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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted as a part of the range finding study to the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422 and US EPA OPPTS 870.3650) and the report contains sufficient information to permit a meaningful evaluation of study results
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Diethylene Glycol Mono Phenyl Ether
- Physical state: liquid
- Analytical purity: 99.92%
- Impurities (identity and concentrations): no information available
- Composition of test material, percentage of components: The purity of the test material was determined to be 99.92%, after correcting for water, by gas chromatography with identification by proton nuclear magnetic resonance and gas chromatography mass spectrometry.
- Lot/batch No.: Lot # 201103261-7-18
Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Portage, Michigan)
- Age at study initiation: approximately 8 weeks at initiation of treatment
- Weight at study initiation:
- Fasting period before study: not applicable
- Housing: housed singly in solid stainless steel cages
- Diet (ad libitum): Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form.
- Water: Municipal water was provided ad libitum
- Acclimation period: Animals were acclimated to the laboratory for at least one week prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes were prepared periodically throughout the study based on stability data. Diets were prepared weekly for two weeks prior to breeding of the P1 adults. Initial concentrations of test material in the diet were calculated from historical body weights and feed consumption data. Subsequently, the concentrates of the test material in the diets were calculated from the most recent body weight and feed consumption data. To avoid potential overdosing during the breeding period, co-housed animals were provided with the female diet, which was of lower concentration. Following breeding, the male diet was prepared weekly until necropsy. During gestation and lactation, females from each dose group were provided with the appropriate dietary concentration of diethylene glycol mono phenyl ether given during breeding.
Duration and frequency of treatment / exposure:
Animals were dosed daily via the diet for the duration of the rangefinding study; 14 days.
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500 and 1000 mg/kg diet
No. of animals per sex per dose:
5 male + 5 female rats/dose
Control animals:
yes, plain diet
Positive control:
not applicable
Details on study design:
- Dose selection rationale: The exposure levels were chosen on the basis of previous toxicity data, limited to an acute oral LD50 of 3526 mg/kg in rats with the objective of estimating a maximum tolerated dose.
- Rationale for animal assignment: random
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine and blood, plasma
- Time and frequency of sampling: Urine samples were obtained from all surviving rats/sex/group on day 13 and blood samples were collected on day 14 at 6:30 a.m., 1:30 p.m and 3:30 p.m. ((± 30 minutes).

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine and blood plasma
- Time and frequency of sampling: Urine samples were obtained from all surviving rats/sex/group on day 13 and blood samples were collected on day 14 at 6:30 a.m., 1:30 p.m and 3:30 p.m. ((± 30 minutes).
- From how many animals: (samples pooled or not): all animals
- Method type(s) for identification: LC-MS (urine) and HPLC-ESI-MS-MS (blood)
- Limits of detection and quantification: Refer to attachments

- Test Material Name: 2-(2-Phenoxyethoxy)acetic acid
Vendor: Ryan Scientific, Inc.
Lot#: R446803
Molecular Formula: C10H12O4
Molecular Weight: 196.2
Purity: NA

Test Material Name: Phenoxyacetic acid
CAS Number: 122-59-8
Vendor: Acros Organics
Lot#: A0282821
Molecular Formula: C8H8O3
Molecular Weight: 152.2
Purity: 99.4% (Reference: Van den Broek, 2012)

Internal Standards
Chemical Name: 13C2-2-(Phenoxyethoxy) acetic acid
Vendor: Chemalong Laboratories, LLC
Lot #: CA207503
This internal standard was used to facilitate quantitation of PEAA during mass spectral analysis.


Chemical Name: D5-Phenoxyacetic acid
Vendor: C/D/N Isotopes Inc.
Lot #: E57P2
This internal standard was used to facilitate quantitation of PAA during mass spectral analysis.

Chemical Name: D5-Diethylene Glycol Phenyl Ether
Vendor: Chemalong Laboratories, LLC
Lot #: CA204402
This internal standard was not used to facilitate quantitation of DiEPH during mass spectral analysis but is included in this report for future assays.

