(6S,7S)-7-Amino-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

. EC directive 92/69, method B.10., considering the draft of the update, DOC.ECB/TM/8. This is equivalent to 2000/32/EC, B.10.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

435-680-1

EC Name:

-

Cas Number:

24701-69-7

Molecular formula:

C9H12N2O4S (Hill formula) C9H12N2O4S (CAS formula)

IUPAC Name:

(6R,7R)-7-amino-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: 7-AMCA
Chemical name: 7-Amino-3-methoxy-cefalosporanic acid
Molecular formula: C9H12N2O4S
CAS No.: 24701-69-7
Supplier: Sponsor
Batch No.: 36423006
Certificate of analysis: Available
Purity: 90.1 % on anhydrous basis (HPLC)
Water content: 0.3 %
Solubility: In water 0.1 g/L at 20 °C
In methanol 0.05 g/L
Appearance: Yellowish powder
Conditions of storage: Refrigerator, in the dark. Protected from moisture
Stability: Stable over ca. 6 months under conditions of storage. Stable over several weeks at room temperature
Date of expiry: October 2000

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes.

Details on mammalian cell type (if applicable):

Whole blood was obtained from healthy males (aged 43 years for experiments "A" and 47 years for experiments "B") into Na-heparinized Vacutainers (Becton-Dickinson). The volunteers were non-smokers and were not suspected of any virus infection nor had been exposed to high levels of radiation or toxic chemicals. Primary cultures were established within one hour after venipuncture by adding 0.7 mL of whole blood to 9.3 mL of complete culture medium

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254 induced rat livers.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 4 to 100 µg/mL.
Concentration range in the main test (without metabolic activation): 4 to 100 µg/mL.
The test substance is of low solubility in aqueous media. 0.1 mg test substance per mL of culture medium were the highest technically feasible concentration in medium.

Vehicle / solvent:

Culture medium: RPMI 1640 medium with L-glutamine.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Four experiments were performed: two of them without and two with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution).
Primary lymphocyte cultures were established from whole blood freshly obtained from male donors. After 48 hours of incubation, the test substance was added.
In the experiments with the use of a metabolic activation system the test substance was washed out three hours later, and the cultures were cultivated for another 15 hours. Colcemid was added for 2 hours, and then cells were fixed and slides prepared.
For cultures without addition of a metabolic activation system, the treatment time was 3 hours in the first experiment and 20 hours in the second experiment.
For each concentration of the test substance two cultures were established.

Evaluation criteria:

Mitotic indices:
The mitotic indices were determined by counting a total of 2000 lymphocytes per cell culture and by recording the number of lymphocytes in any stage of mitosis.
Selection of cultures for analysis:
In all experiments the cultures with the highest test substance concentrations and with the next lower two concentrations were analysed. By analysing 3 consecutive concentrations, an about tenfold range of concentrations was covered in each experiment.
Coding of slides
Slides from the selected treatments were coded by assigning random numbers before analysing for aberrations. Self adhesive labels covered the identification marks on the slides. The code list was not available to the analysing persons until the last slide was analysed.
Chromosome analysis
ZEISS microscopes (Universal R and Photomicroscope III) were used for analysis (magnification factor of about 600 to 1000). For each culture evaluated 100 metaphases were analysed for structural and numerical chromosomal aberrations (except for cultures with obviously high numbers of metaphases with aberrations), i.e. 200 per concentration. Only well spread cells with 44 to 47 chromosomes, polyploid and endoreduplicated cells were acceptable for analysis.

