6-aminopenicillanic 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

Study period:

27 November 2009 to 12 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted to current accepted guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

6-aminopenicillanic acid

EC Number:

208-993-4

EC Name:

6-aminopenicillanic acid

Cas Number:

551-16-6

Molecular formula:

C8H12N2O3S

IUPAC Name:

6-amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Details on test material:

- Name of test material : 6-Aminopenicillanic acid
- Substance type: Off white powder
- Physical state: Solid
- Analytical purity: 98.7 % (w/w)
- Impurities : Any other single impurity 0.07 %, Penicinic acid 0.17 %, Related Substance and Degradation Products 0.85 %
- Purity test date: 29 May 2009
- Lot/batch No.: 09X10247
- Expiration date of the lot/batch: 26 May 2011
- Storage condition of test material: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: Collected from a human volunteer

Details on mammalian cell type (if applicable):

The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal enzyme fraction

Test concentrations with justification for top dose:

PRELIMINARY TEST
4(20)-hour without S9 (µg/mL) : 0, 8.44, 16.88, 33.75, 67.5, 135, 270, 540, 1080, 2160
4(20)-hour with S9 (µg/mL): 0, 8.44, 16.88, 33.75, 67.5, 135, 270, 540, 1080, 2160
24-hour without S9 (µg/mL): 0, 8.44, 16.88, 33.75, 67.5, 135, 270, 540, 1080, 2160

MAIN TEST
4(20)-hour without S9 (µg/mL) : 0, 67.5, 135, 270, 540, 1080, 2160, mitomycin C 0.4
4(20)-hour with S9 (µg/mL): 0, 67.5, 135, 270, 540, 1080, 2160, cyclophosphamide 5
24-hour without S9 (µg/mL): 0, 67.5, 135, 270, 540, 1080*, 2160, mitomycin C 0.2

Vehicle / solvent:

- Vehicle(s)/solvent: Minimal Essential Medium (MEM)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: in the presence of S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: in the absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours exposure in the absence of metabolic activation (S9), 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), and a 24 hours continuous exposure in the absence of metabolic activation.
- Expression time (cells in growth medium): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 µg/ml) was added two hours before the required harvest time
STAIN (for cytogenetic assays): 5% Gurrs Giemsa for 5 minutes

NUMBER OF REPLICATIONS: Performed in duplicate

NUMBER OF CELLS EVALUATED: In total 2000 lymphocyte cell nuclei

DETERMINATION OF CYTOTOXICITY
- Method: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated(E).

Evaluation criteria:

The first 100 consecutive well-spread metaphases (where possible) from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.

The classification criteria is defined in the section Any other information on materials and methods incl. tables under the heading Classification Criteria.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm
- Precipitation:. No precipitate was observed in the parallel blood-free cultures at the end of the exposure, at any dose level in any of the three exposure groups.

RANGE-FINDING/SCREENING STUDIES: Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2160 µg/ml in all three of the exposure groups. The test material induced some evidence of toxicity in the 4(20)-hour exposure groups, and clear dose-related toxicity in the 24-hour continuous exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA: In the 4(20)-hour exposure group in the absence of metabolic activation a very modest statistically significant increase in the frequency of cells with aberrations was recorded at the maximum dose level of 2160 µg/mL. The response which was only just outside the historical control range for this dose group, was compared to a low vehicle control value, did not include any exchange-type aberrations and was not reproduced in the 24-hour continuous exposure group. It was therefore considered to have no toxicological significance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tabulated results are detailed in the attached pdf document, Appendix I

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce a toxicologically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.