Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro Reverse Mutation Assay in bacteria (Ames test), OECD 471: Result: non-mutagenic (negativ)

in vitro Chromosome Abberation, OECD 473, Result: non-clastogenic (negativ)

in vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene, OECD 490, Result: non-mutagenic (negativ)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-05 - 2016-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: the S9 fraction made for this study was pre-prepared using the methodology outlined in the Study Plan. This unplanned deviation is thought not to affect the validity, integrity or the result of the study.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: the S9 fraction made for this study was pre-prepared using the methodology outlined in the Study Plan. This unplanned deviation is thought not to affect the validity, integrity or the result of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: blood of non-smoking volunteers
- Cell cycle length, doubling time or proliferation index: 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.
- Sex, age and number of blood donors if applicable: Preliminary Toxicity Test: male, aged 28 years
Main Experiment: female, aged 23 years
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Number of passages if applicable:
- Methods for maintenance in cell culture if applicable: 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 °C with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 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 °C with 5 % CO2 in humidified air
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
preliminary test: 0, 4.88, 9.77, 19.53, 39.06, 78.13,156.25, 312.5, 625 and 1250 µg/mL (maximal possible dose)
main test: 0, 2.5, 5, 10, 20, 40, 80, 160 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in DMSO and acetone at 250 and 500 mg/mL but was doseable suspension in DMSO at 125 mg/mL in solubility checks performed in-house. Therefore, the maximum practical dose level achieved in the cultures was 1250 µg/mL.
Untreated negative controls:
no
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:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hrs
- Exposure duration: 4 hrs
- Recovery period after washing: 20 hrs
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

FIXATION METHOD
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 pg/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 KC1. 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 KC1 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 °C to ensure complete fixation prior to slide preparation.
- Staining: 5% Giemsa for 5 minutes
Evaluation criteria:
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.

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). 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 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.
• All 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.
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:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
some evidence of toxicity in all of the exposure groups in high doses
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The dose range for the Preliminary Toxicity Test was 4.88 to 1250 µg/mL. The maximum dose was the maximum achievable dose level due to the solubility issues with the test item. 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 the 4(20)-hour exposure groups and at and above 312.5 µg/mL in the 24-hour continuous exposure group. Hemolysis was observed following exposure to the test item at and above 625 µg/mL in the 4(20)-hour exposure groups and at and above 312.5 µg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 625 pg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 312.5 µg/mL. The test item induced some 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 in the main test.

The qualitative assessment of the slides determined that precipitate was similar to thatvobserved 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 40 pg/mL in all exposure groups. No haemolysis was observed in the 4(20)-hour exposure groups but was observed at and above 160 µg/mL in the 24-hour exposure group only.

The mitotic index data for the Main Experiment are given in Table 1 and Table 2. They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the exposure groups. Therefore, the maximum dose level selected for metaphase analysis was the lowest precipitating dose level (40 µg/mL) in all three exposure groups.

The chromosome aberration data are given in attached 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 chemicals 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 test item did not induce any polyploid cells at any dose level in any of the exposure groups.

Table 1: Mitotoc Index - Main experiment (4(20)-hour Exposure Groups)

DoseLevel (µg/mL) 4(20)-Hour Without S9 4(20)-Hour With S9
A B Mean % of Control A B Mean % of Control
0 2.95 5.25 4.1 100 6.2 4.75 5.48 100
2.5 - - - - - - - -
5 2.15 4.6 3.38 82 4.75 5.5 5.13 94
10 3.15 3.4 3.28 80 7.10 4.8 5.95 109
20 5.1 2.25 3.68 90 5.05 3.10 4.08 74
40 3.00 P 3.85 P 3.43 84 6.20 P 4.00 P 5.10 93
80 -P -P - - -P -P - -
160 -P -P - - -P -P - -
MMC 0.4 2.95 1.5 2.23 54 NA NA NA NA
CP 2 NA NA NA NA 3.1 3.5 3.3 60

MMC = Mitomycin C

CP = Cyclophosphamide

P = Precipitate

NA = Not applicable

- = Not assessed for mitotoc index

Table 2: Mitotic Index - Main Experiment (24 -hour Exposure Group)

Dose Level (µg/mL) 24-Hour Without S9
A B Mean % of Control
0 9.05 9.25 9.15 100
5 7.65 6.55 7.10 78
10 8.90 10.75 9.83 107
20 5.20 7.20 6.20 68
40 10.05 P 10.30 P 10.18 111
80 -P -P H - -
160 -P H -P H - -
320 -P H -P H - -
MMC 0.2 1.90 2.10 2.00 22

MMC = Mitomycin C

P = Precipitate

NA = Not applicable

- = Not assessed for mitotoc index

Conclusions:
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. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The test item has been assed in an in vitro GLP-study in accordance with OECD Guideline 473 for 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., 1991).

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 0, 2.5, 5, 10, 20, 40, 80, 160

4(20)-hour with S9 (2%) 0, 2.5, 5, 10, 20, 40, 80, 160

24-hour without S9 0, 5,10, 20, 40, 80,160, 320

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 toxic at high doses but it 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.

