(6R,7R)-7-[((2Z)-2-[({2-[(1,1-dimethylethyl)oxy]-1,1-dimethyl-2-oxoethyl}oxy)imino]-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetyl)amino]-8-oxo-3-(1-pyridiniumylmethyl)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A battery of three in vitro mutagenicity and genotoxicity studies has been completed. The substance did not induce any mutations, either with or without metabolic activation, at upto 5000 µg/plate in the bacterial reverse mutation assay. Similarly, when tested in human peripheral lymphocytes, the substance 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 metabolising system. The test item was, therefore, considered to be non-clastogenic to the human lymphocytes in vitro.

In the mammalian cell gene mutation assay, the test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor. Consequently, it is considered to be non-mutagenic in this assay.

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:

01 August - 27 November 2017

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)

Version / remarks:

Adopted 29 July 2016

Deviations:

yes

Remarks:

Validity, integrity and result of the study were not affected by the deviations from study plan

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI) and Ministry of the Environment (MOE) Guidelines

Version / remarks:

31 March 2011

Deviations:

no

Principles of method if other than guideline:

The required number of cells and concentrations were analyzed except for the ‘A’ culture for the positive control in the 4(20)-hour exposure group in the absence of S9-mix. An
additional 62 cells were scored for chromosomal damage a due to a poor positive response in the first 150 cells. Therefore a total of 212 cells were analyzed.

This unplanned deviation is thought not to affect the validity, integrity or the result of the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not relevant - The study examined gross chromosomal aberrations

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Donor for Preliminary toxicity test: male, aged 28 years
Donor for Main Experiment: female, aged 23 years
non-smokers and not knowingly exposed to high levels of radiation or hazardous chemicals and not knowingly recently suffered from a viral infection

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix Microsomal enzyme fractions

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/ml
Main experiment: 0, 8 (not for the 24h exposure without S9), 16, 32, 64, 128, (192 only for 24h exposure without S9-mix), 256 and 320 μg/ml
Dose levels for the main test were selected after the results of the preliminary one. Mitotic index data was used to estimate the test item toxicity.
Maximum dose for the Preliminary Toxicity Test was the maximum achievable concentration.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental 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 minimal essential medium (MEM) and 10% foetal bovine serum (FBS), 0.1 ml Li-heparin, 0.1 ml phytohaemaglutinin, 0.75 ml heparinised whole blood.

Three exposure groups were used in the Preliminary Toxicity Test:
i) 4h exposure to the test item without S9 mix, followed by a 20h recovery period in treatment-free media
ii) 4h exposure to the test item with S9 mix (2%), followed by a 20h recovery period in treatmentt-free media
iii) 24h continuous exposure to the test item without S9-mix

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

Three exposure groups were used for the Main Experiment:
i) 4h exposure to the test item without S9-mix, followed by 20h culture in treatment-free media prior to cell harvest.
ii) 4h exposure to the test item with S9-mix (2%), followed by 20h culture in treatment-free media prior to cell harvest.
iii) 24h continuous exposure to the test item without S9-mix prior to cell harvest

Evaluation criteria:

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
- 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 analysed except for the 'A' culture for the positive control in the 4(20)-hour in the absence of S9-mix where 212 cells were analysed due to a poor positive response in the first 150 cells.

Criteria for determining the study conclusion
Provided that all acceptibility 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 is 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 frwquency 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

Statistics:

The frequency of cells with aberration 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 observes only with gap-type aberrations were assessed on a case by case basis.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 320 μg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 256 μg/ml levels in the 4(20)-h exposures with and without S9, whereas precipitate were present at and above 192 μg/ml in the 24h continuous exposure group. No haemolysis was observed in any of the exposure groups, either with or without S9-mix.

In the 4(20)-h exposures with and without S9, no dose-related inhibitions of mitotic index were observed. The maximum dose level selected for metaphase analysis was, therefore, the lowest precipitating dose level for both exposure groups (256 μg/ml). In the 24h continuous exposure group, a dose related inhibition of mitotic index was noted, where 13%, 32% and 50% Mitotic Inhibition was observed at 64, 128 and 192 μg/ml respectively. Therefore, maximum dose level selected for metaphase analysis was 192 μg/ml, as this achieved optimum toxicity as defined by the OECD Guideline 473 (55+/- 5%) and was the lowest precipitating dose level

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.

