complex mixture from biological origin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-16 to 2017-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed

EC Number:

941-809-7

IUPAC Name:

Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed

Test material form:

liquid

Method

Target gene:

The test item was assay for the ability to induce micronuclei in human lymphocytes cultured in vitro, after treatment in the absence and presence of S9 metabolism.

Species / strain

Species / strain / cell type:

lymphocytes: human lymphocytes

Details on mammalian cell type (if applicable):

Two batches of human whole blood, provided by Biopredic International (France), were used in this study and had the following characteristics:

Sex Female
Age 35 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 644 IU/mL of whole blood
Collection date 05-Apr-2017
Batch Number SAG025A1E019


Sex Female
Age 35 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 556 IU/mL of whole blood
Collection date 05-Apr-2017
Batch Number SAG025A1E020

Cytokinesis block (if used):

the inhibitor of actin polymerisation: cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction, provided by Trinova Biochem GmbH, from Rats pretrerated with Phenobarbital and 5,6-Benzoflavone

Test concentrations with justification for top dose:

Dose levels for treatment were selected on the basis of the cytotoxicity observed in previous experiments where unacceptable values of frequency of micronucleated cells were observed or the required cytotoxicity was not achieved to select dose levels for scoring micronuclei.

Dose levels of 528, 440, 367, 306, 255, 212, 177, 147, 123, 102 and 85.3 µg/mL were used for the three hour treatment in the absence of S9 metabolism.

Dose levels of 684, 570, 475, 396, 330, 275, 229 and 191 µg/mL were used for the three hour treatment in the presence of S9 metabolism.

Dose levels of 191, 159, 133, 111, 92.1, 76.7, 64.0, 53.3 and 44.4 were used for the continuous treatment in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point.

Vehicle / solvent:

Solutions of the test item were prepared in culture medium.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine 0.080, 0.040 and 0.020 μg/mL

Details on test system and experimental conditions:

Culture media:
The culture medium for the lymphocytes had the following composition:
RPMI 1640 1x (Dutch modification) 500mL
Foetal Calf Serum 100mL
L-Glutamine (200mM) 6.25mL
Antibiotic solution 1.25mL

The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used in the following proportion: 10mL of PHA was added to 500mL of medium.

Preparation of the test cultures and treatment:

Two main experiments were performed including negative and positive controls. Two cultures were prepared at each test point. Lymphocyte cultures were treated approximately fourty-eight hours after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.

Since the test item was solubilized in culture medium, the composition of the treatment media was as follows:

Treatment medium in the presence of S9 metabolism
Test item solution 0.50mL
S9 mix 1.00mL
Culture medium (without PHA) 3.50mL

Treatment medium in the absence of S9 metabolism
Test item or control solution 0.50mL
Culture medium (without PHA) 4.50mL

For the first main experiment, the treatment media was added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time Cytochalasin-B was added to achieve a final concentration of 6 µg/mL.

For the second main experiment, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

Harvesting and slide preparation:
The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed up to approximately 5 mm from the pellet. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this way were dropped onto clean, wet, greasefree glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

Slide evaluation:
The cytokinesis-block proliferation index CBPI was calculated as follows:
CBPI = mononucleated + (2×binucleated) + (3×multinucleated) / total number of cells counted where mononucleated, binucleated and multinucleated are respectively the number of mononucleated cells, binucleated cells and multinucleated cells.
CBPI was used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed. The highest concentration for genotoxicity assessment was selected on the basis of the cytotoxicity as calculated by the CBPI.

The percentage cytotoxicity was evaluated according to the following formula:
% Cytotoxicity = 100 − [100 x (CBPIt-1/CBPIc −1)]

where:
t = test item treated culture
c = vehicle /solvent control culture

The highest concentration for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50 % and 60 %. In the absence of cytotoxicity, the highest treatment level is selected as the highest concentration for scoring.
Two lower concentrations were also selected for the scoring of micronuclei. For the three selected concentrations, for the untreated and for the positive controls (Cyclohosphamide),
1000 binucleated cells per cell culture were scored to assess the frequency of micronucleated cells.
Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was
observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.

The criteria for identifying micronuclei were as follows:
1. the micronucleus diameter was less than 1/3 of the nucleus diameter;
2. the micronucleus diameter was greater than 1/16 of the nucleus diameter;
3. no overlapping with the nucleus was observed;
4. the aspect was the same as the chromatin.

Evaluation criteria:

Acceptance criteria:
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
– Adequate cell proliferation is observed in solvent control cultures.
– The appropriate number of doses and cells is analysed.

Criterion for outcome:
In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
– There is a significant dose effect relationship.

The test item is considered clearly negative if the following criteria are met:
– None of the concentrations shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data.

Statistics:

Statistical analysis:
For the statistical analysis, a modified X^2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

For both experiments, the following dose levels were selected for scoring:

Experiment No.:	S9	Treatment time (hours)	Harvest time (hours)	Concentration (µg/mL)
1	-	3	31-32	212, 147 and 102
1	+	3	31-32	570, 396 and 275
2	-	31	31	133, 92.1 and 64.0

Applicant's summary and conclusion

Conclusions:

The test material did not induce structural and/or numerical chromosomal damage in human lymphocytes. The test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In an in vitro mammalian micronucleus assay (OECD 487), human lymphocytes cultured in vitro were exposed to the test item HH-2015-623 (47.2% solid content) in cell culture medium.Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 32 hours corresponding to approximately 2.0 cell cycles was used. A second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 31 hours. Solutions of the test item were prepared in culture medium.

Dose levels for treatment were selected on the basis of the cytotoxicity observed in previous experiments where unacceptable values of frequency of micronucleated cells were observed or the required cytotoxicity was not achieved to select dose levels for scoring micronuclei.

Dose levels of 528, 440, 367, 306, 255, 212, 177, 147, 123, 102 and 85.3 µg/mL were used for the three hour treatment in the absence of S9 metabolism.

Dose levels of 684, 570, 475, 396, 330, 275, 229 and 191 µg/mL were used for the three hour treatment in the presence of S9 metabolism.

Dose levels of 191, 159, 133, 111, 92.1, 76.7, 64.0, 53.3 and 44.4 µg/mL were used for the continuous treatment in the absence of S9 metabolism.

Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI).

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. All results were within the distribution of historical negative control data. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system.

It is concluded that HH-2015-623 does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 487 for in vitro mutagenicity data.