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

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-18 - 2016-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: The S9 fraction used for this study was pre prepared in bulk using the methodology outlined in the Study Plan. This unplanned deviation is thought not to affect the validity, integrity or the result of the study.
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: The S9 fraction used for this study was pre prepared in bulk using the methodology outlined in the Study Plan. This unplanned deviation is thought not to affect the validity, integrity or the result of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
TK +/-
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 (heterozygous at the thymidine kinase locus) 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.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL main test
4.88 to 1250 μg/mL preliminary toxicity test
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Cell Line
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.

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.

Microsomal Enzyme Fraction
Lot No. PB/βNF S9 10/04/16 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a certificate of S9 efficacy is presented. S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0. 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

Experimental Design and Study Conduct
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. GN78TY), 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. However, the test item was not suitable for dosing at this concentration. The concentration was therefore reduced until a suspension suitable for dosing was achieved at 1250 μg/mL. 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). 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.
Evaluation criteria:
The cell counts were 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.
Statistics:
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.
Key result
Species / strain:
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:
not specified
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 4.88 to 1250 μg/mL.
There was evidence of marked 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 reductions in %RSG values were only observed at dose levels beyond the onset of test item precipitate at 19.53 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.

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

There was no evidence of any marked toxicity following exposure to the test item in any of the three exposure groups, as indicated by the %RSG and RTG values. 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 . Acceptable levels of toxicity were seen with the positive control substances.

Precipitate of the test item was observed at 20 and 40 μg/mL in the 4-hour exposure groups, and at 10, 20, and 40 μg/mL in the 24-hour exposure group, at the end of the exposure periods. Therefore, as sufficient precipitating dose levels were observed (as recommended by the OECD 490 Guideline), the 40 μg/mL dose level was considered to be surplus to requirements and was 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.

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

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:

The GLP-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.

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 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL for all three testing groups.

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.

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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-05 - 2016-07-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: S9 fraction made for this study was pre-prepared in bulk (Certificate of S9 Efficacy has been added as an Appendix)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: S9 fraction made for this study was pre-prepared in bulk (Certificate of S9 Efficacy has been added as an Appendix)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
Microsomal Enzyme Fraction: S9 fraction made for this study was pre-prepared in bulk (Certificate of S9 Efficacy has been added as an Appendix)
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate dose range finding Experiment 1
15, 50, 150, 500, 1500 and 5000 µg/plate dose range finding Experiment 2

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.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.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide, acetone and acetonitrile at 50 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in tetrahydrofuran, therefore, this solvent was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours


Rationale for test conditions:
according to OECD guideline and GLP
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.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

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 precipitate (greasy in appearance) 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 (absence of S9-mix only). Small, statistically significant increases in WP2uvrA revertant colony frequency were also observed in the second mutation test at 15 and 150 μg/plate (presence of S9-mix only). These increases were considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant colony counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.4 times the concurrent vehicle controls.

The vehicle (tetrahydrofuran) 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.

Table 1. Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

 
 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

Wp2uvrA

TA98

TA1537

102

9

19

13

14

112 (105)

17 (12)

25 (20)

16 (14)

11 (11)

101

10

16

14

8

Experiment 2   

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

Wp2uvrA

TA98

TA1537

86

14

12

14

9

115 (105)

9 (12)

19 (16)

17 (18)

13 (11)

114

12

18

24

12

Conclusions:
The test substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published in the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", and 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. The test was conducted in accordance with GLP requirements.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of 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 (tetrahydrofuran) 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 precipitate (greasy in appearance) 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 (absence of S9-mix only). Small, statistically significant increases in WP2uvrA revertant colony frequency were also observed in the second mutation test at 15 and 150 μg/plate (presence of S9-mix only). These increases were considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant colony counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.4 times the concurrent vehicle controls.

Magnesium metaborate 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

Additional information

Gene mutation in bacteria (Ames test)

The test method in the in vitro key study (Thompson, 2016) was designed to be compatible with the guidelines for bacterial mutagenicity testing published in the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", and 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. The test was conducted in accordance with GLP requirements.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of 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.

The vehicle (tetrahydrofuran) 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 precipitate (greasy in appearance) 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 (absence of S9-mix only). Small, statistically significant increases in WP2uvrA revertant colony frequency were also observed in the second mutation test at 15 and 150 μg/plate (presence of S9-mix only). These increases were considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant colony counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.4 times the concurrent vehicle controls.

Magnesium metaborate was considered to be non-mutagenic under the conditions of this test.

Chromosome aberration data (in vitro)

The test item has been assed in the in vitro GLP-compliant key study (Bowles, 2017) in accordance with OECD Guideline 473 for 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., 1991).

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 0, 2.5, 5, 10, 20, 40, 80, 160

4(20)-hour with S9 (2%) 0, 2.5, 5, 10, 20, 40, 80, 160

24-hour without S9 0, 5,10, 20, 40, 80,160, 320

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 toxic at high doses but it 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.

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

Mammalian cell gene mutation data (in vitro)

The GLP-compliant key study (Flanders, 2016) 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.

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 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL for all three testing groups.

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.

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.

Justification for classification or non-classification

Based on the all negative results of recent in vitro studies for the test item, it is considered to be neither mutagenic nor genotoxic. According to the Regulation (EC) No 1272/2008 (CLP Regulation) the test item does not meet the criteria for classification and will not require labelling as a mutagen.