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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
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:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Direct plate incorporation method.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: 7-ACT
Chemical name: (6R, 7R)-7-Amino-3-(2,5-dihydro-2-methyl-5-oxo-6-hydroxy-1,2,4-triazin-3-yl) thiomethyl-ceph-3-em-4-carbonic acid
CAS No.: 58909-56-1
Supplier: Biochemie Ges.m.b.H.
Batch No.: 3201530
Purity: 99.5 % (content related to waterfree substance)
Water content: 1.5 %
Impurities: Heavy metals: < 20 ppm, by-products: < 0.2 % each
Appearance: white powder
pH: 3.97 (1 % aqueous suspension, measured by pH-meter)
Stability of the test substance: stable under conditions of storage
Disintegration: from 180 *C on
Storage: in a refrigerator in the dark
Date of handing over: July 26, 1994



Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Metabolic activation system:
Microsomal fraction of rat liver, induced with Aroclor 1254.
Test concentrations with justification for top dose:
1.2 to 100 µg/plate.

Vehicle / solvent:
Water.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-aminoanthracene; 1,8-dihydroxy-anthraquinone; 4-nitro-o-phenylenediamine; t-butyl-hydroperoxide
Details on test system and experimental conditions:
In a preliminary experiment the strain TA100 was mixed with the test substance and top-agar and plated on standard agar. The growth of the bacterial lawn was recorded. 9 concentrations ranging from 0.8 µg to 5000 µg test substance per plate were used. 7-ACT was toxic at concentrations of 560 µg/plate and above (no growth of bacteria). At 190 µg/plate only single bacterial colonies and no homogeneous bacterial lawn were seen. At 62 µg/plate the growth was still reduced. No toxicity was seen at 21 µg/plate and lower.

The plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was tested without as well as with an external metabolising system (S9-mix). The results were verified by a second, independent experiment.
The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state.
Evaluation criteria:
The plates were counted visually by marking the colonies with a felt tipped pen. From plates with more than about 300 colonies a fraction of the total area was counted visually and the total amount of colonies was calculated. When more than one person was counting, each person counted the same parts of the control group and of each of the dosed groups.
The criteria for a positive result are:
A reproducible statistically significant increase of the number of revertants to more than twice the amount of the spontaneous revertants for at least one of the concentrations.

Statistics:
Means and standard deviation were calculated for the number of mutants in each concentration group.

For comparison of the control group and the test substance groups the analysis of variance was used followed by the test of Scheffé. For comparison of the control group and the positive control group, the t-test was used. p = 0.05 was used as the significance level.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Reference substances:
The positive control substances increased the mutation frequency statistically significantly. As 2-aminoanthracene is non mutagenic without metabolic activation and the mutagenicity of 1,8-dihydroxy-anthraquinone is lower without metabolic activation, the results of these substances demonstrate also the efficiency of the metabolising system.
The mutation frequencies of the negative control groups were in the usual range for the different strains.

Toxicity of the test substance:
The test substance was slightly toxic at the concentration of 100 µg per plate, resulting in reduced numbers of spontaneous revertants. No toxicity could be detected at the lower concentrations. All concentrations could be evaluated.

Mutagenicity of the test substance:
There was no statistically significant increase in the number of mutants in any of the tested bacterial strains with any of the tested concentrations, in both experiments. The addition of an external metabolising system did not change this results.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

7-ACT is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to the concentration of 100 µg per plate which causes slight toxicity.
Executive summary:

7 -ACT was tested at concentrations ranging from 1.2 µg to 100 µg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed. Results:

Positive controls:

All positive control groups showed significantly increased mutation frequencies which demonstrates the sensitivity of the test system.

Test substance:

The test substance was slightly toxic at the concentration of 100 µg per plate, resulting in reduced numbers of spontaneous revertants. No toxicity could be detected at the lower concentrations.

In none of the tested concentrations and with none of the used strains a statistically significant increase of the mutation frequency was obtained. Metabolic activation did not change these results.