Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 January 1999 to 26 May 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to EC and OECD test guidelines, and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Objective of study:
absorption
metabolism
other: biokinetics in blood
Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU 88/302/EEC (part B)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzoflex 2-45
- Substance type: Technical material
- Physical state: Liquid
- Analytical purity: (non-radiolabelled) 99.0%
- Lot/batch No.: Non-radiolabelled: 558260; Radiolabelled: HLS Batch Number SPS/VCL319/12
- Expiration date of the lot/batch: Non-radiolabelled: September 1999
- Radiochemical purity (if radiolabelling): ≥ 99.09% (by TLC); 99.3% (by HPLC)
- Specific activity (if radiolabelling): 28.52 mCi/mmol
- Locations of the label (if radiolabelling): [ring-U-14C-phenyl]
- Storage condition of test material: < - 15°C
Radiolabelling:
yes
Remarks:
ring-U-14C phenyl

Test animals

Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-10 weeks
- Weight at study initiation: approx 200 g
- Fasting period before study: no data
- Housing: stainless steel cages with suspended mesh floors
- Individual metabolism cages: yes
- Diet: LAD 1 ad libitum):
- Water: ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%): RH 40 - 70%
- Air changes (per hr):no data
- Photoperiod: 12hrs dark / 12 hrs light):

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet: Freshly prepared

HOMOGENEITY AND STABILITY OF TEST MATERIAL: Radiolabelled and non radiolabelled DEGDB were mixed together in methanol to achieve the desired specific activity for the dose material The solvent was removed under a stream of nitrogen and the remaining material suspended in corn oil.
Duration and frequency of treatment / exposure:
Excretion: Single doses at a nominal dose level of 50 mg/kg for 120 hours. Similar regime for high dose, 750 mg/kg.
Blood/plasma radioactivity kinetics: Single nominal dose of 50 mg/kg and 750 mg/kg for up to 120 hours
Tissue distribution: Single nominal dose of 50 mg/kg for up to 48 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
Single low dose 50 mg/kg,
Single high dose 750 mg/kg
No. of animals per sex per dose:
4
Control animals:
not specified
Details on study design:
- Dose selection rationale: Low dose (50 mg/kg) and high dose (750 mg/kg).
- Rationale for animal assignment : Equal numbers of male and female to eradicate any difference in responses of the sexes.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum, Adrenal glands, Lungs, Bone marrow (femur), Muscle (skeletal), Brain, Ovaries/prostate, Fat (abdominal), Skin, Gastro intestinal tract (including contents), Spleen, Heart, Thyroid, Kidneys, Uterus/testes, Liver, Residual carcass
- Time and frequency of sampling: 0.25, 0.5, 1, 2, 3, 4, 6, 12, 24, 48, 96, and 120 hours


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, cage washings, solvent extracts and other liquid samples.
- Time and frequency of sampling: 24 hour intervals, urine additionally at 0, 6 and 6 24 hours up to 120 hours into receivers cooled with solid carbon dioxide.
- From how many animals: samples pooled from 8 rats, 4 male and 4 female.
- Method type(s) for identification: HPLC, Liquid scintillation counting, TLC



Results and discussion

Main ADME resultsopen allclose all
Type:
excretion
Results:
Low level (50 mg/kg) - There was no measurable radioactivity detected in the gastrointestinal tract.
Type:
excretion
Results:
High level (750 mg/kg) - There was no measurable radioactivity detected in the gastrointestinal tract.
Type:
other: Blood/plasma concentration
Results:
In general there was much variation between individual rats within each group due presumably to the very rapid absorption into and elimination from the systemic circulation.
Type:
distribution
Results:
The tissue distribution of the radiolabel was variable but not remarkable. There was no substantial difference between male and female animals.
Type:
excretion
Results:
DEGDB was completely metabolised by the rat as no parent compound was observed in urine.

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
Concentrations were measured in pairs of rats (1 male, 1 female) killed at 0.25, 1, 4, 12 and 48 hours after single low level doses.

