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Administrative data

Link to relevant study record(s)

Reference
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25-11-2013 to 11-3-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to compare the metabolism in vitro of the test substance using cryopreserved hepatocytes from rat, rabbit and human. Incubations were conducted with cryopreserved hepatocytes in duplicate for each species at three test substance concentrations (5, 25 and 100 µM) over three incubation times (0, 1 and 4 h). A radio-HPLC method was developed and used to determine the metabolite profiles generated during the hepatocyte incubations with test substance.
GLP compliance:
yes (incl. QA statement)
Remarks:
Huntingdon Life Sciences, Huntingdon Research Centre, Woolley Road Alconbury, Huntingdon Cambridgeshire, PE28 4HS UK
Radiolabelling:
yes
Details on test animals or test system and environmental conditions:
Rat (Sprague-Dawley), rabbit (New Zealand White) and human cryopreserved hepatocytes were obtained from Bioreclamation IVT (formerly Celsis IVT) and delivered stored frozen in liquid nitrogen. All hepatocytes were from male donors.
Route of administration:
other: in vitro application in liquid medium
Vehicle:
ethanol
Details on exposure:
- Species: Rat, rabbit and human
- Test substance concentrations: 5, 25 and 100 μM
- Incubation times: 0, 1 and 4 h
- Number of replicates: 2
- Cell concentration: 1 x 10^6 viable cells per mL of culture medium
- Volume of incubation medium: 1 mL
Duration and frequency of treatment / exposure:
0, 1 and 4 h
Details on study design:
INCUBATION CONDITIONS
- The incubation components were mixed together in glass vials so that each sample contained the following: Foetal calf serum (100 µL), Supplemented Williams’ Medium E (890 µL – volume of hepatocyte suspension), Hepatocyte suspension (volume containing 1 x 10^6 viable cells), Test substance (10 µL of solution in ethanol).
- The vials for the 0 h incubations were sealed immediately after preparation and the reactions stopped.
- At the end of the requisite incubation period, the reactions were stopped by transferring the sealed incubation vials on to solid carbon dioxide. The contents of the vials were allowed to freeze completely (minimum of approximately 10 min) prior to transfer to storage at approximately -20°C pending analysis.
-In addition, viability samples were incubated to assess LDH activity and leakage. These consisted of duplicate 0.5 mL incubations with hepatocytes (no test substance) from each species for 0, 1 and 4 h, together with single 0.5 mL incubations with hepatocytes from each species andtest substance (100 µM) for 1 and 4 h.
- the hepatocyte incubations with test substance were conducted in duplicate for each species (rat, rabbit and human), at each incubation time (0, 1 and 4 h) and at each concentration (5, 25 and 100 µM).

CONTROL INCUBATIONS
- Incubation of test substance for 0, 1 and 4 h in the absence of hepatocytes (singly at each concentration).
- Incubation with hepatocytes for 4 h in the absence of test substance (single sample per species). These samples were not analysed, but were prepared to permit a subsequent assessment of endogenous materials if required.
- Positive control samples incubating [14C]7-EC at a concentration of 50 µM for 4 h with and in the absence of hepatocytes (each conducted in duplicate).

MEASUREMENT OF LDH LEAKAGE
- Duplicate 0.5 mL incubations with hepatocytes (no test substance) from each species for 0, 1 and 4 h, together with single 0.5 mL incubations with hepatocytes from each species and the test substance (100 μM) for 1 and 4 h. The methodology for further treatment was adapted from Howes 1990.