Standard Preparation:
A mixed stock solution was prepared at 10239 µg DiEPH, 10160 µg PEAA and 10101 µg PAA / mL acetonitrile. This mixed stock solution was further diluted with acetonitrile to prepare standards with concentrations between 0.0101 and 10.1 µg/mL. A 100-µL aliquot of mixed internal standard solution (D5-DiEPH at 10.0 µg/mL, D5-PAA at 10.0 µg/mL and 13C2-PEAA at 20.0 µg/mL) was added to each standard for a final volume of 1.0 mL.

Plasma Sample Preparation:
The following was added or performed:
- 20 µL of plasma was added to a micro-centrifuge tube
- 20 µL of acetonitrile
- Briefly vortex-mix samples
- Centrifuge samples at 15,000 rcf for 10 minutes
- 10 µL of supernatant was placed into a limited-volume autosampler vial
- 10 µL of internal standard solution (D5-DiEPH at 10.0 µg/mL, D5-PAA at 10.0 µg/mL and 13C2-PEAA at 20.0 µg/mL) was added
- 80 µL of Milli-Q Water
- Briefly vortex-mix samples
- Samples were analyzed by HPLC/ESI/MS/MS

Fortified Plasma Sample (Spike) Preparation:
The following was added or performed:
- 100 µL of plasma was added to a micro-centrifuge tube
- 100 µL of acetonitrile was added containing DiEPH, PEAA and PAA to achieve plasma concentrations of 0.5, 5 and 50 µg analyte / mL blood.
- Briefly vortex-mix samples
- Centrifuge samples at 15,000 rcf for 10 minutes
- 10 µL of supernatant was placed into a limited-volume autosampler vial
- 10 µL of internal standard solution (D5-DiEPH at 10.0 µg/mL, D5-PAA at 10.0 µg/mL and 13C2-PEAA at 20.0 µg/mL) was added
- 80 µL of Milli-Q Water
- Briefly vortex-mix samples
- Samples were analyzed by HPLC/ESI/MS/MS

Urine Sample Preparation:
The following was added or performed:
- 1000 µL of urine sample was placed in a 1-dram glass vial
- 100 µL of concentrated hydrochloric acid (HCl) was added
- Briefly vortex-mix samples
- Heat samples overnight at 80 °C
- Centrifuge samples at 3400 rpm for 10 minutes
- 100 µL of supernatant was placed into a clean 1-dram glass vial
- 900 µL of Milli-Q Water was added to the vial
- Briefly vortex-mix samples
- The diluted supernatant was further diluted with Milli-Q water to bring the sample in range for analysis (target concentrations between 0.01 – 10 µg/mL)
- 100 µL of internal standard solution (D5-DiEPH at 10.0 µg/mL, D5-PAA at 10.0 µg/mL and 13C2-PEAA at 20.0 µg/mL) was added, the final volume of each sample was 1.0 mL.
- Briefly vortex-mix samples
- Samples were analyzed by HPLC/ESI/MS/MS

Fortified Urine Sample (Spike) Preparation:
The following was added or performed:
- 900 µL of control urine sample was placed in a 1-dram glass vial
- 100 µL of Milli-Q water was added containing DiEPH, PEAA and PAA to achieve urine concentrations of 1, 10, 100 and 1000 µg analyte / mL urine
- 100 µL of concentrated hydrochloric acid (HCl) was added
- Briefly vortex-mix samples
- Heat samples overnight at 80 °C
- Centrifuge samples at 3400 rpm for 10 minutes
- 100 µL of supernatant was placed into a clean 1-dram glass vial
- 900 µL of Milli-Q Water was added to the vial
- Briefly vortex-mix samples
- The diluted supernatant was further diluted with Milli-Q water to bring the sample in range for analysis (target concentrations between 0.01 – 10 µg/mL).
- 100 µL of internal standard solution (D5-DiEPH at 10.0 µg/mL, D5-PAA at 10.0 µg/mL and 13C2-PEAA at 20.0 µg/mL) was added, the final volume of each sample was 1.0 mL.
- Briefly vortex-mix samples
- Samples were analyzed by HPLC/ESI/MS/MS

