PEG.
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Oral Gavage Dose
One group of four male rats received a single oral (target) dose of 0.5 mg 14C-ODA/kg body weight formulated in PEG and a second group of four male rats received a single oral (target) dose of 200 mg 14C-ODA /kg body weight formulated in PEG.
Dose / conc.:
1 other: mg/animal
Remarks:
Dermal application
Four male rats received a cannula in the jugular vein. Rats were allowed to recover from surgery for one day before dosing. Following recovery, each rat received a single dermal application of 1 mg 14C-ODA. The dermal dose was formulated in methanol: polyethylene glycol [PEG]:water (10:20:70) such that 100 µl contained 1 mg of 14C-ODA. Non-labeled ODA was mixed with l4C-ODA such that each rat received 18.8 µCi of 14C radioactivity with a specific radioactivity content of 18.85 µCi/mg and an application concentration of 0.167 (µg/cm2).
No. of animals per sex per dose:
4 males per dose.
Control animals:
no
Positive control:
None.
Details on study design:
- Dose selection rationale: To ensure uniform dosage of radioactive material.
- Rationale for animal assignment (if not random): Random
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled:

The following tissues and organs were collected from the dermal test group:

heart brain
lungs G.I. tract (and contents)
liver skin (application site)
spleen skin (non-application site)
kidneys fat
testes

The remaining carcass was saved and the metabolism units washed with Micro®, acetone, and water. All samples were assayed for radioactivity.

- Time and frequency of sampling:
Oral Gavage Dose
Urine and feces were collected from each rat 12, 24, and 48 hours after dosing.
Dermal Dose
Blood samples were drawn through the cannula at 1, 2,4, 6, 12, and 24 hours following the dermal application.
Animals were sacrificed at 48 hours for subsequent assessment, with subsequent collection of the carcass and organs.

- From how many animals: (samples pooled or not): Representative samples of whole blood, organs, tissues, carcasses, and feces were assayed for radioactivity by combustion with Packard Model 306 Tissue Oxidizers

- Method type(s) for identification:

Absorption:

Liquid scintillation counting was conducted with Mark HI model 6882 Liquid Scintillation Counters (TM Analytic, Elk Grove, Illinois). Samples directly assayed (following additions of 12-15 mL scintillation cocktail) included:
• urine,
• dose solution or dilutions,
• cage washes, and
• HPLC eluant fractions.

Aquasol®-LI scintillation cocktail or Ultima Gold-XR (Packard) was used for analysis.

Representative samples of whole blood, organs, tissues, carcasses, and feces were assayed for radioactivity by combustion with Packard Model 306 Tissue Oxidizers (Packard Instrument Company, Downers Grove, Illinois). Evolved l4C02 was automatically trapped in 7-8 mL of Carbo-Sorb® (Packard) and mixed with 12-13 mL of Permafluor® V (Packard) scintillation cocktail and counted with Mark III model 6882 Liquid Scintillation Counters.

Metabolism

URINE PREPARATION

Urine samples from the low and high (oral) dose groups were thawed and a portion transferred to separate vials and adjusted to pH 6 with IN hydrochloric acid. One-mL samples were incubated overnight at 37°C after addition of a glucuronidase/sulfatase enzyme preparation ((3-giucuronidase, type H-l isolated from Helix pometia and purchased from Sigma Chemical Company). Companion urine samples were also incubated as non-enzyme treated controls.
All urine samples were prepared for HPLC analysis by mixing 0.5 mL methanol with 0.5 mL of the enzyme-treated or non-enzyme-treated urine. These samples were centrifuged for 5 minutes in an Eppendorf model 5413 microfuge (Brinkmann instruments, Inc., Westbury, New York) to remove particulates. The liquid layer was then passed through a 0.45 p: or 0.22 \i Acrodisc® filter (Gelman Science) into a HPLC sample vial (crimp top) and subsequently assayed by HPLC.