ANALYSIS OF 7-ETHOXY[14C]COUMARIN INCUBATIONS
-Following storage at approximately -20°C, the 7-EC samples were centrifuged (18,620 x g, 15 min, 4°C). The resulting supernatants were transferred to clean tubes and evaporated to dryness using a centrifugal evaporator.
-The samples were reconstituted in 40 mM ammonium formate pH 5.0 (1 mL, with one exception where 1.35 mL was used was used to accommodate a heat control) and an aliquot (0.45 mL) of each was taken into a clean tube for use in deconjugation. The remainder of each reconstituted supernatant was transferred to a HPLC vial.
-Deconjugation was conducted by incubating (1 h, 37°C) the aliquots of the reconstituted supernatant with ß-glucuronidase enzyme (2000 units, type H1 from Helix pomatia, also containing sulfatase activity). Samples were then transferred to HPLC vials.
Positive controls for ß-glucuronidase and sulfatase enzyme activities were determined by the production of free phenolphthalein from phenolphthalein glucuronic acid and p-nitrocatechol from p-nitrocatechol sulfate, respectively, upon the addition of 1 M sodium hydroxide, after incubation (1 h, 37°C).
-The percentage of sample radioactivity associated with 7-EC and the known metabolite 7-hydroxycoumarin (7-HC) was determined for untreated and deconjugated samples by HPLC.
Details on dosing and sampling:
SAMPLE PREPARATION
Hepatocyte incubation samples (0, 1 and 4 h) were treated as follows (samples incubated in the absence of hepatocytes were analysed directly without filtration):
-Each sample was allowed to thaw on ice and then was vortex mixed briefly. Duplicate aliquots (50 µL for 5 and 25 µM incubations and 25 µL for 100 µM incubations) were taken into separate scintillation vials. Ultima Gold scintillation cocktail (approximately 5 mL) was added to each vial and the vials were submitted to LSC. An aliquot (approximately 250 µL) was transferred into a syringe body and was then passed through a Pall Acrodisc GHP filter (13 mm, 0.2 µm) directly into an HPLC vial.
-Duplicate aliquots (as above) of the filtrate were taken into separate scintillation vials, Ultima Gold scintillation cocktail (approximately 5 mL) was added to each vial and the vials were submitted to LSC to assess the recovery of radioactivity following filtration.
-Due to a relatively low recovery of radioactivity following filtration of the 0 h samples, the original incubation samples were allowed to thaw on ice and were then vortex mixed briefly. An aliquot (approximately 250 µL) of each was transferred into a microcentrifuge tube and centrifuged (15 min, 10,000 x g). Each supernatant was transferred into a HPLC vial and duplicate aliquots (50 µL for 5 and 25 µM incubations and 25 µL for 100 µM incubations) were taken into separate scintillation vials. Ultima Gold scintillation cocktail (approximately 5 mL) was added to each vial and the vials were submitted to LSC to assess the recovery of radioactivity following centrifugation.
-Samples were stored at approximately -20°C prior to and on completion of analysis and at approximately 10°C during analysis.

INSTRUMENTATION AND ANALYTICAL METHOD
- HPLC system: Waters Alliance 2695XE (including solvent conditioning module, pump, autosampler, column heater)
- Detectors: Waters 2487 UV detector LabLogic ß-RAM radioactivity detector
- Column: Phenomenex Kinetex C18, 2.6 µm, 100 Å, 100 × 4.6 mm
- Guard column: Phenomenex KrudKatcher + SecurityGuard Ultra C18
- Column oven temperature: 50°C
- Mobile phase A: 0.1% (v/v) formic acid in purified water
- Mobile phase B: 0.1% (v/v) formic acid in acetonitrile
- Flow rate: 1.5 mL/min
- Detection: UV at 220 nm and Radioactivity flow detector (with a 500 µL homogenous cell using Monoflow 4 liquid scintillant at 5 mL/min)
- Gradient: see any 'other information on materials and methods'
- Data capture time: 40 min
- Radioactivity measurements were performed by taking aliquots of samples (in duplicate where possible) by volume and mixed directly with Ultima Gold scintillation cocktail (5 mL) for analysis using Wallac 1409 automatic liquid scintillation counters.
Metabolites identified:
no
Details on metabolites:
- The hepatocytes used in the study were shown to be metabolically viable over the incubation periods used.
- The metabolite profiles obtained following incubations of test substance with rat, rabbit and human hepatocytes and in the absence of hepatocytes showed up to 40 regions of radioactivity; assigned E1 to E40 in order of increasing retention time. Not all regions were observed in each radioactivity profile.
- The extent of metabolism following incubations of test substance with rat, rabbit and human hepatocytes generally increased with time and was therefore greatest for the 4 h incubations. See 'any other information on results' for an overview in tabular format.
- Similarities in the radioactivity profiles were observed across species and for the different incubation concentrations used. The profiles were relatively complex and showed a total of up to 40 regions of radioactivity, although not all were observed in all species and at each concentration. The number of regions tended to increase with incubation time. No regions of radioactivity were observed in the human profiles that were not also observed in profiles from at least one other species.
- Eight major metabolites (ie >10% of sample radioactivity) were detected most frequently in the profiles: Regions E17, E19, E20, E21, E30, E32, E34 and E35. Between two and five of these major metabolites were observed in most profiles.
- Overall, metabolism of the test substance was rapid, with between two and five major components typically observed following incubation with rat, rabbit and human hepatocytes. None of the metabolites observed were unique to the human hepatocyte profiles.

Summary of the radioactivity profiles obtained following 4h incubations

Region

RT (min)

 

Rat

Rabbit

Human

No hepatocytes

 

 

5µM

25µM

100µM

5µM

25µM

100µM

5 µM

25 µM

100 µM

5 µM

25 µM

100 µM

E1

0.8

-

-

-

o

o

o

o

-

-

o

-

-

E2

1.1

-

-

-

o

o

o

-

-

-

-

-

-

E3

2.4

-

-

-

-

-

-

-

-

-

-

-

-

E4

3.3

-

-

-

o

o

-

-

-

-

-

-

-

E5

3.8

o

-

o

-

-

-

-

-

-

-

-

-

E6

4.4

-

o

o

-

-

-

-

-

o

-

-

-

E7

5.0

-

-

-

-

-

-

-

-

-

-

-

-

E8

5.6

-

-

-

-

-

-

-

-

-

-

-

-

E9

6.1

o

o

o

o

o

o

-

-

-

-

-

-

E10

6.5

o

o

o

o

o

-

o

-

-

-

-

-

E11

7.5

o

-

o

o

o

o

o

-

-

-

-

-

E12

8.4

o

-

-

-

o

-

-

o

-

-

-

-

E13

9.0

o

-

-

o

-

-

-

-

-

-

-

-

E14

10.3

o

o

o

o

o

o

o

o

o

-

-

-

E15

11.3

o

o

-

o

-

-

o

o

-

-

-

-

E16

11.7

o

o

o

o

o

o

o

o

o

-

-

-

E17

12.6

♦

♦

♦

o

o

o

♦

♦

♦

-

-

-

E18

13.4

o

o

o

o

o

o

-

o

o

-

-

-

E19

13.7

o

o

o

♦

♦

♦

o

-

o

-

-

-

E20

15.0

♦

♦

♦

o

o

o

o

o

o

-

-

-

E21

16.2

o

o

o

♦

♦

♦

o

o

o

-

-

-

E22

17.4

-

-

-

-

-

-

-

o

o

-

-

-

E23

18.4

-

-

-

o

-

-

-

-

-

-

-

-

E24

19.0

-

-

-

-

-

-

-

-

-

-

-

-

E25

21.5

-

-

-

o

o

-

o

o

-

-

-

-

E26

22.3

-

-

-

-

o

-

-

-

-

-

-

-

E27

23.0

o

o

o

o

o

o

o

o

o

o

-

-

E28

24.2

o

♦

♦

o

o

o

o

o

o

-

-

-

E29

25.1

-

-

-

-

-

o

-

-

-

-

-

-

E30

25.6

o

o

♦

o

o

o

♦

♦

♦

-

-

-

E31

26.0

-

-

-

o

o

o

-

-

o

-

-

-

E32

26.6

-

-

-

o

o

o

-

-

o

-

-

-

E33

27.4

-

-

-

-

-

-

-

-

-

o

-

-

E34

27.8

♦

♦

♦

♦

♦

♦

♦

♦

♦

o

o

o

E35

28.6

-

-

-

o

o

o

-

-

o

-

o

o

E36

29.6

-

-

o

o

o

o

o

-

o

-

-

-

E37

30.4

-

-

-

-

-

-

o

-

-

♦

♦

♦

E38

31.6

-

-

-

o

o

-

o

o

-

♦

♦

♦

E39

32.6

-

-

-

-

-

-

-

-

-

-

-

-

E40

36.5

-

-

-

-

-

-

-

-

-

o

o

o

- Not detected or <0.5% of sample radioactivity; o  Minor (0.5% to 10% of sample radioactivity); ♦  Major (10% or more of sample radioactivity)

Executive summary:

In vitro metabolism of the test substance using cryopreserved hepatocytes from rat, rabbit and human was assessed in a GLP study. Incubations were conducted with cryopreserved hepatocytes in duplicate for each species at three test substance concentrations (5, 25 and 100 µM) over three incubation times (0, 1 and 4 h). A radio-HPLC method was used to determine the metabolite profiles generated during the hepatocyte incubations with test substance. The hepatocytes used in the study were shown to be metabolically viable over the incubation periods used. Overall, metabolism of the test substance was rapid, with between two and five major components typically observed following incubation with rat, rabbit and human hepatocytes. Similarities in the radioactivity profiles were observed across species and for the different incubation concentrations used. The profiles were relatively complex and showed a total of up 40 regions of radioactivity, although not all were observed in all species and at each concentration. The number of regions tended to increase with incubation time. None of the metabolites observed were unique to the human hepatocyte profiles.

