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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 February 2008 to 24 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Deviation: no analytical certificate of test item
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
- Type and identity of media: agar
- Properly maintained: purchased
- Periodically checked for Mycoplasma contamination: not applicable (purchased)
- Periodically checked for karyotype stability: not applicable (purchased)
- Periodically "cleansed" against high spontaneous background: not applicable (purchased)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: agar
- Properly maintained: purchased
- Periodically checked for Mycoplasma contamination: not applicable (purchased)
- Periodically checked for karyotype stability: not applicable (purchased)
- Periodically "cleansed" against high spontaneous background: not applicable (purchased)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
5, 1.5, 0.5, 0.15, 0.05 µL/plate for each strain ate in each experiment.
Vehicle / solvent:
None (liquid tgest item).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 9-Aminoacridine ; 2-Nitrofluorene ; Mitomycin C; 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar
First experiments by direct plate incorporation.
Second experiments by preincubation.

DURATION
- Preincubation: 20 min
- Exposure duration: 72 h
Evaluation criteria:
A reproducible increase in the number of revertants compared with the negative controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Biological significance: from a 2.5-fold increase.
Statistics:
Not applicable.
Species / strain:
other: all tested strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test )
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH-S2A (1995) and ICH-S2B (1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The objective of this study was to evaluate the clastogenic and aneugenic potential of CAE by examining its effects on the frequency of micronuclei (or number of binucleate cells with micronuclei - MNBN) in cultured human peripheral blood lymphocytes treated in the absence and presence of rat liver metabolic activation system S9. These micronuclei are produced from whole chromosomes or acentric fragments that are unable to attach to the spindle at mitosis and appear during the next interphase as small darkly staining bodies adjacent to the main daughter nucleus.
Cytochalasin B (Cyto-B), when added to cultures, inhibits cytokinesis (cell division) but not karyokinesis (nuclear division) resulting in the formation of binucleate cells.
Species / strain / cell type:
lymphocytes: cultured human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
Blood from two healthy, non-smoking male volunteers was used for each experiment.
The use of human peripheral blood lymphocytes is recommended because the cells are only used in short-term culture and maintain a stable karyotype.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9) from male Sprague Dawley rats
Test concentrations with justification for top dose:
Following a preliminary cytotoxicity range-finder experiment (from 10.88 to 3000 µg/mL), micronuclei were analysed with four concentrations in the main experiment.
The highest concentration selected for micronucleus analysis following 3+21 hours and 24+0 hours treatment in the absence of S9 was the lowest concentration which induced 55 ± 5% cytotoxicity.
Following 3+21 hours treatment in the presence of S9, the lowest precipitating concentration (precipitate observed by eye at the end of the treatment period) was selected as the highest concentration for micronucleus analysis.

- 3+21 hours without S9 (50, 150, 250 and 325 µg/mL among a range from 50 to 400 µg/mL)
- 3+21 hours with S9 (140, 260, 320 and 400 µg/mL among a range from 60 to 500 µg/mL)
- 24+0 hours without S9 (40, 60, 100 and 130 µg/mL among a range from 20 to 250 µg/mL)
Vehicle / solvent:
CAE was formulated in dimethyl sulphoxide (DMSO).
Details on test system and experimental conditions:
Blood cultures: blood from two healthy, non-smoking male volunteers was used for each experiment. The measured cell cycle time of the donors falls within the range 13 +/- 2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within two days of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing pooled heparinised blood into pre-warmed medium containing heat inactivated foetal calf serum and penicillin / streptomycin. The final volume following addition of S9 mix/KCl and the test item in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide.

Negative control: appropriate negative control (vehicle) cultures were included in the test system under each treatment condition to measure the proportion of micronucleated binucleate cells (MNBN) (within current historical vehicle control normal ranges).
Positive control: mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of S9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of S9. Cells receiving these were sampled in the main experiment at 24 hours after the start of treatment.

