Soybean oil, maleated, ester with triethanolamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 01 August 2016 Experimental completion date 12 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Chromosome Aberration Test in Human Lymphocytes in vitro

Test material

Test material information

Constituent 1

Reference substance name:

Soybean oil, maleated, ester with triethanolamine

EC Number:

285-107-2

EC Name:

Soybean oil, maleated, ester with triethanolamine

Cas Number:

85029-82-9

Molecular formula:

C63H113NO12 to C67H123NO12

IUPAC Name:

Soybean oil, maleated, ester with triethanolamine

Test material form:

other: Clear amber viscous liquid

Details on test material:

100% product
Physical state/Appearance: Clear amber viscous liquid
Expiry Date: 24 June 2018
Storage Conditions: Room temperature in the dark

Specific details on test material used for the study:

Identification: Test item
Physical state/Appearance: Clear amber viscous liquid
Purity: 100% (UVCB)
Expiry Date: 24 June 2018
Storage Conditions: Room temperature in the dark

Method

Target gene:

The purpose of the study was to assess the potential chromosomal mutagenicity of the test item on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.

Species / strain

Species / strain / cell type:

other: Normal human lymphocytes. Human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

The purpose of the study was to assess the potential chromosomal mutagenicity of the test item on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Test were as follows:
0, 5, 10, 20, 40, 80 and 160 µg/mL.

Vehicle / solvent:

The test item was insoluble in MEM and DMSO at 50 mg/mL and 500 mg/mL, respectively. However, the test item was a fine dispersion in DMSO at 500 mg/mL in solubility checks performed in-house. Prior to each experiment, the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Vehicle and positive controls were used in parallel with the test item. The vehicle control used was as follows:
Identity: DMSO
Supplier: Sigma Aldrich
Batch number SZBF3070V
The positive control items were as follows: Absence of S9-mix:
Identity: Mitomycin C (MMC)
CAS No.: 50-07-7
Supplier: Sigma Aldrich
Batch Number: SLBM6258V
Purity: Assume 100%
Expiry Date: 01 May 2019
Solvent: Minimal Essential Medium
Concentration: 0.4 µg/mL for 4-hour exposure and 0.2 µg/mL for the 24-hour exposure
Presence of S9-mix:
Identity: Cyclophosphamide (CP)
CAS No.: 6055-19-2
Supplier: Acros Organics
Batch Number: A0355340
Purity: Assume 97%
Expiry Date: 01 January 2020
Solvent: DMSO
Concentration: 1 µg/mL for 4-hour exposure

Test System and Supporting Information

Cells

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years
in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore, using this average, the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

The details of the donors used are:

Preliminary Toxicity Test: male, aged 28 years Main Experiment: male, aged 28 years
Cell Culture

Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2


in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix

The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/BNF S9 10/04/16 was used in this study. A copy of the S9 Certificate of Efficacy is presented in Appendix 2.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Experimental Design and Study Conduct

Test Item Preparation and Analysis

The test item was considered to be a UVCB and, therefore, the maximum recommended dose was initially set at 5000 µg/mL. The purity of the test item was considered to be 100% and was not accounted for in the test item formulations.

The test item was insoluble in MEM and DMSO at 50 mg/mL and 500 mg/mL, respectively. However, the test item was a fine dispersion in DMSO at 500 mg/mL in solubility checks performed in-house. Prior to each experiment, the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared.

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

The pH and osmolality readings are presented in the following table:

Dose level 0 19.53 39.06 78.13 156.25 312.5 625 1250 2500 5000
µg/mL
pH 7.26 7.26 7.26 7.26 7.27 7.29 7.30 7.31 7.31 7.34
Osmolality 415 413 415 417 411 406 405 401 385 352
mOsm
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


Culture conditions

Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)

After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.
4-Hour Exposure Without Metabolic Activation (S9)

After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-Hour Exposure Without Metabolic Activation (S9)

As the exposure was continuous the cultures were established, at a nominal volume of
9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.


The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

Preliminary Toxicity Test

Three exposure groups were used:

i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.

ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.

iii) 24-hour continuous exposure to the test item without S9-mix.

The dose range of test item used was 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500
and 5000 µg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made.
Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Main Experiment

Three exposure groups were used for the Main Experiment:

i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.

ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest.

iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest.

The dose range of test item used for the main test was 0, 5, 10, 20, 40, 80 and 160 µg/mL.

Cell Harvest

Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads

The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining

When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation of Response

Qualitative Slide Assessment

The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding

The slides were coded using a computerized random number generator.

Mitotic Index

A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage

Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 1.


Data Evaluation

The following criteria were used to determine a valid assay:

• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• A l the positive control chemicals induced a positive response (p≤0.01) and
demonstrated the validity of the experiment and the integrity of the S9-mix.

• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Rationale for test conditions:

The purpose of the study was to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.

Numerical and structural chromosome aberrations are implicated in the pathology of neoplasia (Radman et al. 1982; Cairns, 1981) and also occur in a high proportion of spontaneous abortions and abnormal live births (Chandley, 1981). Furthermore, most carcinogens are capable of inducing such changes in chromosome fidelity. Metaphase analysis in vitro involves recording such structural and numerical aberrations in the chromosomes of exposed cells. Many of these changes are lethal to the cells in which they occur and are therefore not of heritable significance. However, it is assumed that agents capable of inducing gross chromosomal changes also induce more subtle changes (translocations, inversions and small deletions) which are not cell lethal, and therefore represent a hazard. The ability to induce chromosome aberrations also correlates well with the induction of gene mutations (Hollstein et al. 1979).

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:

1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.

2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.

3) There is no concentration-related increase at any dose level A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.

2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.

3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related

When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Results and discussion

Test results

Key result

Species / strain:

other: metaphase chromosomes of normal human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/mL. The maximum dose was the 10 mM concentration.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 78.13 µg/mL in all three exposure groups.

Hemolysis was observed following exposure to the test item at 5000 µg/mL and at above 1250 µg/mL in the 4(20)-hour exposure group in the absence of metabolic activation (S9) and the 24-hour continuous exposure group, respectively. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. No hemolysis was observed in the in the 4(20)-hour exposure group in the presence of metabolic activation.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 625 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9) whereas the maximum dose with metaphases present in the 24-hour continuous exposure was 312.5 µg/mL. The mitotic index data are presented in Table 1. The test item induced marked evidence of toxicity in all of the exposure groups.

The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for all three exposure groups.

Chromosome Aberration Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Group Final concentration of test item (µg/mL)
4(20)-hour without S9 0*, 5, 10*, 20*, 40*, 80*, 160, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 5, 10*, 20*, 40*, 80*, 160, CP 1*
24-hour without S9 0*, 5, 10, 20*, 40*, 80*, 160*, MMC 0.2*

The qualitative assessment of the slides determined that the precipitate was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum dose level in all three exposure groups.

Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 80 µg/mL in the 4(20)-hour exposure groups and at 160 µg/mL in the

* = Dose levels selected for metaphase analysis MMC = Mitomycin C
CP = Cyclophosphamide


24-hour continuous exposure group. No haemolysis was observed at any dose level in any of the exposure groups.

The mitotic index data for the Main Experiment are given in Table 2 and Table 3. They confirm the qualitative observations in that no marked dose-related inhibition of mitotic index was observed. Therefore, the maximum dose level selected for metaphase analysis was the lowest precipitating dose level for each group.

The chromosome aberration data are given in Table 4, Table 5 and Table 6. The assay was considered valid as it met all of the following criteria:

The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

All the positive control chemical induced a demonstrable positive response (p≤0.01) and
confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

The required number of cells and concentrations were analyzed.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 7. The test item did not induce any polyploid cells at any dose level in any of the exposure groups.

Remarks on result:

other: The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system.

Any other information on results incl. tables

Please see attached for tables & appendices

Applicant's summary and conclusion

Conclusions:

The test item, was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

 

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1990).

 

Methods

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls.  In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

 

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate.   The dose levels selected for the Main Test were as follows:

 

Group                                                 Final concentration of test item (µg/mL)

4(20)-hour without S9 

4(20)-hour with S9 (2%)                             0, 5, 10, 20, 40, 80, 160

               

24-hour without S9      

 

Results

 

All vehicle (dimethyl sulphoxide (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

 

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test item was not toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

 

Conclusion

 

The test item, was considered to be non-clastogenic to human lymphocytes in vitro.