Soybean oil, maleated, ester with triethanolamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

L5178Y mouse lymphoma test

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Bacterial Reverse Mutation Test AMES

The test item was considered to be non-mutagenic under the conditions of this test.

Chromosome Aberration Test in Human Lymphocytes in vitro

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

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

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

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

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 / 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.

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.

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.

Please see attached for tables & appendices

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

L5178Y TK +/- Mouse Lymphoma Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 16 August 2016 Experimental completion date 20 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japanese MITI/MHW guidelines for testing of new chemical substances.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: L5178Y TK +/- Mouse Lymphoma Assay

Specific details on test material used for the study:

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

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Lot No. PB/BNF S9 10/04/16 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures.

Test concentrations with justification for top dose:

0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000.
The test item was considered to be a complex mixture (UVCB) therefore the maximum proposed dose level in the solubility test was set at 5000 µg/mL, the maximum recommended dose level

Vehicle / solvent:

Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same test item (Envigo Study No. VY27GX), the test item was accurately weighed and formulated in dimethyl sulfoxide (DMSO)

Untreated negative controls:

other: vehicle controls used

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulphonate and Cyclophosphamide

Remarks:

Ethylmethanesulphonate and Cyclophosphamide

Details on test system and experimental conditions:

Cell Culture

The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.
Cell Cleansing

The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e.
THMG without Methotrexate) before being returned to R10 medium.

Test Item Preparation

Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same test item (Envigo Study No. VY27GX), the test item was accurately weighed and formulated in dimethyl sulfoxide (DMSO) prior to serial dilutions being prepared. The test item was considered to be a complex mixture (UVCB) therefore the maximum proposed dose level in the solubility test was set at 5000 µg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. There was no marked 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 from the Chromosome Aberration Test are in the following table:



µg/mL 0 19.53 39.06 78.13 156.25 312.5 625 1250 2500 5000
pH 7.26 7.26 7.26 7.26 7.27 7.29 7.30 7.31 7.31 7.34
mOsm 415 413 415 417 411 406 405 401 385 352


No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation

Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO) exposure groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBQ0451V, purity 100%, expiry 12.02.17) at 400 µg/mL and 150 µg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0355340,
purity 97%, Expiry 24.11.17) at 1.5 µg/mL was used as the positive control in the presence of metabolic activation. The positive controls were also formulated in DMSO.
Test Procedure

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 19.53 to 5000 µg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.

The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.

ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.

iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (2.44 to 312.5 µg/mL for all three of the exposure groups), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or
0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).

The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Measurement of Survival, Viability and Mutant Frequency
At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL
5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
Plate Scoring

Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Calculation of Percentage Relative Suspension Growth (%RSG)

The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Day 2 Viability (%V)

Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.

P(0) = number of negative wells divided by total wells plated
%V = - ln P(0) x 100 divided by number of cells/well

Calculation of Relative Total Growth (RTG)

For each culture, the relative cloning efficiency, RCE, was calculated:


RCE = %V divided by Mean Solvent Control %V
Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100

Calculation of Mutation Frequency (MF)

MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.


Data Evaluation
The Historical Vehicle and Positive Control data is generated by the Mutant 240C program on a rolling system of the last twenty sets of archived data. The program combines the 4-hour and 24-hour data in the absence of metabolic activation as the acceptability criteria is the same for all three of the exposure groups.
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Rationale for test conditions:

This study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al., 1980; Jotz and Mitchell, 1981).
The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

Evaluation criteria:

See section any other information

Key result

Species / strain:

mammalian cell line, other: mouse lymphoma L5178Y cells

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 Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 19.53 to 5000 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose % RSG (-S9) % RSG (+S9) % RSG (-S9)
(µg/mL) 4-Hour Exposure 4-Hour Exposure 4-Hour Exposure
0 100 100 100
19.53 88 105 90
39.06 96 105 98
78.13 105 88 76
156.25 88 102 77
312.5 72 78 57
625 33 0 0
1250 0 0 0
2500 0 0 0
5000 0 0 0

There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups when compared to the concurrent vehicle control groups. However, the marked reductions in %RSG values were only observed at dose levels beyond the onset of test item precipitate that occurred at
156.25 µg/mL in all three of the exposure groups. The maximum dose level in the subsequent Mutagenicity Test was therefore limited by the onset of test item precipitate to 312.5 µg/mL.

Mutagenicity Test

A summary of the results from the test is presented in Table 1.

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 10.

There was no evidence of any marked dose-related toxicity following exposure to the test item in any of the three exposure groups at non-precipitating dose levels, as indicated by the
%RSG and RTG values (Tables 3, 6, and 9). There was also no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had also not occurred (Tables 3, 6, and 9). Acceptable levels of toxicity were seen with the positive control substances (Tables 3, 6, and 9).

Precipitate of the test item was observed at and above 78.13 µg/mL in the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation, and at and above 156.25 µg/mL in the 4-hour exposure group in the absence of metabolic activation, at the end of the exposure periods. Therefore, as sufficient precipitating dose levels were observed (as recommended by the OECD 490 Guideline), the
156.25 and 312.5 µg/mL dose levels in the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation, and the 312.5 µg/ml dose level in the 4-hour exposure group in the absence of metabolic activation, were considered to be surplus to requirements and were not plated out for viability or 5-TFT resistance.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional (Tables 3, 6, and 9).

The test item did not induce any toxicologically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell at any of the dose levels (including the precipitating dose level), in any of the three exposure groups.

