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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
other: bioaccumulation
Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: Commission Directove 2002/72/EC relating to plastiic materials and articles intended to come into contact with foodstuffs
Version / remarks:
6 August 2002
Principles of method if other than guideline:
The present study was designed to provide data on the possible bioaccumulation of radio-labelled C14 test substance in male and female Wistar rats following a single oral dose of 100 mg/kg body weight. This was achieved by:
- estimating tissue distribution of possible absorbed radioactivity at two time-points
post dose
- estimating the rate and route of excretion of radioactivity
- estimating the radioactivity in blood at various time points post-dose
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
424-650-3
EC Name:
-
Cas Number:
182121-12-6
Molecular formula:
C17H18O2
IUPAC Name:
9,9-bis-(methoxymethyl)fluorene
Test material form:
solid
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Laboratory rats were selected as standard for rodent species. The strain was chosen to comply with the corresponding toxicological
studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: At the time of dosing the animals were 9-10 weeks old.
- Weight at study initiation: At the commencement of the study, the weight variation of the animals did not exceed ± 20% of the average weight (mean females: 177g; males: 272g)
- Housing: During acclimatisation, all animals were housed in macrolon cages with wood shavings (Lignocel 3/4) as bedding, 4 to 5 rats per cage. During the study they were housed individually in open Plexiglas metabolism cages (Techniplast™).
- Diet (e.g. ad libitum): The rats were allowed to have free access to a commercial rat diet. Each batch of this diet was analysed by the supplier (SDS, Special
Diets services, Whitham, England) for nutrients and contaminants.
- Water (e.g. ad libitum): Drinking water was offered ad libitum at all times. The drinking water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 98/83/EC) was supplied by N.V. Hydron, Midden-Nederland.
- Acclimation period: At least 5 days prior to the experimental start date to the laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3°C
- Humidity (%): 40 - 70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

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

2 ml of ethanol was added to one vial of [14C]9,9-bis(methoxymethyl)fluorene and sonicated for 90 seconds, giving a radioactivity concentration of 1 mCi/ml
(37 MBq/ml). 1 ml of this solution was added to 496.9 mg of non-radioalabeled 9,9- bis(methoxymethyl)fluorene, and evaporated to dryness under a stream of nitrogen. Subsequently, corn oil was added to a total weight of 20.0 gram, and the test substance dissolved. This dose solution contained 25.03 mg 9,9-bis(methoxymethyl)fluorene per gram of corn oil corresponding to a radioactivity concentration of 54.82 μCi per gram. of corn oil. The specific activity of the test substance in the dose solution was 2.19 μCi/mg (81.0 kBq/mg). The dose solution was prepared on the Friday before dose administration (Monday, February 20th), and stored in the refrigerator. On the day of dosing, the solution was brought to room temperature, and continuously stirred with a magnetic bar. The actual (radioactivity) concentration was checked at the time of administration. Homogeneity was checked by taking aliquots just before and immediately after dosing. The test substance was administered as a single oral dose by stomach tube. The exact amount given was determined retrospectively by the difference in weight of the stomach tube plus syringe before and after administration of the formulated test substance. The administered dose and time of dosing were recorded for each rat.
Duration and frequency of treatment / exposure:
Single dose by gavage
Doses / concentrations
Dose / conc.:
100 mg/kg bw (total dose)
Remarks:
corresponding to a radioactive dose of 200 μCi/kg bodyweight.
No. of animals per sex per dose / concentration:
8 males and 8 females (4 male/females per group)
Control animals:
no
Details on study design:
- Dose selection rationale: To ensure that food is not contaminated at an unacceptable level, the plastic directive stipulates an overall migration limit
(OML) of 60 mg/kg food. This is based on the assumption that humans consume 1 kg of packaged food contaminated at the OML every day of their life, and have a body weight of 60 kg. This means an exposure to maximally 60 mg of contamination, or 1 mg per kg body weight. Taking into account a toxicological safety factor of 100, this means that animal tests should reveal a no-effect-level (NOEL) of 100 mg/kg body weight.

Two groups are dosed with 100 mg/kg bw..
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, cage wash , tissues
- Time and frequency of sampling:
- Blood samples- (tail blood, 100 Zl) were collected at various
time-points: 30 min, 1h, 2h, 4h, 6h, 8h, 24h, 48h and 96h postdose. At sacrifice (168 h post-dose) blood was collected from the abdominal aorta.
- Urine and faeces were collected at 24 hour intervals.
- At sacrifice (t=24 hours post dose & t=7 days post-dose) the following organs and tissues were collected:
Liver, Brain, Bone (Femur), Kidneys, Testes/Uterus*, Muscle, Lungs, Prostate/Ovaries*, Skin, Heart, Adrenals, Spleen, Fat, Blood/plasma, GI-tract and contents, Residual carcass
*depending on the sex

Radioactivity was determined in all collected samples. All samples to be counted were weighed.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Following oral administration of test substance, the highest concentrations of radioactivity in blood were found around 4 (males) and 6 hours (females) post-dose.
The blood data of the male animals most accurately followed first-order kinetics with a half-life (T1/2) of about 20 hours.