Analysis Conditions for Sample Analyser:
Pump: Agilent 1200 Series; G1312B
Autosampler: Agilent 1200 Series; G1367C
Column Heater: Agilent 1200 Series; G1316B
Mass Spectrometer: Agilent Triple Quad; G6410A
Analytical Column:Phenomenex Kintex Phenyl-Hexyl, 2.1 x 100 mm, 1.7 µm
Guard Column: None
HPLC Eluent - A = Milli Q water + 0.1% formic acid
B = Acetonitrile + 0.1% formic acid
Gradient:
Time (min) Flow Rate (mL/min) % A % B
0 0.400 98 2
0.5 0.400 98 2
8 0.400 60 40
9 0.400 0 100
10 0.400 0 100
10.5 0.400 98 2
14 0.400 98 2

Injection Volume: 5 µL
Column Temp: 35 °C
Source: ESI
Mode: MRM
Analyte Q1 Mass (amu) Q3 Mass (amu) Dwell (ms) Polarity CE (V)
PEAA 197 121 200 Positive 10
13C2-PEAA 199 121 200 Positive 10
DiEPH 183 121 200 Positive 6
D5-DiEPH* 188 121 200 Positive 6
PAA 151 93 200 Negative 10
D5-PAA 156 98 200 Negative 10
* This transition was collected but not used

Fragmentor:75 V
Dry Gas Heater: 350 °C
Dry Gas Flow: 12 L/min
Nebulizer Pressure: 30 psi
Sheath Gas Temp: 400 °C
Sheath Gas Flow: 12 L/min
Capillary Voltage:4000 V
Statistics:
Standard statistical methods were employed

Results and discussion

Preliminary studies:
not applicable
Main ADME results
Type:
metabolism
Results:
The relevant biomarkers were identified as (DiEPH), 2-(2-Phenoxyethoxy) acetic acid (PEAA) and Phenoxyacetic acid (PAA).

Toxicokinetic / pharmacokinetic studies

Details on absorption:
not applicable
Details on distribution in tissues:
not applicable
Details on excretion:
not applicable
Toxicokinetic parametersopen allclose all
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: Blood plasma: Half-life values based on 2-(2-Phenoxyethoxy) acetic acid (PEAA) blood levels were approximately 7.4 hours, on average, and ranged 3.9-15.6 hours
Test no.:
#2
Toxicokinetic parameters:
half-life 1st: Blood plasma: As Phenoxyacetic acid (PAA) was present in 50 per cent of the blood samples, systemic exposure (AUC24h) and elimination half-life (t½) values could not be calculated.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Qualitative Metabolic Biomarker Identification - A series of analytical screening experiments were conducted to determine the relevant biomarkers in rat plasma and urine following dietary administration of DiEPh at dose levels of 0, 250, 500, or 1000 mg/kg/day. The proposed metabolic scheme of DiEPh presented in the attachment Figure 1 which shows the likely metabolic pathways and related biomarkers that were screened. These target analytes included: ethylene glycol (EG), diethylene glycol (DEG), phenoxy ethanol (PE), phenol, hydroxylethoxy acetic acid (HEAA), diglycolic acid (DGA), glycolic acid (GA), and oxalic acid (OA), diethylene glycol mono phenyl ether (DiEPh), phenoxyethoxy acetic (PEAA), and phenoxy acetic acid (PAA). These screening experiments identified the relevant biomarkers as DiEPh, PEAA, and PAA. The levels of the other potential biomarkers were low relative to both background PEAA or PAA levels. Therefore, no definitive quantitative data was generated for EG, DEG, PE, HEAA, DGA, GA, or OA.

Quantitative Toxicokinetic Assessment - DiEPh was not present at levels above the lower limit of quantitation (LLQ) in most of the blood samples. The two metabolites, PEAA and PAA, were present at higher levels in the blood. PEAA was present well above the limit of quantitation and in nearly all of the blood samples from exposed animals, while PAA was present in 50 percent of the blood samples For this reason, systemic exposure (AUC24h) and elimination half-life (t½) values could only be calculated for PEAA. PEAA systemic exposure (AUC24h) values were dose-proportional in both males and females. Half-life values based on PEAA blood levels were approximately 7.4 hours, on average, and ranged 3.9-15.6 hours.

