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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not given. The final report was signed on Feb. 11, 2014.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted in the spirit of GLP, but no claim of GLP compliance was made.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Objective of study:
absorption
metabolism
bioaccessibility
Principles of method if other than guideline:
The study consists of the following 3 in vitro tests that are in wide use for studying intestinal absorption and metabolism of pharmaceuticals and other chemicals:

1. Small intestinal absorption of the test compound is studied using an everted rat proximal small intestine in vitro. The everted intestinal sacs are place in flasks filled with “Fed-State”-Simulated Intestinal Fluid that contains the test compound. After incubation period of 1 h the buffer inside the sacs (serosal side of the intestine) is collected and analysed for the presence of the test compound.

2. The test compound is incubated with rat liver microsomal fraction. Serial samples of the reaction mixture are taken and analysed for the presence of the test compound and its metabolites.

3. The test compound is incubated with rat hepatocytes. Serial samples of the reaction mixture are taken and analysed for the presence of the test compound and its metabolites.
GLP compliance:
no
Remarks:
The study was conducted in the spirit of GLP, but no claim of GLP compliance was made.

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): NEXBTL renewable diesel
- Substance type: UVCB

Certificate of analysis presented in Appendix 5.

Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: 1. Han/Wistar 2 and 3. Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Sources:
1. Harlan, Bicester, UK
2. Microsomes obtained from BD Gentest, USA
3. Cryopreserved hepatocytes obtained from In-Vitro Technologies GmbH, Germany

- Age at study initiation: 1. 8-12-weeks; 2.-3. not given
- Weight at study initiation: not given
- Fasting period before study: not given
- Housing: not given
- Individual metabolism cages: not given
- Diet (e.g. ad libitum): not given
- Water (e.g. ad libitum): not given
- Acclimation period: not given

ENVIRONMENTAL CONDITIONS
not given

IN-LIFE DATES: not given

Administration / exposure

Route of administration:
other: in vitro
Vehicle:
other: 1. “Fed-State”-Simulated Intestinal Fluid, 2. Reaction mixture, 3. Cell culture medium (10% foetal calf serum Leibowitz CL 15)
Duration and frequency of treatment / exposure:
1. In vitro exposure, samples at 1 h
2. In vitro exposure, samples prior to starting the reaction and at 2, 5, 10, 15, 20 and 30 min
3. In vitro exposure, samples at 0, 15, 30, 60, 90 and 120 min
Doses / concentrations
Remarks:
Doses / Concentrations:
1. 2 µl/ml
2. 10 mM
3. 10 mM
No. of animals per sex per dose:
1. 3
2. –
3. -
Control animals:
no
Positive control:
1. A mixture of the alkenes n-decane, n-dodecane, n-hexadecane, n-octadecane and n-eicosane (2 µl/ml)
Details on study design:
- Dose selection rationale:
1. Not given
2.-3. To achieve analytical sensitivity
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma, serum or other tissues, cage washes, bile.
- Time and frequency of sampling: 1. 1 h

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, tissues, cage washes, bile
- Time and frequency of sampling:
2. Prior to starting the reaction and at 2, 5, 10, 15, 20 and 30 min
3. At 0, 15, 30, 60, 90 and 120 min

- Method type(s) for identification: 2.-3. GC-FID, LC-MS
- Limits of detection and quantification: No data
Statistics:
No data.

Results and discussion

Preliminary studies:
no
Main ADME resultsopen allclose all
Type:
absorption
Results:
1
Type:
metabolism
Results:
2
Type:
metabolism
Results:
3

Toxicokinetic / pharmacokinetic studies

Details on absorption:
1. The rat everted intestinal sac incubations showed that no hydrocarbon constituents of NEXBTL renewable diesel were present in the serosal fluid of the intestinal sac at levels greater than the limit of detection (for the main constituents C15-C18 approximately 1% of the starting concentration). This implies that the main constituents of NEXBTL renewable diesel are not readily absorbed across the gut wall and are therefore not highly bioavailable. The lower carbon chain length alkanes present in NEXBTL renewable diesel were also not detected in the serosal fluid from the intestinal sacs. Studies with the positive control (n-alkane mixture) indicated that n-decane (C10) and to lesser extent n-dodecane (C12) were absorbed into the serosal fluid. However, n-hexadecane (C16), n-ocatdecane (C18) and n-eicosane (C20) were not detected, implying that longer chain alkanes are not absorbed.

