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)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance has been screened for mutagenicity in the Ames bacterial reverese mutation assay and the Mouse Lymphoma Gene mutation assay. There were no indications of any genotoxic potential in either assay. The chromosome aberration assay was waived give that a suitable in vivo micronucleus assay is available.

Link to relevant study records

Reference

Reference 1

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

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:

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.

Target gene:

Histidine or tryptophan

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.

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.

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2018- 24 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: Ceftazidime dihydrochloride
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

Target gene:

Thymidine Kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction

Test concentrations with justification for top dose:

The dose range of test item used in the main test was selected following the results of a
preliminary toxicity test at a concentration range of 7.81 to 2000 µg/mL. The maximum
dose level used in the Mutagenicity Test was up to the maximum recommended dose level of
2000 µg/mL. The dose levels plated for viability and expression of mutant colonies were as
follows:
4-hour without S9: 125, 250, 500, 750, 1000, 2000 µg/ml
4 hour with S9 (2%): 125, 250, 500, 750, 1000, 2000 µg/ml
24 hour without S9: 31.25, 62.5, 125, 250, 500 µg/ml

Vehicle / solvent:

R0 culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

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.

3.2.3 Microsomal Enzyme Fraction
Lot No. PB/NF S9 14/12/17 and 29/09/18 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
certificates of S9 efficacy are presented in Appendix 2.
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.

Rationale for test conditions:

Standard for test guideline

Evaluation criteria:

Standard for test guideline based on comparison of Mutation Frequency against Global Evaluation Factor

Statistics:

Plate count data from the viability and mutation frequency plates and the daily cell count data
will be analyzed using a dedicated computer program (Mutant 2.40 York Electronics) which
applies the statistical analysis method recommended by the UKEMS (Robinson et al., 1989).
The statistical package used indicates the presence of statistically significant increases and
linear trend events.

Species / strain:

mouse lymphoma L5178Y cells

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

6.1        Preliminary Cytotoxicity Test

 

The dose range of the test itemused in the preliminary toxicity test was 7.81 to 2000 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

 

Dose (mg/mL)	%RSG(-S9) 4-Hour Exposure	%RSG(+S9) 4-Hour Exposure	%RSG(-S9) 24-HourExposure
0	100	100	100
7.81	120	97	90
15.63	105	83	99
31.25	91	89	81
62.5	113	66	111
125	96	66	97
250	110	81	58
500	85	81	48
1000	89	73	4
2000	80	67	1

 

There was evidence ofmarked dose-related reductions in theRelative Suspension Growth (%RSG) of cells treated with the test itemin the 24-hour exposure group when compared to the concurrent vehicle control groups. No precipitate was observed. Therefore, the dose levels selected for the main test was upto the maximum recommended dose level of 2000 µg/mL.

6.2 Mutagenicity Test

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

As was seen previously, there was evidence of marked dose related toxicity following

exposure to the test item in the 24-hour exposure group only (Tables 3, 6, and 9).  In both 4hour

exposure groups the test item was exposed up to the maximum recommended dose level of

2000 µg/mL.  Optimum levels of toxicity were achieved in the 24-hour exposure in the absence

of metabolic activation at 500 µg/mL.  There was evidence of marked reductions in viability

(%V) in the 24-hour exposure group only, indicating that residual toxicity had occurred (Table

9).  Acceptable levels of toxicity were seen with the positive control substances (Tables 3, 6, and 9).  The concentrations

of 750, 1000 and 2000 µg/mL in the 24hour exposure in the absence of metabolic activation were not plated out for 5-TFT resistance

and viability due to excessive toxicity.

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 increases in the mutant frequency

x 10 e-6 per viable cell at any of the dose levels, in any of the three exposure groups.  The GEF

value was not exceeded in all three of the exposure groups.

KEY TO TABLES

$        =      Cell counts (x105cells/ml). Set up on previous day to 2 x 105cells/ml unless otherwise stated in parenthesis.

 %RSG  =      Relative Suspension Growth

 RTG     =      Relative Total Growth

 %V      =      Viability Day 2

§ or #   =      Positive wells per tray,96 wells plated unless otherwisestated in parenthesis

 A,B      =      Replicate cultures

 CP        =      Cyclophosphamide

 EMS    =      Ethylmethanesulphonate

 MF§     =      5-TFT resistant mutants/10e6 viable cells 2 days after exposure

 Nv        =      Number of wells scored, viabilityplates

 Yv        =      Number of wells without colonies, viability plates

Ym       =      Number of wells without colonies, mutation plates

Nm       =      Number of wells scored,mutation plates

Ø          =      Not plated surplus to requirements

NP        =      Not plated for viability or 5-TFT resistance

Table 1

 

Treatment (µg/ml)		4 hours – S9				Treatment (µg/ml)		4 hours +S9
		%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	114.37		0		100	1.00	117.83
31.25	Ø	93				31.25	Ø	112
62.5	Ø	90				62.5	Ø	103
125		86	0.87	120.22		125		104	1.03	120.63
250		106	1.04	124.90		250		107	0.98	119.48
500		100	0.87	145.91		500		88	0.72	119.10
750		92	0.95	126.48		750		105	0.97	108.85
1000		109	1.18	119.09		1000		117	1.08	130.02
2000		98	0.96	118.70		2000		120	1.11	134.14
MF threshold for apositiveresponse : 240.37					MF threshold for apositiveresponse : 243.83
EMS 400		83	0.62	1343.40		CP 1.5		85	0.58	876.29

 

Treatment (µg/ml)	24-hours-S-9
	%RSG	RTG	MF§
0 31.25 62.5 125 250 500 750    Ø 1000   Ø 2000   Ø	100 98 96 84 48 15 3 1 0	1.00 0.95 0.88 0.77 0.51 0.17	150.65 125.07 181.04 152.74 193.52 274.59
MF threshold for apositiveresponse : 276.65
EMS 150	62	0.33	1427.33

 

Table3            SummaryAnalysis:Mutagenicity Test (-S9) 4-Hour Exposure

 

Treatment (µg/ml)	SG	%RSG	%V	RTG	MF§
0 31.25       Ø 62.5         Ø 125 250 500 750 1000 2000	14.77 13.20 12.68 11.80 14.54 14.61 12.30 15.66 14.28	100 93 90 86 106 100 92 109 98	90.77      90.38 89.59 77.81 93.66 98.08 88.81	1.00      0.87 1.04 0.87 0.95 1.18 0.96	114.37      120.22 124.9 145.9 126.4 119.0 118.7
Treatment (µg/ml)	SG	%RSG	%V	RTG	MF§
400	11.69	83	67.27	0.62	1343.40

 

GEF =126,therefore MF threshold for a positive response= 240.37

Table 6            Summary Analysis:Mutagenicity Test (+S9) 4-HourExposure

 

Treatment (µg/ml)	SG	%RSG	%V	RTG	MF§
0 31.25       Ø 62.5         Ø 125 250 500 750 1000 2000	14.49 16.51 15.41 15.05 15.95 12.66 14.86 16.85 18.11	100 112 103 104 107 88 105 117 120	101.93        100.94 93.66 85.83 95.38 93.66 94.51	1.00        1.03 0.98 0.72 0.97 1.08 1.11	117.83        120.63 119.48 119.10 108.85 130.02 134.14
PositiveControl CP
Treatment (µg/ml)	SG	%RSG	%V	RTG	MF§
1.5	12.19	85	68.80	0.58	876.29

  

GEF =126,therefore MF threshold for a positive response= 243.83

Table9            SummaryAnalysis:Mutagenicity Test (-S9) 24-HourExposure

 

Treatment (µg/ml)	SG	%RSG	%V	RTG	MF§
0 31.25 62.5 125 250 500 750              Ø 1000             Ø 2000             Ø	95.03 93.96 93.54 83.45 58.09 24.81 5.86 3.00 1.52	100 98 96 84 48 15 3 1 0	85.83 83.01 76.59 75.40 72.54 54.93	1.00 0.95 0.88 0.77 0.51 0.17	150.65 125.07 181.04 152.74 193.52 274.59
PositiveControl EMS
Treatment (µg/ml)	SG	%RSG	%V	RTG	MF§
150	63.70	62	41.63	0.33	1427.33

 

 

GEF =126,therefore MF threshold for a positive response= 276.65

Conclusions:

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 (GEF) of 126 x 10e-6
, consequentlyit is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Intraperitoneal exposure of CD-1 mice to upto 2500 mg/kg of the test substance did not result in an increase in erythrocyte micronuclei 48 hours after exposure.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not applicable

Remarks:

The study was conducted before the respective guideline was adopted

GLP compliance:

no

Remarks:

The study was conducted before GLP was adopted. Syudy conducted according to internal company Quality Assurance systems

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Cephalosporin GR 20263 (pentahydrate), Toxicology reference number
TM 3/14a, 3/14b was provided by the Pharmacy Division, Glaxo Group
Re.search Ltd, Greenford, under their reference number EPND 2/6
(original batch no. CRI 016).

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

Seventy male Charles River CDl mice, ex Charles River (UK) Ltd,
Manston Road, Margate, Kent. were housed in NKP plastic cages with
sawdust bedding. The mice were allocated to 14 groups of 5 animals,
one group per cage. S.Q.C. Rat/Mouse No. 1 expanded and modified
diet (BP Nutrition Ltd) was provided ad libitum. Tap water from
the domestic water supply was provided ad libitum from plastic con­
tainers. The weight of the mice fell within the range 20.9 to
33. 6g. Groups were identified by means of non-toxic indelible ink
marks on the dorsal skin surface of each mouse.

Route of administration:

intraperitoneal

Vehicle:

0.9% w/v saline

Details on exposure:

Solutions of ceftazidime were prepared: 1.455g or 0.582g of GR 2026 3
pentahydrate .were. mixed. with 0.145g or 0.0582g of sodium carbonate
respectively and made up to 5 ml with sterile water for injection.
These 25% (w/v) and 10% (w/v) solutions (expressed as anhydrous
betaine) were sterilised by passage through 0.45µm membrane filters.
The solutions were then stored at 25•c in a water bath for 24 hours
prior to administration to the test animals. A negative control
solution consisting of sterile saline (0. 9% w/v sodium chloride)
was also stored under identical conditions.

Fresh solutions of ceftazidime in sterile water for injection
were also prepared in an identical manner immediately before
administraton to the test animals. Saline stored at 4°C was used
as the negative control, for these freshly prepared ceftazidime
solutions. This saline solution will be .referred to as fresh
saline to distinguish it from saline stored at 25°C.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

One dose

Post exposure period:

24 and 48 hours

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 500 mg/kg bw (total dose)

No. of animals per sex per dose:

five

Control animals:

yes, concurrent vehicle

Positive control(s):

5 animals recievd 50 mg/kg bw cyclophosphamide

Tissues and cell types examined:

femur bone marrow

Details of tissue and slide preparation:

Both femurs were removed from each mouse and the bone marrow
aspirated into foetal bovine serum in order to wash the marrow
cells. The resulting cell suspensions were centrifuged and the
supernatants discarded. Smears were prepared from drops of the
resuspended cell pellets. After differential staining in May­
Grunwald and Giemsa, the smears were examined for micronuclei.

Evaluation criteria:

One thousand immature (polychromatic) erythrocytes were examined
per animal. The number of micronucleated immature erythrocytes was
recorded. The ratio of mature/immature erythrocytes, obtained by
counting the number of red cells observed in fields containing a
total of 250 polychromatic erythrocytes, was also recorded.

Statistics:

arc-sine transformation was used to stabilize inter­
animal variation prior to statistical analysis (Kilian et al, 1977; Mostella et al 1961).
arc-sine of square root ( no of micronucleated cells/total number of immature cells).
Bartlett's test (Snedecor et al, 1967), was used to test for
homogeneity of error variances. As the variances were not signi­
ficantly different, Dunnett's test (Dunnett, 1955) was used to
compare the treated groups with the negative controls.
The mean, standard deviation and standard error of the mean were
calculated for each group. All ceftazidime-treated groups were
compared with the appropriate saline negative control by Dunnett's
t-test; the cyclophosphamide positive control was compared with the
fresh saline negative control. Despite the arc-sine transformation,
the variance of the positive control treated mice is usually
somewhat greater than those of the other groups and was therefore
analysed separately to avoid bias in favour of the test compound.

ii) Ratio of Mature to Immature Erythrocytes
The ratio of mature to immature erythrocytes was determined for
each animal. The mean, standard deviation and standard error of
the mean were calculated for each group. Dunnett's t-test was
then used on the untransformed data to test the differences between
group means from animals receiving stored and fresh ceftazidime and
their appropriate saline controls. The cyclophosphamide dose group
and the fresh saline control were compared separately.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

The concentrations of pyridine present after storage of ceftazidime

solutions for 24 hours at 25°C were found to be 0.82% w/w and 0.72% w/w

in the 250 mg/ml and 100 mg/ml solutions respectively. The concentra­

tions of pyridine in fresh solutions of ceftazidime were 0.13% w/w and

0.11% w/w in the 250 mg/ml and 100 mg/ml solutions respectively.

The mean results after treatment of each group with stored

or fresh solutions of ceftazidime or saline are given in the table below.

These show the mean number of micronuclei scored for each group per thousand

polychromatic erythrocytes for each expression time. The tables also

contain the transformed data and the ratios of mature to immature

erythrocytes. The data for the positive control, cyclophosphamide, is also given for both expression times.

Treatment	24 hours			48 hours
	Mean micronucleated cells/1000	Arc-sine transformed	Ratio mature/immature erythrocytes	Mean micronucleated cells/1000	transformed	Ratio mature/immature per 250 cells
Fresh preparation
Control	1	0.024	0.88	0.4	0.013	0.97
1000 mg/kg	0.8	0.026 ns	1.02	1.4	0.028 ns	0.93
2500mg/kg	0.8	0.022 ns	1.01	0.8	0.022 ns	0.92
Stored preparation
Control	0.8	0.022	0.86	0.8	0.022	0.98
1000 mg/kg	0.6	0.015 ns	0.95	1	0.024 ns	0.96
2500 mg/kg	1	0.032 ns	1.08	0.6	0.011 ns	1.13
50 mg/kg cyclophosphamide	26.2	0.162*	1.13	25.2	0.159*	1.41

*= P<0.05 by Dunnett's t-test ns= not significant

The table below gives the statistical analysis of the ratios of

mature to immature erythrocytes in test and control groups. A signi­

ficant reduction in the proportion of immature cells was observed

with the largest dose ( 2500 mg/kg) of stored ceftazidime solution 24

hours after dosing. This may indicate mild marrow depression following

very large doses of ceftazidime since reductions also occurred with

fresh solutions, although they were not statistically significant.

Treatment	24 hours			48 hours
	Ratio mature/immature erythrocytes	Standard error	T value	Ratio mature/immature erythrocytes	Standard error	T value
Fresh preparation
Control	0.88	0.039	-	0.97	0.057	-
1000 mg/kg	1.02	0.064	1.73	0.93	0.017	0.63
2500mg/kg	1.01	0.058	1.63	0.92	0.056	0.69
50 mg/kg cyclophosphamide	1.13	0.057	3.56*	1.41	0.032	6.72*
Stored preparation
Control	0.86	0.019	-	0.98	0.095	-
1000 mg/kg	0.95	0.045	2.23	0.96	0.076	0.1
2500 mg/kg	1.08	0.011	5.49*	1.13	0.078	1.27
50 mg/kg cyclophosphamide	1.13	0.057	4.53*	1.41	0.032	4.32*

Cyclophosphamide (50 mg/kg) also affected cell maturity at both expres­

sion times. A significant reduction in the proportion of immature to

mature erythrocytes was observed which was more marked 48 hours art er­

dosing. These effects are to be expected with therapeutic doses of a

cytostatic drug such as cyclophosphamide.

Conclusions:

In the micronucleus test, neither ceftazidime, nor the pyridine
generated through degradation of the antibiotic during storage in
solution, induced a significant increase in detectable chromosome
damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based upon the lack of mutagenic potential observed in the in vitro Ames and MOuse Lymphoma saays, and the lack of effects observed in an in vivo mouse micronucleus assay, the substance demonstrates no genotoxic potential; the classification criteria for Germ Cell Mutagenicity as set out in 1272/2008/EC are not met.