HPLC ANALYSIS AND METABOLITE PURIFICATION

HPLC analyses were conducted with a Perkin-Elmer Series 4 Liquid Chromatograph (Perkin-Elmer, Norwalk, Connecticut). Radioactivity was monitored and the relative percent of each radioactive peak was determined with a Ramona-D radioactive flow monitor (Raytest U.S.A., Inc., Pittsburgh, Pennsylvania).
The following HPLC conditions were used for analysis of 14C-ODA, dose solutions, urine samples, and metabolites:

1. Columns:
a) C-8 Adsorbosphere® guard column, 21.5 mm
b) Zorbax®C-8, 21.5 mm id x 25 cm preparative column; serial number EY1119; particle size diameter: 5 |im

2.Solvent Flow Rate: 5.0 mL/min

3.Column Temperature: Ambient

4. Solvents:
a) Acetonitrile
b) 0.1 M ammonium acetante adjusted to pH 3.3 with concentrated HC1

5. Gradient System:

1) 10:90 Acetonitrile: 0.1 M pH 3.3 ammoniumacetate isocratically for 10 min
2) 10:90 to 50:50 Acetonitrile: 0.1 M pH 3.3 ammonium acetate over a 25 min linear gradient
3)50:50 Acetonitrile: 0.1 M pH 3.3 ammonium acetate isocratically for 15 min
4)50:50 to 10:90 Acetonitrile: 0.1 M pH 3.3 ammonium acetate linear gradient for 10 min
5)10:90 Acetonitrile: 0.1 M pH 3.3 ammonium acetate isocratically for 10 min prior to next injection

Urine collected from the 200 mg/kg oral gavage dose group (PEG formulation) during the 0- to 12-hour collection interval was subsequently pooled and discreet fractions of the HPLC eluant collected using a Foxy Fraction Collector (Isco, Inc., Lincoln, Nebraska). The resulting metabolite fractions were concentrated by evaporation under nitrogen, or extracted with methylene chloride and concentrated by evaporation with nitrogen and submitted for mass spectral analysis.

- Limits of detection and quantification: 40 ppb (estimate)
- Other:
Statistics:
Please refer to attached tabulated data below for details of statistical means.

Results and discussion

Preliminary studies:
None. The study did not include a preliminary assessment based on the reported data.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Distribution of Radioactivity Following Dermal Application

Tables 3 and 4 (attached) summarize the distribution of dermally applied 14C-ODA (1 mg per rat). Approximately 7.3% (73 (µg) of the applied dose was absorbed, whereas 70% of the applied dose was accounted for as non-absorbed radioactivity. Total material balance at the 24 hours post-dose sacrifice ranged from 70.1 % to 87% accounting for a mean of 77.4% of the total applied dose, with a loss of approximately 22% due possibly to the volatilization of the test compound.

Approximately 3% (2 pg) of the dermally absorbed l4C-ODA was excreted in the urine by the 24-hour post-application sacrifice (Table 4). Approximately 2.46% was excreted in the 12- to 24-hour post-application urine sample. The tissues and carcass retained approximately 7.65% of the absorbed dose at the 24-hour post-application sacrifice; 87% of the absorbed dose was retained in the applied skin.
Details on distribution in tissues:
Tissue Distribution Following Dermal Application

The concentrations of radioactivity in organs and tissues are presented as ug equivalents of 14C-ODA per g of tissue (Table 5 and Figure 3, attached). The liver contained the highest concentrations of 14C-ODA (0.214 µg/g) followed by the kidneys (0.052 ug/g). Whole blood concentrations averaged 0.023 pg equivalents per mL, whereas plasma concentrations averaged 0.029 ug equivalents per mL at sacrifice. The lowest concentrations were seen in the brain (0.005 µg/g) and in the fat (0.007 pg/g).



Plasma Time-Course Concentrations Following Dermal Application

Plasma concentrations of total 14C-ODA at 1, 2,4, 6, 12, and 24 hours following dermal exposure are presented in Table 6 and Figure 4 (attached). Maximum plasma concentration of 14C-ODA was 0.0342 µg equivalents per mL of plasma at the 12-hour time point.
Details on excretion:
Urinary Excretion Following Oral Dosing
Urinary excretion, expressed as a percent of dose, for the 0.5 and 200 mg/kg oral doses of 14C-ODA are presented in Table 1 (attached) and mean cumulative percent urine excretion in Figure 1(attached). The total 14C excreted in the urine through 48 hours were similar for 0.5 and 200 mg/kg dose (approximately 35% and 28%, respectively). However, the 0-12 hour average percent values from the 0.5 mg/kg group were much greater than the 200 mg/kg group (10.7% compared to 3.6%).