Harvesting: at the defined sampling time, cultures were centrifuged, the supernatant removed and discarded and cells resuspended in hypotonic solution (KCl) at 37°C for 4 minutes to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v:v). The fixative was changed by centrifugation and resuspension. This procedure was repeated as necessary until the cell pellets were clean.
Slide preparation: lymphocytes were kept in fixative at 2-8°C for a minimum of 3 hours before being centrifuged and resuspended. Cell suspensions were then gently spread onto multiple clean, dry microscope slides. After several processing steps (such as air-drying, staining and washing with PBS), the slides were stored protected from light at room temperature prior to analysis.
Evaluation criteria:
After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
For valid data, the test item is considered to induce clastogenic and/or aneugenic events if:
- a statistically significant increase in the frequency of MNBN cells at one or more concentrations is observed.
- an incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates is observed.
- a concentration-related increase in the proportion of MNBN cells is observed.
The test item is considered positive in this assay if all of the above criteria are met.
The test item is considered negative in this assay if none of the above criteria are met.
Results which only partially satisfied the above criteria are dealt with on a case-by-case basis. Evidence of a concentration-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations.
Statistics:
The proportion of MNBN cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. Additionally, the number of micronuclei per binucleate cell was obtained and recorded.
Species / strain:
lymphocytes: cultured human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
CAE does not induce micronuclei
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
tested up to cytotoxic concentrations for 3+21 hours and 24+0 hours (without S9) and when tested up to the limit of solubility for 3+21 hours (with S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cytotoxicity range-finder: a selection of random fields (10.88 to 3000 µg/mL) was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

Micronucleus experiment: treatment of cells with CAE for 3+21 hours and 24+0 hours in the absence of S9 resulted in frequencies of MNBN cells that were similar to and not significantly higher than those observed in concurrent vehicle controls for all concentrations analysed (Table 1). The MNBN cell frequency of all CAE treated cultures fell within normal ranges.
Treatment of cells with CAE for 3+21 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.01) than those observed in concurrent vehicle controls at an intermediate concentration (260 μg/mL; Table 1). The MNBN cell frequency marginally exceeded the normal range in a single culture at 260 μg/mL (1.20% compared to normal range of 0.10-1.10%), but no such increase was observed in the replicate culture at this concentration or at any other concentration analysed under this treatment condition and there was no evidence of a concentration-related increase in MNBN cell frequency. Therefore, this isolated observation was considered of no biological relevance.
Remarks on result:
other: strain/cell type: cultured human peripheral blood lymphocytes
Remarks:
Migrated from field 'Test system'.

Table 1: Main micronucleus experiment – results summary

Treatment

Concentration (μg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical control range (%) #

Statistical significance

3+21 hour, 

Vehicle

-

0.55

0.10 - 0.95

-

 without S9

50.00

3

0.35

 

NS

 

150.00

18

0.80

 

NS

 

250.00

31

0.20

 

NS

 

325.00

51

0.35

 

NS

 

MMC, 0.80

ND

14.55

 

p ≤ 0.001

 

 

 

 

 

 

3+21 hour,

Vehicle

-

0.15

0.10 - 1.10

-

with S9

140.00

7

0.40

 

NS

 

260.00

20

0.75

 

p ≤ 0.01

 

320.00

34

0.25

 

NS

 

400.00

47*

0.25

 

NS

 

CPA, 12.50

ND

3.75

 

p ≤ 0.001

 

 

 

 

 

 

24+0 hour,

Vehicle

-

0.45

0.10 - 1.10

-

without S9

40.00

6

0.70

 

NS

 

60.00

16

0.40

 

NS

 

100.00

33

0.30

 

NS

 

130.00

53

0.30

 

NS

 

VIN, 0.02

ND

6.95

 

p ≤ 0.001

NS: Not significant

ND: Not determined

#: 95thpercentile of the observed range

*:Lowest precipitating concentration observed by eye at the end of treatment incubation period

Conclusions:
It is concluded, that under the conditions of the test, CAE did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to cytotoxic concentrations for 3+21 hours and 24+0 hours in the absence of a rat liver metabolic activation system (S9) and when tested up to the limit of solubility for 3+21 hours in the presence of S9.
Executive summary:

The test item CAE was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of rat liver metabolic activation system (S9).

CAE was formulated in dimethyl sulphoxide (DMSO) and the highest concentrations used were determined following a preliminary cytotoxicity range-finder experiment.

Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test item concentrations for micronucleus analysis were selected by evaluating the effect of CAE on the replication index (RI). Therefore micronuclei were analysed with four concentrations in the main experiment:

- 3+21 hours without S9 (50, 150, 250 and 325 µg/mL among a range from 50 to 400 µg/mL)

- 3+21 hours with S9 (140, 260, 320 and 400 µg/mL among a range from 60 to 500 µg/mL)

- 24+0 hours without S9 (40, 60, 100 and 130 µg/mL among a range from 20 to 250 µg/mL)

Appropriate negative control (vehicle) cultures were included in the test system under each treatment condition; and the proportion of micronucleated binucleate cells (MNBN) in these cultures fell within current historical vehicle control (normal) ranges.

Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of S9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of S9. Cells receiving these were sampled in the main experiment at 24 hours after the start of treatment; all compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid.

Treatment of cells with CAE for 3+21 hours and 24+0 hours in the absence of S9 resulted in frequencies of MNBN cells that were similar to (and not significantly higher than) those observed in concurrent vehicle controls at all concentrations analysed. The MNBN cell frequency of all treated cultures fell within the normal ranges.

Treatment of cells with CAE for 3+21 hours in the presence of S9 resulted in frequencies of MNBN cells that were significantly higher (p ≤ 0.01) than those observed in concurrent vehicle controls only at the low intermediate concentration of 260 μg/mL: marginal exceed of 1.20% compared to normal range of 0.00-1.10%. However no such increase was observed in the replicate culture at this concentration or at any other concentration analysed under this treatment condition. In conclusion there was no evidence of a concentration-related increase in MNBN cell frequency and the isolated observation at 260 μg/mL was considered of no biological relevance.

It is concluded that CAE did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to cytotoxic concentrations for 3+21 hours and 24+0 hours in the absence of a rat liver metabolic activation system (S9) and when tested up to the limit of solubility for 3+21 hours in the presence of S9.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Adequacy of study:
weight of evidence
Executive summary:

CAE is defined as an UVCB substance (substance of unknown or variable composition). Its detailed chemical characterisation is presented in Table 1 (see PDF document attached).

The final complex is composed of glycerides (46.3%), free fatty acids and unsaponifiables such as alkyl furans, alkyl triols. More particularly alkyl furans are the result of chemical reaction during the heat treatment and are not found naturally in the fruit.

 

CAE was not experimentally tested for all requested toxicological end-points.

Glycerides and free fatty acids are very well-known substances which are not of toxicological concern. On the contrary, alkyl furans and alkyl triols, respectively named fraction H and fraction I, which are less well known, could raise questions about the toxicological profile of CAE.

 

CAE is an intermediate in the synthesis of PIAD, which is one of the active substances of the medicinal product PIASCLEDINE 300 mg, hard capsules (French marketing authorization No.3214954). Indeed PIASCLEDINE 300 mg is composed of Avocado oil unsaponifiables (100 mg) and soybean oil unsaponifiables (200 mg) identified as PIAD and PISD, respectively. The mixture of both active substances (one third of PIAD and two thirds of PISD) is identified as PIAS.

 

According to their chemical characterisations (see Table 2 in PDF document attached), apart for glycerides and free fatty acids, avocado fraction of PIAS and PIAD have compositions similar to CAE, containing Fraction H (alkyl furans) and Fraction I (alkyl triols) in equal or higher proportions, and for which genetic toxicity studies were conducted. Indeed on the occasion of the development of the medicinal product PIASCLEDINE 300 mg, in vitro and in vivo toxicity studies were performed on PIAS.

 

In order to avoid unnecessary experimentation, especially on animals, some existing genetic toxicological data from PIAS studies can be extrapolated to CAE (read-across); and one in vitro study on the genotoxic profile of the substance CAE was performed in order to confirm its mutagenic profile.

 

PIAS - In vitro studies

Ames tests (Forichon A., 1994, No.8894 and Simar Meintieres S., 2005, No.FSR-IPL050702/PIAS)

Two separate studies were performed and were carried out on 5 strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the 1ststudy or TA 102 in the 2nd), with and without metabolic activation, and compared with positive and negative controls. PIAS did not induce any significant or reproducible increase in the number of revertants at concentrations of 8 μg to 5000 μg/plate (1ststudy from 1994) or 15 to 3000 µg/plate (2ndstudy from 2005). PIAS is considered as non-mutagenic under these test conditions, in those two separate Ames' tests.

