3-acetoxymethylen-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Genetic toxicity: in vitro

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:

March 28, 1995 to June 22, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study (OECD 471) with acceptable restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-acetoxymethylen-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

EC Number:

213-485-0

EC Name:

3-acetoxymethylen-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Cas Number:

957-68-6

Molecular formula:

C10H12N2O5S

IUPAC Name:

(6R,7R)-3-[(acetyloxy)methyl]-7-amino-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 of test material (as cited in study report): 7-ACA
- Physical state: white powder
- Analytical purity: 95%-102% (HPLC)
- Batch No.: 3701586
- Expiration date of the batch: stable under conditions of storage
- Storage condition of test material: in a refrigerator in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

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 pg to 5000 pg test substance per plate were used.
"7-ACA" was toxic at concentrations of 5000 pg/plate (no growth of bacteria). At
1700 pg/plate a reduced bacterial lawn was seen. The lower concentrations allowed normal
growth of the bacteria.

So it was decided for the first experiment to use 2000 pg "7-ACA" per plate as the highest concentration which would possibly be slightly toxic, and to use 4 lower concentrations, obtained by subsequent dilution to one third (one part solution + two parts solvent). As the obtained toxicity was not clearly pronounced in the first experiment, a higher concentration, 5000 pg per plate, was added in the second experiment for all strains. 5000 pg per plate is the highest recommended concentration for the Ames test according to the OECD guideline.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4NOPD: 4-Nitro-o-phenylene-diarnine. tBHPO: t-Butyl-hydroperoxide. 2AA: 2-Amino-anthracene. DHA: 1,8-Dihydoxy-anthraquinone.

Details on test system and experimental conditions:

Reference substances:
• 2-Aminoanthracene, (Sigma, article A-1381. Practical grade).
• Sodium azide (Aldrich, Gillingham, England, article 19,993-1, 99%).
• 4-Nitro-o-phenylenediamine (Sigma, article N-9504).
• 1,8-Dihydroxy-anthraquinone (Danthron, Sigma, article D-5636, 95%).
• t-Butyl-hydroperoxide (Sigma, article B-2633, 70% aqu. solution).
1,8-Dihydroxy-anthraquinone and 2-aminoanthracene are mutagenic only when activated by a metabolising system; 4-nitro-o-phenylenediamine, t-butyl-hydroperoxide and sodium azide are directly acting mutagens.

Test and reference substance solutions - preparation
250 mg of test substance were dissolved in 4.0 ml of sterile H20. 1.0 ml of 1 Mol/1 NaOH was added to solve the test substance. This solution contained 5000 ug in 100 ul and was used for the highest concentration group. From this solution 0.6 ml were combined with 0.9 ml of sterile water. The resulting solution contained 2000 ug in 100 ul and was used for the second concentration group. The lower concentrations were obtained by subsequent dilution to one third each (one volume of solution plus two volumes of H2O).

4-Nitro-o-phenylenediamine, 1,8-dihydroxy-anthraquinone and 2- aminoanthracene were prepared from stock solutions in DMSO (dimethylsulfoxide). 4-Nitro-o-phenylenediamine was diluted with H20, 1,8-dihydroxy-anthraquinone with 80% DMSO and 2-aminoanthracene with 50 % DMSO.
Sodium azide and t-butyl-hydroperoxide were dissolved in sterile water.

Test system:
Bacterial strains of salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535, obtained in Nov. 1988 from Prof. Bruce N. Ames, Berkeley, California.

The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
The strains were tested for ampicillin resistance, UV - sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances on December 1994 (TA97a, TA98, TA100 and TA102) and on January 1995 (TA1535). The bacteria were stored frozen since that time.

The main genetic properties of these strains are:

Strain: his-Mutation: rfa: uvrB: pkMlOl:
TA97a D6610 yes yes yes
TA98 D3052 yes yes yes
TA100 G46 yes yes yes
TA102 G428 yes no yes
TA1535 G46 yes yes no

D6610 and D3052 are frameshift mutations, G46 is a base pair mutation, G428 is an ochre-mutation.

The rfa mutation leads to a reduced lipopolysaccharide barrier in the cell wall and allows larger molecules to pass the cell wall. Bacteria with this mutation are sensitive to crystal violet.

UvrB results in a loss of the DNA-excision repair system. This increases the sensitivity to mutagenic influences. Bacteria with this mutation are sensitive to UV light.

The plasmide pkMlOl also disturbs the ability of the bacteria to repair genetic damage and therefore increases its sensitivity to mutagens. Bacteria with this plasmide are resistant against ampicillin.

Justification for the bacterial strains used:
The OECD guideline requires at least the strains TA1535, TA1537 or TA97a (or TA97), TA98 and TA100 for the test. TA102 is recommended because it detects some mutagens which are not detected or are detected poorly by the standard set of tester strains.

Conditions of cultivation:
A small amount from each of the frozen bacterial cultures was transferred with a spatula to ten ml of nutrient broth. They were incubated overnight (about 16 hours) at 37 °C and then used for the exposure.

Metabolic system:
The microsomal fraction of homogenised livers of rats treated once with 500 mg/kg of aroclor 1254 was used. The treatment was on Sept. 9,1994 (for the first experiment) and April 12, 1995 (for the second experiment). 5 days after treatment, the feed was withdrawn for one night. The livers of the animals were removed and homogenised in cold 0,15 mol/1 KC1. 3 ml of homogenate were obtained per gram of liver. Then the homogenate was centrifuged for 10 minutes at 9.000 x g. The supernatant contained the microsomes. Portions of one ml of the microsomes were stored in liquid nitrogen. Immediately before use they were thawed and mixed with the cofactor solution.

Exposure
Groups, concentrations, number of samples:

high concentration 1 5000 ug/plate 3 samples
concentration 2 2000 -"- 3 -"-
concentration 3 667 -"- 3 -"-
concentration 4 222 -"- 3 -"-
concentration 5 74 3 -"-
low concentration 6 25 -"- 3
control (H20) 100 ul/plate 6 -"-
positive control * * 3 -"-

* Positive-controls (ug per plate):

Strain: without S9 with S9
TA97a 4NOPD, 20 ug 2AA, 3 pg
TA98 4NOPD, 5 ug 2AA, 3 ug
TA100 Sodium-azide, 5 pg 2AA, 3 ug
TA102 tBHPO, 50 ug DHA, 50 pg
TA1535 Sodium-azide, 5 pg 2AA, 3 us

4NOPD: 4-Nitro-o-phenylene-diarnine. tBHPO: t-Butyl-hydroperoxide. 2AA: 2-Amino-anthracene. DHA: 1,8-Dihydoxy-anthraquinone.

All concentrations of the test substance were tested without as well as with an external metabolising system (S9-mix). The results were verified by a second, independent experiment.

Exposure technique:
The exposure is 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.
For each sample the following solutions were combined:
• 0.1 ml of the overnight culture of the bacteria,
• 0.5 ml of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 ml of the appropriate test- or reference substance solution and
• 2 ml of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solified, the plates were incubated at 37 °C until the colonies were visible (2 days)

Evaluation criteria:

Counting of colonies:
The plates were counted visually by marking the colonies with a felt tipped pen. From plates with more then about 300 colonies a fraction of the total area was counted visually and the total amount of colonies was calculated.

Statistics:

Means and standard deviation were calculated for the number of mutants in every concentration group.
For comparison of the control group and the test substance groups the analysis of variance was used followed by the test of Scheffe. 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.
The criteria for a positive result are:
A reproducible statistically significant increase of the number of revertants to more then twice the amount of the spontaneous revertants for at least one of the concentrations.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

Bacteria:
The properties of the strains are listed in Table 7. The used strains of Salmonella typhimurium showed the expected genetic properties and were sensitive against several mutagenic chemicals.

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.

Test substance:
The means and standard deviations of the combined results are shown in Table 1 to Table 5, the counts of the individual plates in Table 6.

Toxicity:
The test substance was toxic at the concentration of 5000 pg per plate, resulting in reduced numbers of spontaneous revertants. At 2000 pg per plate, the number of revertants was nearly normal and no toxicity could be detected at the lower concentrations. All concentrations could be evaluated.

Mutagenicity:
This is the parameter of major interest, because a substance is called mutagenic in the Ames test when a reproducible statistically significant increase in the number of revertants to more than twice the amount of the spontaneous revertants for at least one of the concentrations occurs.

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

Applicant's summary and conclusion

Conclusions:

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

According to these results, "7-ACA" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to the concentration of 5000 pg per plate which is the limit of toxicity as well as the limit concentration for the Ames test.

Executive summary:

Method:

 "7-ACA" was tested for mutagenic action with the"Salmonella typhimurium Reverse Mutation Assay"(Ames Test). The study was conducted in accordance with the OECD-guideline 471.

 

The test substance was tested at concentrations ranging from 25 ug to 5000 ug 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 2000 ug per plate in the first experiment, resulting in reduced numbers of spontaneous revertants. As the toxicity however was pronounced only in strain TA100, a further, higher concentration was added at the second experiment for all strains exept TA100. This concentration - 5000 ug per plate - was clearly toxic. 5000 ug per plate is also the limit concentration for the Ames test.

 

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.

 

"7-ACA" is thereforenon-mutagenic in the Ames testwith the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 ug per plate which is the limit of toxicity and the limit of this kind of test.