Ceratonia siliqua, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Sep 2017 - 28 Dec 2017

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

- Physical appearance: Dark brown to black liquid
- Storage of test material: At room temperature

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
- Sex, age and number of blood donors if applicable:
Blood was collected from healthy adult, non-smoking volunteers
The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:
Dose-range finding study: age 22, AGT = 13.2 h
First cytogenetic assay (presence of S9-mix): age 31, AGT = 13.2 h
First cytogenetic assay (absence of S9-mix): age 25, AGT = 13.2 h
Second cytogenetic assay: age 29, AGT = 14.2 h


Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.


- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.

Cytokinesis block (if used):

Cytochalasine B (5 μg/mL) for 24 hours (1.5 times normal cell cycle)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without S9-mix, 3 hr exposure; 27 hr harvest: 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL
Without S9-mix, 24 hr exposure, 24 hour harvest 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL
With S9-mix, 3 exposure; 27 hr harvest: 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL

First cytogenetic test:
Without S9-mix, 3hr exposure; 27 hr harvest: 0, 1250, 2500 and 5000 μg/mL
With S9-mix, 3hr exposure; 27 hr harvest: 0, 1250, 2500 and 5000 μg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure: 0, 1250, 2500 and 5000 μg/mL

A concentration of 5000 µg/mL showed no precipitation in the culture medium and was used as the highest concentration of Carob Bean Extract.

Vehicle / solvent:

- Vehicle: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle:
DMSO is recommended in international OECD guidelines to be used as solvent.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hanks’ Balanced Salt Solution without calcium and magnesium.

True negative controls:

no

Positive controls:

yes

Remarks:

Without Metabolic Activation

Positive control substance:

mitomycin C

Remarks:

Mitomycin C was used as a direct acting clastogen at a final concentration of 0.25 and 0.38 µg/mL for a 3 hour exposure period and 0.15 and 0.23 µg/mL for a 24 hour exposure period.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hanks’ Balanced Salt Solution without calcium and magnesium.

True negative controls:

no

Positive controls:

yes

Remarks:

Without Metabolic Activation

Positive control substance:

other: Colchicine

Remarks:

Colchicine was used as a direct acting aneugen at a final concentration of 0.1 µg/mL for a 3 hour exposure period and 0.05 µg/mL for a 24 hour exposure period

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hanks’ Balanced Salt Solution without calcium and magnesium.

True negative controls:

no

Positive controls:

yes

Remarks:

With Metabolic Activation

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide was used as an indirect acting clastogen, requiring metabolic activation, at a final concentration of 10, 12.5, 15 and 17,5 µg/mL for a 3 hour exposure period.

Details on test system and experimental conditions:

Environmental conditions:
Humidity: 37 - 89%
CO2: 5.0 ± 0.5%
Temperature: 34.5 - 37.1°C

METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period: 3 hour
- Exposure duration:
Without and with S9-mix: 3 hour treatment, 27 hour harvest time
The exposure period was followed by centrifugation, rinsing, a second centrifugation and incubation for another 24 hours with cytokinase block.
Without S9-mix: 24 hour treatment, 24 hour harvest time.
The exposure period was followed by immediate fixation without rinsing.



ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B

STAIN: 5% (v/v) Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloridesolution. Immediately after, ethanol: acetic acid (Merck) fixative (3:1 v/v) wasadded. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet werefixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96%(v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafterslides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper

NUMBER OF CELLS EVALUATED: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).

Evaluation criteria:

A test was considered acceptable if it met the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

To evaluate the statistical significane of induction, the Chi-square test is performed ( Chi-square test, onesided, p < 0.05).

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: no, the pH and osmolarity at a concentration of 5000 μg/ml were 7.627 and 374 mOsm/kg respectively (compared to 7.806 and 432 mOsm/kg in the solvent control).

- Definition of acceptable cells for analysis:
Cells could be scored when they had seperate main nuclei of approximately equal size, when the nuclear boundaries were able to be distinguished or when the main nuclei were linked by nucleoplasmic bridges. Trinucleated, quadranucleated, or multinucleated cells or cells where main nuclei were undergoing apoptosis were not scored.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, at the 3 hours exposure time, Carob Bean Extract induced a
statistically significant increase in the number of binucleated cells with micronuclei at dose
levels 1250 and 5000 µg/mL. However, the number of binucleated cells with micronuclei
was within the 95% control limits of the distribution of the historical negative control
database. In addition a negative Cochran Armitage trend test was obtained (p = 0.101).
Therefore, this increase is considered not biologically significant


FIRST CYTOGENETIC ASSAY
All dose levels were selected for scoring:
Without S9-mix:
In the presence of S9-mix, Carob Bean Extract did not induce a statistically significant or
biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

With S9-mix:
In the absence of S9-mix, at the 3 hours exposure time, Carob Bean Extract induced a
statistically significant increase in the number of binucleated cells with micronuclei at dose
levels 1250 and 5000 µg/mL. However, the number of binucleated cells with micronuclei
was within the 95% control limits of the distribution of the historical negative control
database. In addition a negative Cochran Armitage trend test was obtained (p = 0.101).
Therefore, this increase is considered not biologically significant


SECOND CYTOGENETIC ASSAY
All dose levels were selected for scoring:
Without S9-mix:
The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.



ACCEPTABILITY:
The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei.
The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei and cyclophosphamide also showed a statistically significant increase in the number of mononucleated cells
In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

There can be concluded that the test conditions were adequate and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Tables: See Attached Background material

Applicant's summary and conclusion

Conclusions:

An in vitro micronucleus assay in cultured peripheral human lymphocytes, performed according to OECD 487 and GLP principles, showed that Carob Bean Extract is not clastogenic or aneugenic in human lymphocytes

Executive summary:

An in vitro micronucleus assay in cultured peripheral human lymphocytes with Carob Bean Extract was performed according to OECD 487 and GLP principles. The solvent and positive controls, both with and without metabolic activation, functioned properly within the acceptabillity range. Based on the results it can be concluded that the test conditions were adequate and that the metabolic activation system functioned properly. The dose levels at which the test substance was exposed were 0, 1250, 2500 and 5000 μg/mL. No significant genetoxicity or cytotxicity was observed at all dose levels and therefore it can be concluced that Carob Bean Extract is not clastogenic or aneugenic in human lymphocytes under the experimental conditions.