The numbers of small and large colonies and their analysis are presented in Tables 4, 7, and 10.
All tables attached

Remarks on result:

other: The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group                                                 Concentration of test item (µg/mL) plated for mutant frequency

4-hour without S9                                          4.88, 9.77, 19.53, 39.06, 78.13, 156.25

4-hour with S9 (2%)                                      2.44, 4.88, 9.77, 19.53, 39.06, 78.13

24-hour without S9                                        2.44, 4.88, 9.77, 19.53, 39.06, 78.13

 

Results

 

The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

 

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

AMES

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 29 June 2016 Experimental completion date 15 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Test item
Physical state/Appearance: Clear amber viscous liquid

Purity: 100%
Expiry Date: 16 June 2018
Storage Conditions: Room temperature in the dark

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine dependent

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: a uvrA- DNA repair deficiency

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg /plate.The dose range was amended following the results of Experiment 1 and was15 to 5000 µg/plate.

The test item was tested using the following method. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

In solubility checks performed in-house the test item was noted to be immiscible in sterile distilled water, acetone and dimethyl sulphoxide at 50 mg/mL but fully miscible in dimethyl formamide at the same concentration in solubility checks performed in-house. Dimethyl formamide was selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 9-Aminoacridine (9AA) & 4-Nitroquinoline-1-oxide (4NQO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA) & Benzo(a)pyrene (BP)

Details on test system and experimental conditions:

Study Controls

The solvent (vehicle) control used was dimethyl formamide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.

The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.
Direct acting compounds in the absence of S9-mix:

Identity: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)
CAS No.: 4245-77-6
Batch number: 67F-3700
Purity: Not available*
Expiry date: 18 September 2017
Solvent: DMSO
Concentration: 2 µg/plate for WP2uvrA 3 µg/plate for TA100
5 µg/plate for TA1535

* ENNG treated as 100% despite no data sheet being available. The reference item has been used for an extended period of time as it still responds very well as a positive control

Identity:
9-Aminoacridine (9AA)
CAS No.: 90-45-9
Batch number: S32398-438
Purity: 99.9%
Expiry date: 01 October 2017
Solvent: DMSO
Concentration: 80 µg/plate for TA1537
Identity: 4-Nitroquinoline-1-oxide (4NQO)
CAS No.: 56-57-5
Batch number: 030M1206
Purity: 100%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 0.2 µg/plate for TA98

Indirect acting compounds in the presence of S9-mix:

Identity: 2-Aminoanthracene (2AA)
CAS No.: 613-13-8
Batch number: STBB1901M9
Purity: 97.5%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration:
1 µg/plate for TA100
2 µg/plate for TA1535 and TA1537 10 µg/plate for WP2uvrA
Identity: Benzo(a)pyrene (BP)
CAS No.: 50-32-8
Batch number: 090M1400V
Purity: 96%
Expiry date: 12 October 2017
Solvent: DMSO
Concentration: 5 µg/plate for TA98

The sterility controls were performed in triplicate as follows:

Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

Microsomal Enzyme Fraction

The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/BNF S9 10 April 2016 was used in this study. A Copy of the S9 Certificate of Efficacy is presented in Appendix 2.



S9-Mix and Agar

The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.


Media

Top agar was prepared using 0.6% Bacto agar (lot number 5054857 12/19) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot number 41826 07/16).

Test System and Supporting Information

Bacteria

The five strains of bacteria used, and their mutations, are as follows:

Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitution
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from:

• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1758279 10/20) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct

Test Item Preparation and Analysis

In solubility checks performed in-house the test item was noted to be immiscible in sterile distilled water, acetone and dimethyl sulphoxide at 50 mg/mL but fully miscible in dimethyl formamide at the same concentration in solubility checks performed in-house. Dimethyl formamide was selected as the vehicle.

The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl formamide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. No correction was made for purity. Dimethyl formamide is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium
alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Dose selection

The test item was tested using the following method. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation

0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing
0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation

The procedure was the same as described previously, except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring

All of the plates were incubated at 37 ± 3 C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 µg/plate because of a test item film.
Test for Mutagenicity: Experiment 2 – Plate Incorporation Method

Experiment 1 was repeated using the plate incorporation method in the presence and absence of metabolic activation.

Dose selection

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 µg/plate.

Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

Without Metabolic Activation

The procedure was the same as described previously


With Metabolic Activation

The procedure was the same as described previously

Incubation and Scoring

All of the plates were incubated at 37 ± 3 Deg C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 µg/plate because of a test item film.

The reverse mutation assay may be considered valid if the following criteria are met:

All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:

TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2014 and 2015 are presented in Appendix 1.
There should be a minimum of four non-toxic test item dose levels. There should be no evidence of excessive contamination.

Rationale for test conditions:

The purpose of the study was to evaluate the test item for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.

The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel, 1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.

Since many compounds do not exert a mutagenic effect until they have been metabolized by enzyme systems not available in the bacterial cell, the test item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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

Key result

Species / strain:

E. coli WP2 uvr A

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:

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A test item film was noted at and above 1500 µg/plate, this observation did not affect the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. Small, statistically significant increases in TA100 revertant colony frequency were observed in the second mutation test at 150 µg/plate (presence of S9-mix only). This increase was considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle control.

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Remarks on result:

other: The test item was considered to be non-mutagenic under the conditions of this test.

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg /plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate.

 

Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

 

Results

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A test item film was noted at and above 1500 µg/plate, this observation did not affect the scoring of revertant colonies.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. Small, statistically significant increases in TA100 revertant colony frequency were observed in the second mutation test at 150 µg/plate (presence of S9-mix only). This increase was considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle control.

 

Conclusion

The test item was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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

The test item was considered to be non-mutagenic under the conditions of the AMES test, in vitro gene mutation study in bacteria

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in the L5178Y TK +/- Mouse Lymphoma Assay