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 analysed except for the 'A' culture for the positive control in the 4(20)-h in the absence of S9 -mix where 212 cells were analysed due to a poor positive response in the first 150 cells.

For detailed results, please refer to the attached document CQ_CA_tables and figures.pdf

Conclusions:

The substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in ehtier the absence or presence of a liver enzyme metabolising system. The test item was, therefore, considered to be non-clastogenic to the human lymphocytes in vitro.

Executive summary:

1.1 Introduction

This report describes the results of an in vitro study for the detection of structurla chromosomal aberrations in cultured mammalian cells. It supplements microbial systams insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.

1.2 Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three 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 metabolising 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 to 320 μg/ml. The dose levels selected for the Main Test were as follows:

• Group 4(20)-hour with and without S9: 0, 8, 16, 32, 64, 128, 256, 320 μg/ml
• Group 24 -hour without S9: 0, 16, 32, 64, 128, 192, 256, 320 μg/ml

1.3 Results

All vehicle (dimethyl sulfoxide (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 to human lymphocytes but did not induce any statistially 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.

1.4 Conclusion

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August 2017 - 24 October 2017

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:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 7 December 2017 - 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.

Type of assay:

comet assay

Target gene:

thymidine kinase, L5178Y TK+/-3.7.2c mouse lymphoma assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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 the time.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 (Lot No. PB/βNF, 30/06/2017 and 20/08/2017)

Test concentrations with justification for top dose:

A preliminary toxicity test was performed on cell cultures at 5x10^5 cells/ml, using a 4h exposure period both with and without metabolic activation, and at 1.5x10^5 cells/ml using a 24h exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 μg/ml for all three exposure groups. 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 μm/ml, whichever is the lowest. When the test item is a UVCB th 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.

For the main test, the exposures were performed in duplicate, both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (9.75, 19.5, 39, 78, 156, 208, 260 and 312 μg/ml) for the 4h exposure groups (with and w/o metabolic activation) of 1x10^ cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals.
The 24h exposure group in the absence of metabolic activation was exposed to 8 concentrations as well (19.5, 39, 78, 156, 234, 312, 468, 624 μg/ml). Cell density was 0.3x10^6 cells/ml.

Maximum proposed dose level in the solubility test was initially 5000 μg/ml, the maximum recommended dose level for UVCBs, and no correction for the purity of the item was applied. Due to the test item not forming a suspension suitable for dosing at 500 mg/ml, the test item was formulated at 250 mg/ml and dosed at 1% for a maximum achievable dose of 2500 μg/ml.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Positive controls were formulated in DMSO

Details on test system and experimental conditions:

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196C. Cells were routinely cultured in RPMI 1640 medium with Glutamaz-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 37C with 5% CO2 in the 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 30/06/2017 and 20/08/2017 were used in this study and were 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 know mutagens in the Ames test. 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 the main Mutagenicity Test.

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 tests performed in-house for the Human Lymphocyte Chromosome Aberration Test performed on the same test item, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. 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. 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.

Preliminary toxicity test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 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 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 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 10^5 cells/mL, unless the mean cell count was less than 3x10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultureswere 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.

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 10^6 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 10^6 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 (9.75 to 312 µg/mL for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 19.5 to 624 µg/mL in the 24-hour exposure group in the absence of metabolic activation), 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.

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 10^5 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 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. On Day 2 of the experiment, the cells were counted, diluted to 10^4 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.

Rationale for test conditions:

Not mentioned

Evaluation criteria:

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. 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 markedly less than 10% are usually 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.

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At and above 260μg/ml at 4h group

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 312 μg/ml at 4h group

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 312 μg/ml at 24h group

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

(Tables mentioned in this section can be found in the attached CQ_MLA_Tables_2017.pdf)

Preliminary cytotoxicity test
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all of the three exposure groups when compared to the concurrent vehicle control groups. The onset of toxicity was very sharp in all three of the exposure groups indicating that achieving optimum levels of toxicity would be difficult. Precipitate of test item was observed at and above 312.5 µg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and at and above 625 µg/mL in the 24-hour exposure group in the absence of metabolic activation. Therefore, the maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of
test item-induced toxicity and test item precipitate in all three of the exposure groups.