 

Mammalian cell gene mutation test (Stankowski L.F., 1995, No.PH 314-PHA-001 -94)

PIAS was tested on cultures of Chinese hamster ovary cells (CHO) in order to assess the potential of gene mutation on the HGPRT locus, at concentrations of 10 to 1000 μg/mL, with or without metabolic activation.

A significant increase in the mutation frequency was observed during one test at the concentrations of 600 μg/mL (with S9) and 300 μg/mL (without S9).

No dose-effect relationship was observed and the positive results were considered to be due to spontaneous mutations.

PIAS was thus devoid of mutagenic potential in this test.

 

Chromosomal aberration on human lymphocytes

Two separate genotoxicity studies were performed to induce in vitro chromosome aberration on cultures of human lymphocytes.

In the first study (Forichon A., 1994, No. 8994), concentrations of 16 to 5000 μg/mL of PIAS did not result in chromosome aberrations either with or without metabolic activation.

In the second study (Nesslany F., 2006, No.FSR-IPL050703/PIAS), 5 separate tests sessions were performed, using respectively short, medium and long treatment durations, without S9-mix, and short treatment duration with S9-mix at two concentrations (5% and 10%).

PIAS was used at concentrations between 7.8 and 125 μg/mL. In the conditions of this test, Piascledine presented no clastogenic activity with and without metabolic activation after a 4-hour short treatment, and after 20-hour and 44-hour continuous treatments without metabolic activation.

In addition, there was no numerical aberration at any of the concentrations tested, with and without metabolic activation.

Under these experimental conditions, PIAS was considered as a non-clastogenic and non-aneugenic agent, in cultured human lymphocytes.

 

PIAS - In vivo studies

 

Micronucleus tests

The mutagenic potential of PIAS was evaluated by means of the micronucleus test performed in mice (Forichon A., 1994, No.9094) and in rats (Nesslany F., 2006, No.FSR-IPL 050704/PIAS).

In the first study, PIAS was administered orally at the dose level of 5000 mg/kg b.w. to groups of 10 mice (5 males and 5 females), sacrificed 24, 48 and 72 hours post-dose.

The test did not reveal any mutagenic activity on 1000 polychromatic erythrocytes per animal compared with a positive control group (cyclophosphamide). Only a decrease in the number of polychromatic erythrocytes was observed in the animals examined 48 hours after administration.

In the second study, the test product was administered as single oral doses of 2000 mg/kg b.w. to groups of 20 Sprague-Dawley rats (10 males and 10 females), with sampling times at 24 h and 48 h post-dosing. PIAS induced no genotoxic activity under these experimental conditions.

 

In conclusion, consistent negative results from two sets of genotoxicity tests (1994 and 2005-2006) were obtained in the various in vitro and in vivo studies which aimed at detecting gene mutations, chromosome aberrations and primary DNA changes, thus confirming that PIAS is devoid ofmutagenic potential.

 

CAE – genotoxicity

 

Based on the conclusions above, CAE is not expected to be genotoxic. However while concentration in alkyl furans is similar in both mixtures (respectively 18.1 and 15-26%), concentration in alkyl triols is higher in CAE than in PIAS, with respectively 7.0 and 1.0-5.0% (see Table 2).

 

Therefore, in order to confirm that this higher content in alkyl triols has no incidence on the genotoxic profile of CAE, the mutagenic potential of CAE was evaluated by means of an in vitro micronucleus assay (according to OECD 487) using duplicate human lymphocyte cultures.

In this study (Watters G., 2012, No.8257732), it was concluded that CAE did not induce micronuclei, confirming that CAE has no genotoxic potential.

 

Conclusion on CAE

Based on the negative results from the whole range of genotoxic tests on PIAS and confirmed by an in vitro micronucleus assay using CAE on human lymphocyte cultures, it can be concluded that CAE is devoid of mutagenic potential.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification