Pyridinium, 1-[[(6R,7R)-7-[[(2Z)-(2-amino-4-thiazolyl)[(1-carboxy-1-methylethoxy)imino]acetyl]amino]-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-, chloride, hydrochloride (1:1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 March 2018 to 05 April 2018

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

73547-70-3

Molecular formula:

C22-H22-N6-O7-S2.2 H-Cl

Specific details on test material used for the study:

Physical state/Appearance: White powder
Batch Number: G317308
Purity: 99.2% w/w (as dihydrochloride)
Expiry Date: 26 October 2018
Storage Conditions: Approximately 4 °C in the dark
No correction for purity was required.

Method

Target gene:

Histidine or tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

WP2uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The maximum concentration was 5000 ug/plate (the OECD TG 471 maximum recommended dose level). Ten concentrations of the test item (0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Experiment 1 – Plate Incorporation Method
The maximum concentration was determined by the Preliminary Toxicity Test and was the toxic limit of the test. Ten concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method
Dose concentrations were selected as follows:
All Salmonella strains (with and without S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate).
WP2uvrA (with and without S9): 0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 µg/plate).
Ten dose level concentrations were selected in Experiment 1 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item.

Test for Mutagenicity: Experiment 2 – Plate Incorporation Method
The test item induced toxicity at dose levels that could be considered to be at the limit of the sensitivity of the assay. Therefore, with Sponsor approval, the assay was repeated using the same methodology (plate incorporation) given the uncertainty of achieving a dose response curve using the pre-incubation method
The dose range used for Experiment 2 was determined by the results of Experiment 1 as follows:
Salmonella strains TA100 (without S9) and TA98 (with and without S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Salmonella strain TA1537 (with and without S9): 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 µg/plate.
Salmonella strain TA100 (with S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
E.coli strain WP2uvrA and Salmonella strain TA1535 (with and without S9): 0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5 µg/plate).

Vehicle / solvent:

The vehicle control used was as follows:
Identity: Sterile distilled water
Supplier: Baxter
Batch number, (purity), expiry: 17H09BA1A (N/A) July 2020
17K20BA1A (N/A) October 2020 (Experiment 2 only)

vehicle (sterile distilled water) control plates
The negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene,

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.

Strains TA1535 and TA1537
Results will be judged as positive if the increase in mean revertant counts per plate at the peak of the dose response is greater than or equal to 3.0-times the mean concurrent solvent control value.

Strains TA98, TA100 and WP2uvrA
Results will be judged as positive if the increase in mean revertant counts per plate at the peak of the dose response is greater than or equal to 2.0-times the mean concurrent solvent control value.

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Ceftazidime dihydrochloride 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 by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to ten 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 a preliminary toxicity test and ranged between 0.0005 and 500 g/plate, depending on bacterial strain type and presence or absence of S9-mix.  The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations.  Although the dose range was the same as Experiment 1, some tester strain ranges were amended for Experiment 2, depending on bacterial strain type and presence or absence of S9-mix.  Additional dose levels and an expanded dose range were selected in both experiments in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item.

The vehicle (sterile distilled water) 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 preliminary toxicity test was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.  The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the frequency of revertant colonies of both of the tester strains (TA100 and WP2uvrA). In Experiment 1, the maximum concentration of the test item was limited by toxicity.  The

test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the frequency of revertant colonies of all of the tester strains in both the absence and presence of metabolic activation (S9-mix). The test item induced toxicity at dose levels that could be considered to be at the limit of the sensitivity of the assay.  Therefore, with Sponsor approval, the assay was repeated using the same methodology (plate incorporation) given the uncertainty of achieving a dose response curve using the pre-incubation method. In Experiment 2, the toxic limit of the test item was again employed as the maximum concentration.  The test item induced an almost identical toxic response to the first experiment with visible reductions in the growth of the bacterial background lawns and/or substantial reductions in the frequency of revertant colonies of all of the tester strains noted in both the absence and presence of metabolic activation (S9-mix). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in any of the experiments performed. 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 either Experiment.  

Ceftazidime dihydrochloride was considered to be non-mutagenic under the conditions of this test.