Mutagenicity Test
There was evidence of marked dose related toxicity following exposure to the test item in all three of the exposure groups, as indicated by the %RSG and RTG values. There was also evidence of a modest reduction in viability (%V) in the 4h exposure group in the absence of metabolic activation, indicating that residual toxicity had occurred (Table 3). Base on the RTG and %RSG values observed, optimum levels of toxicity were not achieved in the 4h and 24h exposure groups in the absence of metabolic activation, due to the very steep toxicity curve of the test item, a dose level that marginally exceeded the upper limit of acceptable toxicity was plated for viability and 5-TFT resistance in each of these exposure groups as sufficient numbers of cells were available at the time of plating (Tables 3 and 9). The excessive toxicity observed at and above 260 μg/ml in the 4h exposure group in the absence of metabolic activation, at 312 μg/ml in the 4h exposure group in the presence of metabolic activation and at and above 312 μg/ml in the 24h exposure group in the absence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. 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 260 μg/ml in the 4h exposure groups in both the absence and presence of metabolic activation and at and above 468 μg/ml in the 24h exposure group in the absence of metabolic activation at the end of the exposure periods.

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 increases in the mutant frequency at any of the dose levels (including the dose levels that achieved or marginally exceeded optimum levels of toxicity) 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.

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 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guidance.

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 4h exposure groups both in the absence and presence of metabolic activation (2% S9) and a 24h 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:

Group	Concentration of Substance CQ (μg/ml) plated for viability and mutant frequency
4h without S9	9.75, 19.5, 39, 78, 156, 208
4h with S9 (2%)	19.5, 39, 78, 156, 208, 260
24h without S9	19.5, 39, 78, 156, 234

Results

The maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of test item-induced toxicity and test item precipitate. The vehicle control cultures had mutant frequency values that were acceptbale for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frwquency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising 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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-22 May 2018

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:

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: CQ
Physical state/Appearance: White powder
Batch Number: G317974
Purity: Mixture (treated as 100%)
Expiry Date: 31 July 2018
Storage Conditions: Approximately 4 °C in the dark
No correction for purity was required.

Target gene:

histidine (S.typhimurium strains)
Tryptophan (E.coli (WP2uvrR))

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa-; uvrB- ; pKM101 (TA98 and TA100)

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fractions (CD Sprague-Dawley) ; phenobarbital /B-Naphtha induced.

Test concentrations with justification for top dose:

The maximum concentration was 5000 µg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations
of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate)

Vehicle / solvent:

Identity: Dimethyl sulphoxide
Supplier: Fisher Scientific
Batch number, (purity), expiry: 1736851 (>99%) expiry: Apr 2023

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Rationale for test conditions:

Acceptability Criteria
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), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per
plate in the vehicle and untreated controls (negative controls). Typical 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
These values will also be confirmed against current in-house historical control profiles to
further validate acceptability. Although the number of spontaneous revertants can be
expected to fall within the ranges, they may occasionally fall outside these.
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 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 (S9-mix).

A minimum of five concentrations of test item is required, the highest usually being at the
toxic or maximum recommended dose limit (5000 µg/plate). If the test item is non-toxic to
the bacterial strains, it will, if possible, be tested at a maximum of 5000 µg/plate regardless of
solubility.
There should be no evidence of excessive contamination.

Evaluation criteria:

If the results of the experiments are clearly negative or positive, the study will be concluded as
such. Reproducibility of any apparent effect may be taken into account, particularly when the
results are considered weakly positive or when dose-dependent elevations in revertant colony
numbers, not satisfying the criteria for a positive response, are observed.
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. A fold increase greater than two times the concurrent solvent control for TA100,
TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if
accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

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. Values that the program concludes are
statistically significant but are within the in-house historical vehicle/untreated control range
will be considered negative.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium 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

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

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.  

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 (S9-mix), are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and

Table 5 for Experiment 2.  (see attachment)

Experiment 1 (plate incorporation) – Table 2 and Table 3

Controls

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 vehicle (dimethyl sulphoxide) 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 and without metabolic activation.  Thus, the

sensitivity of the assay and the efficacy of the S9-mix were validated.

