Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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).

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals 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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.