The percentage of dose in the 12- to 24-hour urine samples from the 0.5 and 200 mg/kg groups were approximately 11.2% and 7.6%, respectively; the percents of dose excreted in the 24- to 48-hour urine samples from the 0.5 and 200 mg/kg groups were approximately 13.4% and 16.9%, respectively.

Fecal Excretion Following Oral Dosing

Table 2 (attached) summarizes faeces excretion for the 0.5 and 200 mg/kg oral doses of ,4C-ODA. The cumulative percentage of dose excreted (Figure 2 attached) were 28.7% and 13.4% for the 0.5 and 200 mg/kg dose groups, respectively. Over 19% of the 0.5 mg/kg dose was eliminated by 24 hours post dosing as compared to only 6% for the 200 mg/kg dose group during the same time period.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
LC-MS-MS of the urine collected from ODA-dosed-rats indicated that the major component was N-acetyl-hydroxy ODA. The two other prominent metabolites were N-acetyl ODA and N.N'-diacetyl ODA. Other minor components were N,N'-diacetyl ODA and N,N'-diacetyl-hydroxy ODA. The site of hydroxylation could not be determined by LC-MS-MS, therefore, nmr experiments are needed to confirm these structures.

Any other information on results incl. tables

HPLC/Radioactivity Flow Analysis of Urine Samples

 

HPLC UV/Ramona chromatogram ofl4C-ODA is presented in Figure 5 (attached). Radioactive metabolite profiles in urine were characterized by HPLC/radioactivity-flow analysis and are presented in Figures6and 7 (attached). Urine from rats dosed at the 200 mg/kg level contained five urinary metabolites. No parent ODA was present. Overnight incubation with glucuronidase/sulfatase enzymes produced little change in the urinary profile, except for the appearance of a metabolite eluting at approximately 26 minutes and the reduction of metabolite eluting at approximately 12 minutes (Figure 7, attached).

 

Serial fractions from the high-dose urine were collected and submitted for mass spectral analysis. Of the six fractions analyzed, only the metabolites eluting at approximately 41 and 45 minutes were definitively identified as monoacetylated ODA (N-ODA) and diacetylated ODA (N,N'-ODA), respectively. Structural representations of ODA, N-ODA, and N,N'-ODA are presented in Figure 8 (attached).

 

None of the urine samples from the dermal penetration group contained sufficient radioactivity for meaningful HPLC/radioactivity flow analysis.

 

Distribution of Radiolabeled Urinary Metabolites

The relative and absolute percent distributions for the observed urinary metabolites are presented in Tables 7-9 (attached).

 

In the 0- to 12-hour urine samples from the 0.5 mg/kg group, monoacetylated ODA (N-ODA) and diacetylated ODA (N,N'-ODA) represented approximately 3.8% and 20.7% of the total urinary radioactivity, respectively, corresponding to 0.39% and 2.13% of the total dose, respectively. In the 12- and 24-hour urine samples, only N,N'-ODA was detected, representing 17.7% of the total urinary radioactivity, corresponding to 2% of the total dose. In the 24- to 48-hour urine samples, once again only N,N'-ODA was detected representing approximately 14.1% of the total urinary radioactivity corresponding to 1.83% of the total dose.