Assuming that the parent compound in the faeces was excreted directly, and that no metabolism occurred in the intestines, the absorption of test substance was high: 88% in males and 96% in females.
Details on distribution in tissues:
Residues of radioactivity were determined in organs and tissues of both sexes after 24 and 168 hours following dose administration. After 24 hours, 2.4 - 3.9% of the radioactivity was present in excised tissues, organs and residual carcass. After 7 days, this percentage was 0.6 – 1.0%.

After 24 hours, the highest concentrations of radioactivity, excluding GI tract+contents, were found in liver, kidney, adrenals, ovaries and fat. After 168 hours, the highest concentrations were found in adrenals, ovaries and fat.

The data show that especially in fat, adrenals, uterus, ovaries, muscle, skin, fat and residual carcass, depletion of radioactivity was rather low. The lowest rate of depletion was found in fat tissue, which also contained the highest radioactivity concentrations after 7 days (7.1 – 10.7 μg/g).
Details on excretion:
The recovery in individual rats was between 97.9 and 105.6%.
A clear difference was found between males and females regarding excretion in urine and faeces. In males, more radioactivity was excreted in faeces in comparison with urine. In females, however, the majority of radioactivity was excreted in urine. In addition to the amount of radioactivity excreted in urine and faeces, a sex effect was also found with respect to the rate of excretion: females excreted radioactivity more rapidly than males.

Metabolite characterisation studies

Metabolites identified:
yes
Remarks:
measured but not identified
Details on metabolites:
Urine contained a relatively large number of metabolites, containing one major metabolite eluting at 13.3 minutes. In faeces, three major peaks were found. The peak eluting at 42.7 minutes was the parent compound, which was confirmed by co-elution. No parent compound was observed in urine. In faeces, the parent compound could be detected in males up to 3 days after the dose administration, whereas in females it was detected only the first 24 hours.

Any other information on results incl. tables

No study- or test substance-related signs of toxicity or unusual behaviour were observed.

Table 1 Calculated pharmacokinetic parameters

Parameter

 

Males

Females

Cmax

3.75 μg.g-1

3.27 μg.g-1

Tmax

4.0h

5.7h

T1/2 (α)

20.5h

7.6h

T1/2 (β)

-

58.4h

AUC

127 μg.g-1h

70.2 μg.g-1h


Table 2 Total excretion of radioactivity in urine and faeces

Parameter

 

Males

Females

Urine

44.0

72.0

Faeces

53.7

28.8

 

Table 3 Tissue distribution and depletion in tissues containing the highest radioactivity levels (μg/g).

Tissues

Males

Females

24h

168h

24h

168h

Adrenals

5.1

3.3

11.6

5.0

Prostate

3.2

0.7

-

-

Uterus

-

-

2.3

1.2

Ovaries

-

-

6.3

3.3

Skin

2.0

0.8

3.4

1.5

Fat

9.5

7.1

20.1

10.7

 

Table 4 Total amount of radioactivity present as metabolites or parent compound in urine and faeces as % of the administered dose (only major metabolites).

Metabolite

Males

Females

Urine

Faeces

Urine

Faeces

13.6 min

26.0

-

42.5

-

19.3 min

2.8

24.4

3.6

13.0

34.0 min

0.2

12.6

1.0

9.7

parent

-

12.3

-

4.0

Applicant's summary and conclusion

Conclusions:
- Blood kinetics demonstrated a difference between males and females regarding the calculated AUC. In females the AUC was about twice as low as in males, indicating a more rapid elimination in females. This was confirmed by the excretion pattern.
- Absorption of radioactivity was high: 88% in males and 96% in females.
- A clear difference was observed between males and females regarding the excretion pattern in urine and faeces: in females, excretion of radioactivity in urine was much higher than in faeces (72% versus 29%, respectively), while in male animals excretion in urine was lower than in faeces (44% versus 54%, respectively).
- In some of the organs/tissues, elimination of radioactivity seemed to be rather slow, especially in fat. In fat tissue, 75% (males) and 53% (females) of the radioactivity found after 24 hours was still present after 7 days. This could indicate bioaccumulation. Therefore, further investigations (repeated oral dose study) might be required.