DiEPh, PEAA, and PAA all were present at concentrations above the LLQ in all urine samples from treated animals. On average, only 1.3 percent of the dose was excreted as DiEPh in the 24 hour urine, whereas 39.3 percent of the dose was excreted as PEAA and 4.3 percent of the dose was excreted as PAA. Urine levels of all three analytes were dose-proportional. These data suggest DiEPh was rapidly metabolized at all dose levels. A major portion of the dose was metabolized to PEAA (≥ 39.3% of the administered dose, 87.5% of the recovered dose). A smaller portion was metabolized to PAA (≥ 4.3% of the administered dose, 9.6% of the recovered dose). There was no apparent saturation of absorption, distribution or elimination at any dose level. Thus, DiEPh exhibits linear kinetics up to, and including, 1000 mg/kg/day

Bioaccessibility

Bioaccessibility testing results:
no information available

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Under the conditions of the study, there was no apparent saturation of absorption, distribution or elimination at any dose level and DiEPh exhibits linear kinetics up to, and including, 1000 mg/kg/day. DiEPh was rapidly metabolized at all dose levels with the major metabolites detected PEAA (≥ 39.3% of the administered dose) and PAA (≥ 4.3% of the administered dose). 50% of PEAA was present in blood samples with half-life average values of approximately 7.4 hours.
Executive summary:

Selected rat plasma and urine samples were screened for the relevant biomarkers following dietary consumption of rodent feed prepared to deliver diethylene glycol mono phenyl ether DiEPH doses of 0, 250, 500 and 1000 mg/kg/day. The relevant biomarkers were identified as (DiEPH), 2-(2-Phenoxyethoxy) acetic acid (PEAA) and Phenoxyacetic acid (PAA). Plasma and urine (following acid hydrolysis) samples were analyzed for these biomarkers by high performance liquid chromatography (HPLC), negative/positive ion electrospray ionization (-/+ESI), with mass spectrometry detection operating in multiple reaction ion monitoring (MRM) mode. PAA and PEAA were quantified using stable isotope internal standard calibration and diethylene glycol phenyl ether was analyzed using external standard calibration.

Qualitative Metabolic Biomarker Identification - A series of analytical screening experiments were conducted to determine the relevant biomarkers in rat plasma and urine following dietary administration of DiEPh at dose levels of 0, 250, 500, or 1000 mg/kg/day. The proposed metabolic scheme of DiEPh presented in the attachment Figure 1 which shows the likely metabolic pathways and related biomarkers that were screened. These target analytes included: ethylene glycol (EG), diethylene glycol (DEG), phenoxy ethanol (PE), phenol, hydroxylethoxy acetic acid (HEAA), diglycolic acid (DGA), glycolic acid (GA), and oxalic acid (OA), diethylene glycol mono phenyl ether (DiEPh), phenoxyethoxy acetic (PEAA), and phenoxy acetic acid (PAA). These screening experiments identified the relevant biomarkers as DiEPh, PEAA, and PAA. The levels of the other potential biomarkers were low relative to both background PEAA or PAA levels. Therefore, no definitive quantitative data was generated for EG, DEG, PE, HEAA, DGA, GA, or OA.

Quantitative Toxicokinetic Assessment - DiEPh was not present at levels above the lower limit of quantitation (LLQ) in most of the blood samples. The two metabolites, PEAA and PAA, were present at higher levels in the blood. PEAA was present well above the limit of quantitation and in nearly all of the blood samples from exposed animals, while PAA was present in 50 percent of the blood samples For this reason, systemic exposure (AUC24h) and elimination half-life (t½) values could only be calculated for PEAA. PEAA systemic exposure (AUC24h) values were dose-proportional in both males and females. Half-life values based on PEAA blood levels were approximately 7.4 hours, on average, and ranged 3.9-15.6 hours.

DiEPh, PEAA, and PAA all were present at concentrations above the LLQ in all urine samples from treated animals. On average, only 1.3 percent of the dose was excreted as DiEPh in the 24 hour urine, whereas 39.3 percent of the dose was excreted as PEAA and 4.3 percent of the dose was excreted as PAA. Urine levels of all three analytes were dose-proportional. These data suggest DiEPh was rapidly metabolized at all dose levels. A major portion of the dose was metabolized to PEAA (≥ 39.3% of the administered dose, 87.5% of the recovered dose). A smaller portion was metabolized to PAA (≥ 4.3% of the administered dose, 9.6% of the recovered dose). There was no apparent saturation of absorption, distribution or elimination at any dose level. Thus, DiEPh exhibits linear kinetics up to, and including, 1000 mg/kg/day.