 

In the 0- to 12-hour urine samples for the 200 mg/kg group, N-ODA and N,N'-ODA represented 6.3% and 6.9% of the total urinary radioactivity, respectively, corresponding to 0.23% and 0.24% of the total dose, respectively. In the 12- to 24-hour urine samples, N-ODA and N,N'-ODA represented 7.2% and 14.2% of the total urinary radioactivity, respectively, corresponding to 0.56% and 1.05% of the total dose, respectively. Urinary metabolite profiles from the 24-48 hour interval of the 200 mg/kg dose group were not generated since mass spectral analysis of the 0-12 hour interval had confirmed the structure of two marker metabolites of interest, N-ODA and N,N'-ODA, which were present also in the 12-24 hour urine sample.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results It is not possible to predict the bioaccumulation potential based on the study as there is no indication on the systemic absorption after oral ingestion since i.v. is not available and the mass balance data is not closed; urine elimination, fecal eliminat
The study was designed to characterize the urinary metabolites of ODA and to determine the relative distribution and abundance following oral and dermal exposure. Specifically, the 0.5 mg/kg oral dose level and the 1 mg dermal exposures were chosen because they represented potential low-level workplace exposures. The high oral dose was chosen to allow definitive characterization of urinary metabolites which might be observed in the low dose oral and dermal exposure groups.

When comparing cumulative urinary excretion data (total radioactivity) from both oral dose groups, data from the 0.5 mg/kg low dose oral group suggests first-order elimination kinetics. On the other hand, urinary excretion data from the 200 mg/kg dose group is more suggestive of zero-order elimination kinetics. Furthermore, when comparing the 0.5 and 200 mg/kg oral doses, metabolite ratio differences between monoacetylated-ODA (N-ODA) and diacetylated-ODA (N,N'-ODA) are evident and appear at much higher concentrations as a percent of the total dose eliminated in the 0.5 mg/kg dose group urine with N,N'-ODA appearing in the greatest relative abundance. Minor urinary profile changes were observed with respect to the abundance of either metabolite of interest, N-ODA or N,N'-ODA, following glucoronidase/ sulfatase (G/S) hydrolysis of urine from the high dose oral gavage group. The minor urinary profile changes suggests that conjugation by glucuronide or sulfate is not a major process for excreting ODA. Although enzyme hydrolysis of urine from the 0.5 mg/kg dose group was not performed, minimal profile alterations of the 200 mg/kg dose group urine suggests that N,N'-ODA, being present in the urine at all post-dose collection time-points, would not be effected by G/S enzyme hydrolysis.

Only two of the six major metabolites fractions from the high dose oral gavage group, which appear in 12-hour urine (non-hydrolyzed), were definitively characterized by mass spectral analysis. Direct mass spectral analysis of high dose urine offered support to the conclusion that multiple metabolites of ODA were present in the uncharacterized fractions and that no parent ODA was present . Following enzyme treatment of the high dose group 12-hour urine, HPLC analysis revealed the addition of a peak which appeared at about 26 minutes that was not characterized by mass spectral analysis. Treatment of urine samples with 2N sodium hydroxide offered no additional conclusive evidence to support existing mass spectral characterizations.

The dermal application study show ODA is largely retained at the site of application. Three percent of the absorbed dose (cumulative) was present in the urine at the 24-hour sacrifice and over 87% of the absorbed dose was retained in the skin. This suggests a potential for continued release of ODA or ODA-metabolites from the skin reservoir into the systemic circulation. This continued release or depot phenomenon is further supported by blood-plasma concentrations of total radioactivity which persist after removal of excess dermal dose at six hours until the 24-hour sacrifice. Unfortunately, urine samples collected from the dermal absorption group contained insufficient radioactivity for meaningful HPLC/radioactivity flow analysis.

The overall conclusion on bioaccumulation potential of the substance is that it cannot be determined on the basis of this study alone.

Executive summary:

On the basis of this study alone, it is not possible to predict the overall potential for bioaccumulation.

 

The dermal application study show ODA is largely retained at the site of application.. This suggests a potential for continued release of ODA or ODA-metabolites from the skin reservoir into the systemic circulation. This continued release or depot phenomenon is further supported by blood-plasma concentrations of total radioactivity which persist after removal of excess dermal dose at six hours until the 24-hour sacrifice. Unfortunately, urine samples collected from the dermal absorption group contained insufficient radioactivity for meaningful HPLC/radioactivity flow analysis.

 

When comparing cumulative urinary excretion data (total radioactivity) from both oral dose groups, data from the 0.5 mg/kg low dose oral group suggests first-order elimination kinetics. On the other hand, urinary excretion data from the 200 mg/kg dose group is more suggestive of zero-order elimination kinetics.