Statistics:

The Chi2-Test (two-tailed, p=0.05) was used for the comparison between the negative control and the 7-AMCA cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher's Exact Test was used. Chi2-Test or Fisher's Exact Test were also used for the comparison between the negative and the positive controls.
The following parameters were evaluated:
• the number of metaphases with numerical aberrations ( < 46 chromosomes, > 46 chromosomes, tetraploidy, endoreduplication)
• the number of metaphases with structural aberrations, excluding gaps
• the number of chromatid-type aberrations, excluding gaps
• the number of chromosome-type aberrations, excluding gaps
• the number of gaps (chromatid- and isochromatid-type).
If there were more than one structural aberration of the same type within one metaphase, these aberrations were referred to as ,multiple". For statistical comparisons of the numbers of a specific aberration, such aberrations were counted as two aberrations.
A reproducible increase in the number of metaphases with aberrations or a concentration related increase in this number is considered as a positive result. However, a result can also be regarded as positive when other than merely statistical considerations, for example the kind of aberrations observed, are taken into account.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Concurrent positive controls: The mitotic indices were between 66.4 and 88.2 %of the negative controls. The positive control substances caused clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating the validity of both experiments.
Additional statistically significant differences to the negative controls like the number of gaps, the number of chromatid-type aberrations, etc., are indicated in the attached Tables.

Mitotic index: 7-AMCA did not cause marked cytotoxicity in any experiment performed at any concentration tested. The mitotic indices of the test substance treated cultures were between 85.5 and 103.5 % of those of the corresponding negative controls.

Structural aberrations: No statistically significant increases in the number of metaphases with structural aberrations were noted at any concentration analysed compared to the concurrent negative controls, neither after treatment for 3 hours nor for 20 hours and neither without nor with the use of a metabolic activation system. All figures were within the range of historical negative controls.

Numerical aberrations: A statistically significantly higher number of metaphases with less than 46 chromosomes was noted in one experiment with a metabolic activation system at each tested concentration. This result was not confirmed by a second experiment with a metabolic activation system and was therefore regarded as a random event. There were no indications for a raise in numerical aberrations in the test substance treated cultures in any other experiment.

Gaps: No statistically significant increases in the number of gaps were noted at any concentration analysed compared to the concurrent negative controls, neither after treatment for 3 hours nor for 20 hours and neither without nor with the use of a metabolic activation system. All figures were within the range of historical negative controls.

Remarks on result:

other: other: treatment length 3 h.

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test substance did not induce chromosomal aberrations in cultured human lymphocytes.

Executive summary:

Possible mutagenic properties of 7-AMCA were investigated by means of an in vitro mammalian chromosome aberration test in human lymphocytes, according to the EC-method B.10.

Methods

Four experiments were performed: two of them without and two with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution).

Primary lymphocyte cultures were established from whole blood freshly obtained from male donors. After 48 hours of incubation, the test substance was added. In the experiments with the use of a metabolic activation system the test substance was washed out three hours later, and the cultures were cultivated for another 15 hours. Colcemid was added for 2 hours, and then cells were fixed and slides prepared. For cultures without addition of a metabolic activation system, the treatment time was 3 hours in the first experiment and 20 hours in the second experiment.

For each concentration of the test substance two cultures were established. One negative control (medium) and one positive control (methanesulfonic acid methyl ester (MMS) for cultures without metabolic activation system and cyclophosphamide (CP) for cultures with a metabolic activation system) were set up concurrently in each experiment.

The concentrations of 7-AMCA ranged from 0.004 to 0.1 mg/mL.

100 metaphases were analysed for structural and numerical chromosomal aberrations, i.e. 200 per concentration.

Results

Cytotoxicity:

7-AMCA did not cause marked cytotoxicity in any experiment.

Numerical aberrations, gaps:

There were no indications for a raise in numerical aberrations in the test substance treated cultures in any other experiment. Neither with nor without the use of a metabolic activation system a statistically significant increase in the number of gaps compared to the concurrent negative controls was noted at any of the concentrations analysed in any experiment performed.

Structural aberrations:

No statistically significant increases in the number of metaphases with structural aberrations were noted at any concentration analysed, neither after treatment for 3 hours nor for 20 hours and neither without nor with the use of a metabolic activation system. The figures were also within the range of historical negative controls.

Conclusion

7-AMCA did not cause any marked cytotoxic effect at any treatment. Under the conditions of this study there was no relevant evidence that 7-AMCA did induce chromosomal aberrations in cultured human lymphocytes. The conclusion is based on a lack of a statistically significant increase of metaphases with structural aberrations in any experiment and on a lack of a concentration-dependent increase of metaphases with structural aberrations.