These findings demonstrate that the experimental model was successful in showing uptake of lower carbon chain length alkanes by the gut sac model, while an absence of uptake of C16 and above is consistent with other unpublished studies conducted in this laboratory on relevant structures. Together, these findings demonstrate the validity of the model used. They also provide an explanation for the absence of detectable C15-C18 alkanes in serosal fluid from gut sac incubations containing NEXBTL renewable diesel, while suggesting that uptake of the ≤C14 fraction may occur.
Details on distribution in tissues:
Not applicable.
Transfer into organs
Transfer type:
other: intestinal mucosa/intestinal serosa
Observation:
slight transfer

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
2.-3. No metabolic loss of any of the constituents of NEXBTL renewable diesel could be detected in the samples from the rat liver microsomal and the rat cryopreserved hepatocyte incubations. Nor was the metabolite formation of chosen representative constituents of NEXBTL renewable diesel (C10, C12, C14, C16, C18 and C20 n-alkanes) detected in the samples from the rat liver microsomal and the rat cryopreserved hepatocyte incubations. This could be due to the limits of detection for the lower carbon chain length alkane metabolites in the LC-MS system and the very small amounts of lower carbon chain length alkanes present in incubations performed using NEXBTL renewable diesel.

The absence of detectable metabolism of NEXBTL renewable diesel constituents recorded in this study is consistent with the published data showing a negative correlation between metabolic clearance and chain length of n-alkanes (Anand et al., 2007). The results are also consistent with published data showing virtually no biotransformation of related alkane structures by rat and human microsomal fractions in vitro and by rats in vivo (reviewed by INRA, 2012). This could be due to the limits of detection for alkane metabolites in the LC-MS system used in this investigation.


References

Anand S.S., Campbell J.L., Fisher J.W. In-vitro rat hepatic metabolism of n-alkanes: nonane, decane and tetradecane. Int. J. Toxicol. 2007; 26: 325-329.

INRA. In vitro metabolic study on alkanes in hepatic microsomes from humans and rats. Report from Institute National de la Recherche Agronomique – INRA. European Food Safety Authority Supporting Publications 2012: EN-263, 64 pp. Available from www.efsa.europa.eu

Bioaccessibility

Bioaccessibility testing results:
Resuts of the everted intestinal sac model imply that NEXBTL renewable diesel is not readily bioavailable.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
The main constituents of NEXBTL renewable diesel are not readily absorbed across the gut wall and are therefore not highly bioavailable as indicated by studies with the rat everted intestinal sac model. No metabolic loss of any of the constituents of NEXBTL renewable diesel could be detected in the samples from the rat liver microsomal and the rat cryopreserved hepatocyte incubations. Nor was the metabolite formation of chosen representative constituents of NEXBTL renewable diesel (C10, C12, C14, C16, C18 and C20 n-alkanes) detected in the samples from the rat liver microsomal and the rat cryopreserved hepatocyte incubations.
Executive summary:

This in-vitro study was performed to fulfill requirements of non-EU legislation. The objective of this study was to develop screening-level information that would assist in an estimation of the extent to which the hydrocarbon species present in NEXBTL renewable diesel would be absorbed and metabolised in vivo. The aims were to determine

  1. the rat small intestinal absorption potential of NEXBTL renewable diesel
  2. the rat liver microsomal metabolism of NEXBTL renewable diesel and to identify principal metabolites formed, if any
  3. to determine the rat hepatocyte metabolism of NEXBTL renewable diesel and identify principal metabolites formed, if any

NEXBTL renewable diesel comprises of a range of branched and linear alkanes of differing carbon chain lengths (C10-C20) with the main constituent located at C15-C18 according to the GC-FID analysis.

The results from the rat everted intestinal sac incubations showed that no hydrocarbon constituents of NEXBTL renewable diesel were present in the serosal fluid of the intestinal sac at levels greater than the limit of detection (for the main constituents C15-C18 approximately 1% of the starting concentration). This implies that the main constituents of NEXBTL renewable diesel are not readily absorbed across the gut wall and are therefore not highly bioavailable. The lower carbon chain length alkanes present in NEXBTL renewable diesel were also not detected in the serosal fluid from the intestinal sacs.

Additional gut sac incubations were conducted to determine whether intestinal uptake was likely to vary with changes in alkane chain length. The results showed that n-decane (C10) and to lesser extent n-dodecane (C12) were absorbed into the serosal fluid. However, n-hexadecane (C16), n-ocatdecane (C18) and n-eicosane (C20) were not detected, implying that longer chain alkanes are not absorbed. These findings suggest that uptake of alkanes by the gut sac model varies with carbon number, and support the absence of detectable uptake of C15-C18 alkanes in serosal fluid from gut sac incubations containing the “whole” NEXBTL renewable diesel. They also suggest that some uptake of the ≤C14 fraction may occur.

No metabolic loss of any of the constituents of NEXBTL renewable diesel could be detected in the samples from the rat liver microsomal and the rat cryopreserved hepatocyte incubations. Nor was the metabolite formation of chosen representative constituents of NEXBTL renewable diesel (C10, C12, C14, C16, C18 and C20 n-alkanes) detected in the samples from the rat liver microsomal and the rat cryopreserved hepatocyte incubations.