Results

The maximum dose level of the test item in the first experiment was selected as the OECD

TG 471 recommended dose level of 5000 µg/plate.  

There was no visible reduction in the growth of the bacterial background lawn or any

substantial decreases in revertant colony frequency at any dose level, either in the presence or

absence of metabolic activation (S9-mix).

A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate in

both the presence and absence of metabolic activation (S9-mix).  This observation did not

prevent the scoring of revertant colonies.  

There were no biologically relevant 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 (plate incorporation method).  Minor

statistical values were noted (TA98 at 1500 µg/plate in the absence of S9-mix), however this

response was within the in-house historical vehicle/untreated control ranges and was,

therefore considered of no biological relevance

Experiment 2 (pre-incubation) – Table 4 and Table 5

Controls

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 vehicle (dimethyl sulphoxide) 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 and without metabolic activation.  Thus, the

sensitivity of the assay and the efficacy of the S9-mix were validated

Results

The maximum dose level of the test item in the second experiment was the same as for

Experiment 1 (5000 µg/plate).  

There was no visible reduction in the growth of the bacterial background lawn or any

substantial decreases in revertant colony frequency at any dose level, either in the presence or

absence of metabolic activation (S9-mix).

A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate in

both the presence and absence of metabolic activation (S9-mix).  This observation did not

prevent the scoring of revertant colonies.

There were no biologically relevant 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 2 (pre-incubation method).  Minor statistical

values were noted (WP2uvrA at 15 µg/plate in the absence of S9-mix), however this response

was within the in-house historical vehicle/untreated control ranges and was, therefore

considered of no biological relevance.

Conclusions:

CQ was not mutagenic in the bacterial mutation assay, when tested in either the presence or
absence of S9-mix. The maximum concentration was 5000 µg per plate, the maximum
concentration in accordance with current guidelines in the presence and absence of S9-mix
respectively.

Executive summary:

The purpose of this study was to assess the potential of CQ to induce gene mutations (base

pair substitutions and frameshift mutations) in vitro in bacterial strains of Salmonella

typhimurium (TA98, TA100, TA1535, TA1537) and Escherichia coli (WP2uvrA).

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,

the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012;

77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse

Mutation Test.

Study Design

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli

strain WP2uvrA were treated with the test item using both the Ames plate incorporation and

pre-incubation methods 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 (plate incorporation) was based on OECD TG 471 and was

1.5 to 5000 µg/plate.  The experiment was repeated on a separate day (pre-incubation

method) 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 concentrations per bacterial strain were selected in Experiment 2 in order to

achieve both four non-toxic dose levels and the potential toxicity of the test item following

the change in test methodology.

Results

The vehicle (dimethyl sulphoxide) 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 and 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 OECD

TG 471 recommended dose level of 5000 µg/plate.  There was no visible reduction in the

growth of the bacterial background lawn or any substantial decreases in revertant colony

frequency at any dose level, either in the presence or absence of metabolic activation

(S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was

employed in the second mutation test (pre-incubation method).  Similarly, there was no

visible reduction in the growth of the bacterial background lawn or any substantial decreases

in revertant colony frequency at any dose level, either in the presence or absence of metabolic

activation (S9-mix).  

A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate in

both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This

observation did not prevent the scoring of revertant colonies.  

There were no biologically relevant 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 (plate incorporation method).  Minor

statistical values were noted in Experiment 1 (TA98 at 1500 µg/plate in the absence of

S9-mix), however this response was within the in-house historical vehicle/untreated control

ranges and was, therefore considered of no biological relevance.

Similarly, no significant biologically relevant 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 (pre-incubation method).  Minor

statistical values were noted in Experiment 2 (WP2uvrA at 15 µg/plate in the absence of

S9-mix), however this response was within the in-house historical vehicle/untreated control

ranges and was, therefore considered of no biological relevance.

Conclusion

CQ  was not mutagenic in the bacterial mutation assay, when tested in either the presence or

absence of S9-mix.  The maximum concentration was 5000 µg per plate, the maximum

concentration in accordance with current guidelines in the presence and absence of S9-mix

respectively.

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

Based on the negative results obtained in a battery of three in vitro genotoxicity assay, there is no requirement to classify the substance as a Germ Cell Mutagen in accordance with 12/72/2008/EC.