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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 November 2019 - 16 January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
see also prinicple of test method
Principles of method if other than guideline:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
GLP compliance:
yes (incl. QA statement)
Remarks:
certified by Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Reference substance name:
sodium hydrogen octadecanoic acid (2R)-2-octadecanamidopentanedioate
EC Number:
939-201-1
Molecular formula:
unspecified
IUPAC Name:
sodium hydrogen octadecanoic acid (2R)-2-octadecanamidopentanedioate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): BASF SE, Ludwigshafen, Germany
- Lot/batch number of test material: 0020798322
- Purity, including information on contaminants, isomers, etc.: 96.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneity given. The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Due to the use of culture medium as vehicle, the verification of the stability of the test substance in the vehicle is not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Stock formulations of the test item and serial dilutions in culture medium were prepared freshly before treatment and used within two hours of preparation.
- Final concentration of a dissolved solid, stock liquid or gel: please refer to section "Test concentrations with justification for top dose".

FORM AS APPLIED IN THE TEST: dilution in culture medium

OTHER SPECIFICS
- Physical state / Appearance: solid, white
- Molecular weight: 435 g/mol

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Normal cell cycle time (negative control): not specified

For lymphocytes:
- Sex, age and number of blood donors: one male donor (20 years old) for Experiment I and one male donor (21 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection.
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: yes, PHA

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Phenobarbital/ß-naphthoflavone induced rat liver S9
- source of S9: The S9 was prepared and stored according to the currently valid version of the ICCR-Roßdorf GmbH SOP for rat liver S9 preparation.
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation used for this study was 30.2 mg/mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-Aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
- Exp. I: 13.4; 23.4; 40.9; 71.6; 125; 219; 384; 671; 1175; 2056 µg/mL (with and without S9 mix)
- Exp. II: 5.7; 8.6; 12.9; 19.3; 29.0; 43.5; 65.3; 98.0; 171; 300 µg/mL (without S9 mix)

- Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. With regard to the molecular weight and the purity of the test item, 2056 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 13.4 to 2056 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 71.6 μg/mL and above in the absence of S9 mix and at 125 μg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Due to the precipitation data, 300 μg/mL were chosen as top treatment concentration for Experiment II.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqueous solvents (culture medium)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
- Justification for percentage of solvent in the final culture medium: not specified
Controlsopen allclose all
Untreated negative controls:
other: corresponding to solvent control
Negative solvent / vehicle controls:
yes
Remarks:
solvent control (culture medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
other: corresponding to solvent control
Negative solvent / vehicle controls:
yes
Remarks:
solvent control (culture medium)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: Demecolcine
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE: Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 hours, stimulation for proliferation by the addition of the mitogen PHA (3 µg/mL) to the culture medium.
- Exposure duration/duration of treatment: 4 hours (Exp. I); 20 hours (Exp. II)
- Harvest time after the end of treatment (sampling/recovery times): Recovery time: 16 hours (Exp. I only) and another 20 hours after addition of Cytochalasin B (Exp. I and II); Harvest time: 40 hours after start of treatment with the test item

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: Cytochalasin B (4 μg/mL), 20 hours exposure period
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): In each experimental group two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. The frequency of micronucleated cells was reported as % micronucleated cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- A preliminary cytotoxicity test (Exp. I) was performed to determine the concentrations to be used in the main experiment (Exp. II). Cytotoxicity is characterized by the percentages of reduction in the cytokinesis-block proliferation index (CBPI) in comparison to the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
CBPI = ((Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)) / Total number of cells
Cytostasis % = 100 – 100 [(CBPI(Test item) – 1) / (CBPI(Solvent control) – 1)]
Evaluation criteria:
1. Acceptability Criteria: The micronucleus assay will be considered acceptable if it meets the following criteria:
- The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval).
- The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data compared with the concurrent solvent control and produce a statistically significant increase.
- Cell proliferation criteria in the solvent control are considered to be acceptable.
- All experimental conditions were tested unless one exposure condition resulted in a clearly positive result.
- The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
- The criteria for the selection of top concentration are consistent with those described.

2. Interpretation of Results: A test item can be classified as non-clastogenic and non-aneugenic if:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).
A test item can be classified as clastogenic and aneugenic if:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
Statistics:
Statistical significance was confirmed by the Chi square test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control. A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both, biological and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: corresponding to solvent control
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: No relevant influence on osmolarity or pH was observed.
- Precipitation and time of the determination: In Experiment I, precipitation of the test item in the culture medium was observed at 71.6 µg/mL and above in the absence of S9 mix and at 125 µg/mL and above in the presence of S9 mix at the end of treatment. In Experiment II in the absence of S9 mix no precipitation was observed at the end of treatment.
- Definition of acceptable cells for analysis: please, refer to the section "Evaluation criteria"

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value: 0.965 in Exp. I without S9 mix; 0.838 in Exp. I with S9 mix; 0.767 in Exp. II without S9 mix

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes, Demecolcine (100 ng/mL), MMC (0.8 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI and distribution of mono-, bi- and multi-nucleated cells: yes
o In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II in the absence of S9 mix after continuous treatment, clear cytotoxicity (cytostasis of 56.1%) was observed at the highest evaluated concentration.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture: yes
In this study in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

HISTORICAL CONTROL DATA: please refer to Table 1 in the section "Any other information on materials and methods incl. tables"

Any other information on results incl. tables

Table 1: Summary of results

 

Exp.

Preparation

interval

Test item

concentration

in µg/mL

Proliferation

index

CBPI

Cytostasis

in %*

Micronucleated cells

in %**

95% Ctrl limit

Exposure period 4 h without S9 mix

I

40 h

Solvent control1/#

1.64

 

0.45

0.01 – 1.20

 

 

Positive control2

1.54

15.8

8.05S

 

 

 

23.4#

1.72

n.c.

0.50

 

 

 

40.9#

1.67

n.c.

0.60

 

 

 

71.6P/#

1.50

23.0

0.43

 

Trend test: p-value 0.965

Exposure period 20 h without S9 mix

II

40 h

Solvent control1

1.94

 

0.20

0.00 – 1.14

 

 

Positive control3

1.53

43.3

4.20S

 

 

 

19.3

1.84

10.9

0.35

 

 

 

43.5

1.65

31.1

0.05

 

 

 

98.0

1.41

56.1

0.20

 

Trend test: p-value 0.767

Exposure period 4 h with S9 mix

I

40 h

Solvent control1

1.72

 

0.75

0.00 – 1.24

 

 

Positive control4

1.50

30.9

4.15S

 

 

 

40.9

1.75

n.c.

0.35

 

 

 

71.6

1.70

3.2

0.90

 

 

 

125P

1.66

7.8

0.70

 

Trend test: p-value 0.838

 

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

P Precipitation occurred at the end of treatment

S The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1   Culture medium

2   MMC 0.8 µg/mL

3   Demecolcine 100 ng/mL

4   CPA 17.5 µg/mL

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.
Executive summary:

The test item, suspended in culture medium, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

 

 

Without S9 mix

With S9 mix

Exp. I

Exp. II

Exp. I

Stimulation period

48 hrs

48 hrs

48 hrs

Exposure period

4 hrs

20 hrs

4 hrs

Recovery

16 hrs

-

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Total culture period

88 hrs

88 hrs

88 hrs

 

In each experimental group two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2056 µg/mL of the test item) was chosen with regard to the molecular weight and the purity of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II in the absence of S9 mix after continuous treatment, clear cytotoxicity (cytostasis of 56.1%) was observed at the highest evaluated concentration.

In this study in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.