At 0.25 hours after dosing highest concentrations were in the gastrointestinal tract (418.4, 393.3 µg equiv/g) and kidney (129.1, 86 66 µg equiv/g. Intermediate levels were observed in the liver (12.14, 7.77 µg equiv/g), plasma (23.33, 18.53 µg equiv/g) thyroid (12.24, 19.19 µg equiv/g)
and whole blood (16.53, 9.73 µg equiv/g). Concentrations in the remaining tissues were in the range 1.16-8.94 µg equiv/g.

At 1 hour after dosing concentrations in tissues had generally decreased. Highest concentrations were in the gastrointestinal tract (110.5, 161.8 µg equiv/g) and kidney (68.75, 40.06 µg equiv/g). Intermediate levels were observed in the fat (11.91, 1.32 µg equiv/g), spleen (13.83, 2.71 µg equiv/g) and thyroid (12.73, 2.04 µg equiv/g). Concentrations in the remaining tissues were in the range 0.33-8 51 µg equiv/g.

At 4 hours after dosing concentrations in tissues had generally decreased further. Apart from the prostate and thyroid, highest concentrations were in the gastrointestinal tract (153.5, 134.8 µg equiv/g). The apparently high levels in these tissues in the male rat (61.84 and 57.10 µg equiv/g respectively) should be regarded as anomalous as they were considerably out of line with the concentrations at other times. Intermediate levels were observed in the fat (0.42, 4.85 µg equiv/g), kidney (4.20, 6.08 µg equiv/g) and lung (2.72, 0.23 µg equiv/g). Concentrations in the remaining tissues were in the range <0.04-1.98 µg equiv/g.

At 12 hours after dosing highest concentrations were in the gastrointestinal tract (4.59, 8.06 µg equiv/g). Intermediate levels were observed in the kidney (0.86, 1.70 µg equiv/g) and skin(0.70, 2.05 µg equiv/g). Concentrations in the remaining tissues were in the range <0.04-0.79 µg equiv/g.

At 48 hours after dosing concentrations ofradioactivity in most tissues had decreased to around the limit of detection (0.04-0.43 µg equiv/g. The highest tissue concentrations were observed in the gastrointestinal tract (0.42, 0.83 µg equiv/g) and skin (0.54, 1.52 µg equivg). Measurable
concentrations in the remaining tissues were in the range 0.23- 0.24 µg equiv/g.
Details on excretion:
Low level (50 mg/kg) - After single low level doses of 14C DEGDB to rats (4 male, 4 female) means of 104.9% (male) and 102.2% (female) dose were excreted in the urine (including cagewash) during 0-120 hours. Most ofthis radioactivity was excreted in the 0-24 hour urine: 103.9% (male) and 100.2% (female) dose. During the 5 days after dosing, means of 0.33% (male) and 1.22% (female) dose were excreted in the faeces with most of this in the 0-24 hour faecal samples: 0.30% (male) and 0.84% (female) dose. No measurable radioactivity was detected in expired air. After sacrifice at 120 hours, radioactivity in the remaining carcass was below the limit of quantification in males and 0.35% dose in females. There was no measurable radioactivity detected in the gastrointestinal tract. Thus means of 105.2% (male) and 103.8% (female) dose were recovered after 5 days.

High level (750 mg/kg) - After single high level doses of 14C DEGDB to rats (4 male,4 female) means of 99.79% (male) and 102.1% (female) dose were excreted in the urine( including cagewash) during 0-120 hours. Most ofthis radioactivity was excreted in the 0-24 hour urine 97.55% (male) and 100.4% (female) dose. During the 5 days after dosing means of 0.59% (male) and 0.41% (female) dose were excreted in the faeces with most of this in the 0-24 hour faecal samples: 0.57% (male) and 0.32% (female) dose. No measurable radioactivity was detected in expired air. After sacrifice at 120 hours, radioactivity in the remaining carcass accounted for 0.25% (male) and 0.29% (female) dose. There was no measurable radioactivity detected in the gastrointestinal tract. Thus means of 100.6% (male) and 102.7% (female) dose were recovered after 5 days.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Benzoic acid, Hippuric acid and Diethylene glycoI monobenzoate.

Any other information on results incl. tables

Virtually all of a 50 mg/kg and a 750 mg/kg single oral dose of14C DEGDB was absorbed and excreted in the urine mostly within 24 hours of dosing. Only 0.33 - 1.22 % of the dose was excreted in the faeces whilst up to 0.35% dose was retained in the carcass after 120 hours. There was no appreciable difference in the pattern of excretion of radioactivity between dose levels or between male and female rats.

There was a rapid clearance of radioactivity from the systemic circulation with highest measured concentrations at 0.25 (50 mg/kg or

1.0 (750 mg/kg) hours after dosing. There was a disproportionately higher systemic exposure of radioactivity at 750 mg/kg compared to that at 50 mg/kg. The areas under the blood plasma concentration curves (AUCt) were on average 3 and 4 -fold higher for plasma and whole blood respectively after a 750 km/kg dose than would be predicted from a linear relationship from the 50 mg/kg dose.

The tissue distribution of the radiolabel was variable but not remarkable. After a single oral dose at 50 mg/kg, concentrations of radioactivity in tissues were generally highest at the time of the highest measured plasma concentration (0.25 hours). Apart from unabsorbed material in the gastrointestinal tract, highest concentrations of radioactivity were in kidney 86.66 - 129.1 µg equiv/g, thyroid 12.24 - 19.19 µg equiv/g and liver 7.77 - 12.14 µg equiv/g. Concentrations in these tissues were consistent with the measured concentrations in plasma (18.53 - 23.3 µg equiv/g after 0.25 hours). Concentrations in other tissues after 0.25 hours lay in the range 1 - 9 µg equiv/g. At 48 hours after dosing concentrations of radioactivity in most tissues had decreased to around the limit of detection There was no substantial difference between male and female animals.

DEGDB was completely metabolised by the rat as no parent compound was observed in urine. Dose dependent metabolism was observed at the higher dose level of 750 mg/kg. The major metabolite in urine was identified chromatographically as hippuric acid. Hippuric acid represented 94 - 97% of a 50 mg/kg dose and 85 - 88% of a 750 mg/kg dose. A glucuronide of benzoic acid represented 1% of a 50 mg/kg dose and 6 - 9% of a 750 mg/kg dose.

The metabolism of DEGDB therefore appears to proceed via hydrolysis of the ester bonds to form benzoic acid which is then conjugated with glycine (major pathway) or glucuronic acid (minor pathway) prior to excretion.

Doses within the range of 500 to 750 mg/kg are suggestive of saturating the normal conjugation and excretion mechanism of diethylene glycol dibenzoate. Any toxicity observed in this dose range or higher needs to be considered carefully since the normal metabolism of this substance is altered.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
Virtually all of single oral doses of 50 and 750 mg/kg of [ring-U-14C-phenyl] DEGDB administered to Sprague Dawley CD rats were absorbed, metabolised and excreted in the urine within 24 hours of administration. DEGDB was metabolised via hydrolysis of the ester bonds to benzoic acid; this free acid was then conjugated with either glycine (major pathway) or glucuronic acid (minor pathway) prior to excretion.
Executive summary:

A study was performed to investigate the metabolism of the14C radiolabelled test substance DEGDB when orally dosed to male and female Sprague Dawley (CD) rats. The study was conducted to EU and to OECD test guidelines and to GLP. Virtually all of single oral doses of 50 and 750 mg/kg of ring [U-14C phenyl] DEGDB administered to the rats were absorbed metabolised and excreted in the urine within 24 hours of administration. DEGDB was metabolised via hydrolysis of the ester bonds to benzoic acid; this free acid was then conjugated with either glycine (major pathway) or glucuronic acid (minor pathway) prior to excretion.