Description of key information

No experimental toxico-kinetic data are available for assessing adsorption, distribution and excretion of the substance but there is some in vitro metabolism in hepatocytes. Based on effects seen in the human health toxicity studies and physico-chemical parameters the substance is expected to be readily absorbed via the oral and inhalation route and somewhat lower via the dermal route. Using the precautionary principle for route to route extrapolation the final absorption percentages derived are: 50% oral absorption, 50% dermal absorption and 100% inhalation absorption.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
50
Absorption rate - inhalation (%):
100

Additional information

First the in vitro metabolism study will be summarised and thereafter the toxico-kinetic expert statement.

In vitro metabolism in hepatocytes

In vitro metabolism of the test substance using cryopreserved hepatocytes from rat, rabbit and human was assessed in a GLP study. A radio-HPLC method was used to determine the metabolite profiles generated during the hepatocyte incubations with test substance. Overall, metabolism of the test substance was rapid, with between two and five major components typically observed following incubation with rat, rabbit and human hepatocytes. Similarities in the radioactivity profiles were observed across species and for the different incubation concentrations used. The profiles were relatively complex and showed a total of up 40 regions of radioactivity, although not all were observed in all species and at each concentration. The number of regions tended to increase with incubation time. None of the metabolites observed were unique to the human hepatocyte profiles.

Toxico-kinetic information on Floralozone

Introduction

Floralozone (CAS #67634-14-4 and #67634-15-5) is phenyl-propyl aldehyde. To this propyl aldehyde is a dimethyl group is attached in the alpha position. This dimethyl propyl chain is attached to an ethyl-phenyl-group either on the ortho- or para-position. Floralozone is a liquid with a melting point of -20°C, a boiling point of 260°C, awater solubility of 40 mg/L, a vapour pressure of 0.43 Pa and a log Kow of 4.1. The molecular weight is 190 g/mol.

Absorption

Oral route: In an acute oral toxicity study, 4 out of 10 exposed animals died at a concentration of 5000 mg/kg bw, which indicates at least partial oral absorption of the substance. Based on physico-chemical properties; a relatively low molecular weight (190 g/mol), the moderate log Kow (4.1) and water solubility (40.0 mg/L) absorption through the gut is expected. According to Martinez and Amidon (2002) the optimal log Kow for oral absorption falls within a range of 2-7. This shows that Floralozone is likely to be absorbed orally and therefore the oral absorption is expected to be much higher than 50%.

Dermal route: The available study on dermal acute toxicity does not provide evidence for dermal absorption since no animals died upon exposure to 5000 mg/kg bw. Based on the physico-chemical characteristics of the substance, being a liquid, its relatively low molecular weight (190), moderate to relatively lipophilic log Kow (4.1) and moderate water solubility (40 mg/L), it is suspected that (some) dermal absorption is likely to occur. The optimal molecular weight and log Kow for dermal absorption is < 100 and in the range of 1-4, respectively (ECHA guidance, 7.12, Table R.7.12-3). The substance is just outside optimal range and therefore the skin absorption is not expected to exceed the oral absorption.

Inhalation route: Absorption via the lungs is also indicated based on these physico-chemical properties. Though the inhalation exposure route is thought minor, because of its low volatility (0.43 Pa), the octanol/water partition coefficient (4.1), indicates that inhalation absorption is possible. The blood/air (BA) partition coefficient is another partition coefficient indicating lung absorption. Buist et al. 2012 have developed BA model for humans using the most important and readily available parameters:

 

Log PBA = 6.96 – 1.04 Log (VP) – 0.533 (Log) Kow – 0.00495 MW.

