(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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August - 27 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

1-{[(6R,7R)-7-[(2Z)-2-({[1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl]oxy}imino)-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetamido]-2-carboxylato-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl}pyridin-1-ium

EC Number:

617-560-2

Cas Number:

84377-83-3

Molecular formula:

C45H44O7S2.5/2(C3H7NO)

IUPAC Name:

1-{[(6R,7R)-7-[(2Z)-2-({[1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl]oxy}imino)-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetamido]-2-carboxylato-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl}pyridin-1-ium

additive 1

Reference substance name:

N,N-dimethylformamide

EC Number:

200-679-5

EC Name:

N,N-dimethylformamide

Cas Number:

68-12-2

Molecular formula:

C3H7NO

IUPAC Name:

N,N-dimethylformamide

Test material form:

solid: particulate/powder

Method

Target gene:

Not relevant - The study examined gross chromosomal aberrations

Species / strain

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

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.