For Floralozone the B/A partition coefficient would result in:

Log P (BA) = 6.96 – (-0.367) – (0.533 x 4.1) – ( 0.00495 x 190) = 5.05

 

This means that Floralozone has a tendency to go from air into the blood. It should, however, be noted that this regression line is only valid for substances which have a vapour pressure > 100 Pa. Despite Floralozone being somewhat out of the applicability domain and the exact B/A may not be fully correct, it can be seen that the substance will be readily absorbed via the inhalation route and will be close to 100%.

Distribution

The moderate water solubility of the test substance (40 mg/L) would limit distribution in the body via the water channels. The log Kow (4.1) would suggest that the substance is able to pass through biological cell membranes. Due to the observed fast metabolism the substance as such would not accumulate in the body fat.

Metabolism

There is an in vitro metabolism study with Floralozone, which indicates fast conversion of the substance in the liver resulting in many different metabolites and therefore the metabolic profile is considered to be relatively complex. No human specific metabolites were identified in the study covering rat, rabbit and human hepatocytes. The first metabolite to occur is the acid: the oxidized Floralozone at the aldehyde spot. This is supported with available data on oxidative degradation (Marteau et al. 2013) and metabolic simulations with OECD QSAR Toolbox (version 3.4) and Toxtree (version 2.6.13 (Figure 1). Toxtree in addition also predicts minor Cytochrome P450 catalyzed oxidative metabolites. The conversion into the acid is expected to occur vial all routes because of the available oxygen in the blood.

 

 

 

 

Figure 1: Anticipated major metabolic pathway illustrated for the para-isomer of Floralozone. Conversion to the carboxylic acid is predicted as primary metabolic route by OECD Toolbox and Toxtree.

 

Excretion

The log Kow of 4.1 of Floralozone indicates that the key route will be the kidneys. Floralozone is converted into an acid and this acid will be dissociated at the body’s pH of 7.4 in view of its calculated pKa, of 4.6 (SPARC). Also the other metabolites are anticipated to be excreted via the urine because these are also more hydrophilic compared to Floralozone as a parent.

Discussion

The substance is expected to be readily absorbed, orally and via inhalation (although the exposure for the latter route is expected to be low based on the low vapour pressure), based on the human toxicological information and physico-chemical parameters. The substance also is expected to be absorbed dermally based on the physico-chemical properties. The molecular weight and the log Kow are higher than the favourable range for dermal absorption but significant absorption is likely. The IGHRC (2006) document of the HSE and mentioned in the ECHA guidance Chapter 8 will be followed to derive the final absorption values for the risk characterisation.

Oral to dermal extrapolation: Floralozone is absorbed orally and is fast metabolized in the liver (see metabolism paragraph). Via the oral and dermal route the Floralozone-acid will be key substance/metabolite that enters the liver. Since the absorption will be slower via the skin and the substance will also pass the liver, it will be assumed that the oral absorption will equal dermal absorption. The toxicity of the dermal route will therefore not be underestimated. Using the asymmetric handling of uncertainty the oral absorption will be considered 50% (though likely to be higher) and the dermal absorption will be considered also 50% (not expected to exceed the oral route).

Oral to inhalation extrapolation:Though Floralozone is not a volatile liquid the inhalation exposure will be considered. Via the oral and the inhalation route the Floralozone-acid will be the key substance/metabolite that enters the liver. In the absence of bioavailability data it is most precautionary that 100% of the inhaled vapour is bioavailable. For inhalation absorption 100% will be used for route to route extrapolation, because this will be precautionary for the inhalation route.

 

References

Buist, H.E., Wit-Bos de, L., Bouwman, T., Vaes, W.H.J., 2012, Predicting blood:air partion coefficient using basis physico-chemical properties, Regul. Toxicol. Pharmacol., 62, 23-28.

 

Martinez, M.N., And Amidon, G.L., 2002, Mechanistic approach to understanding the factors affecting drug absorption: a review of fundament, J. Clinical Pharmacol., 42, 620-643.

 

Marteau C., Ruyffelaere F., Aubry J.M., Penverne C., Favier D., Nardello-Rataj V., 2013,Oxidative degradation of fragrant aldehydes. Autoxidation by molecular oxygen, Tetrahedron, 69:10, 2268-2275

 

IGHRC, 2006, Guidelines on route to route extrapolation of toxicity data when assessing health risks of chemicals,http://ieh.cranfield.ac.uk/ighrc/cr12[1].pdf