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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 October 1998 to 23 October 1998
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recent EU test guidance in compliance with GLP.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
according to guideline
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
according to guideline
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): T/9601
- Physical state: Powder
- Analytical purity: 94.7
- Lot/batch No.: HI-295
- Expiration date of the lot/batch: May 2001
- Stability under test conditions: Confirmed over 4 hours hours in DMSO
- Storage condition of test material: darkness at approximately 5 C in a refrigerator


Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat liver
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50, 160, 500, 1600, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 160, 500, 1600, 5000 µg/plate
Vehicle / solvent:
Solvent: DMSO
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Without metabolic activation: sodium azide (TA 100, TA 1535), 9-aminoacridine hydrochloride hydrate (TA 1537), 2-nitrofluorene (TA98), 1-methyl-3-nitro-1-nitrosoguanidine (WP2uvrA) With metabolic activation: 2-aminoanthracene – all strains.
Details on test system and experimental conditions:
Preparation and storage of a liver homogenate fraction (S9)

The S9 fraction was prepared by the department conducting the study according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was centrifuged at approx. 9000 g for 10 minutes. The supernatant was the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix

Sufficient S9 fraction was thawed immediately at room temperature before each test. One volume of S9 fraction (batch no. 98/5 for the experiment, protein concentration 24.8 g/l) was mixed with 9 volumes of the S9 cofactor solution, which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the S9-mix were:
8 mM MgCI2 33 mM KCI 5 mM glucose-6-phosphate 4 mM NADP 100 mM phosphate buffer pH 7.4


The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A.. The strain of £ coli was obtained from the National Collection of Industrial Bacteria, Aberdeen, Scotland.
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored at approx. -80 °C. The different bacterial strains are checked half-yearly with regard to their respective biotin, histidine and/or tryptophan requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled as described (2, 3).

Assay procedure

The mutation test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The mutation experiment also assessed the toxicity of the test substance by evaluation of the bacterial lawn in order to select a suitable range of dose levels for a second mutation test. The highest concentration was usually 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 pg/plate were used.
A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
If the total number of concentrations selected for evaluation in the plate incorporation test does not allow for a statement of genotoxicity of the test substance to be made, an additional plate incorporation test, based on the toxicity results of the first test, has to be performed. If negative or equivocal results obtained, a second mutation experiment was performed on the basis of toxicity results in the plate incorporation test as a preincubation test.

For mutagenicity testing top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coli histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:

0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound suspension (suspended in DMSO)

After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose). After incubation for approximately 48 hours at approx. 37 deg C in the dark, colonies (his* and trp* revertants) were counted with an automatic colony counter (Artec counter Model 880). The counter was calibrated for each test by comparison of manual count data of three control plates with automatic data of the colony counter. A correction factor was determined to compensate for differences between manual and automatic count. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present.
Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.
As above.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Highest concentration 1600 µg/plate
Vehicle controls validity:
other: Historical data
Untreated negative controls validity:
not valid
Positive controls validity:
other: Historical data
Additional information on results:
Sterility checks and control plates

Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test materia! and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control.

Solubility and toxicity

The test compound was suspended in DMSO and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 ug/plate. Further dilutions of 1600, 500, 160 and 50 ug/plate were used in the mutation experiment as the plate incorporation method.

Visible precipitation of the test compound on the plates was observed at 500 ug/plate and above.

Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 ug/plate and lower doses.
The test compound proved to be not toxic to the bacterial strains in the mutagenicity experiment.


In the mutation test T-9601 was tested for mutagenicity with the same concentrations as described in section 5.3. The number of colonies per plate with each strain as well as mean values of 3 plates are given.
In the absence of the metabolic activation system the test compound induced a significant and dose-dependent increase in the number of revertant colonies with the bacterial strains TA 98, TA 100, TA 1537. In the presence of metabolic activation the test compound resulted in relevant increases in the number of revertant colonies with the Salmonella strains TA 98, TA 100, TA 1535, TA 1537. No increases of the revertants were induced at the Eschericia coli strain WP 2uvrA with and without S9-mix.

All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.


The results lead to the conclusion that T-9601 is mutagenic in these bacterial test systems with and without an exogenous metabolizing system.
Remarks on result:
other: other: preliminary test
Migrated from field 'Test system'.

Applicant's summary and conclusion

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

The results lead to the conclusion that the substance is mutagenic in these bacterial test systems with and without an exogenous metabolizing system
Executive summary:

Study data conducted to EEC-Guideline B.13 & B14 of the Directive 92/68/EEC, OECD Guidleines for Testing of Chemicals 471 and US.EPA; OPPTS 870.5100 Heath Effects Test Guidelines in compliance with GLP.

The results lead to the conclusion that the susbstance is mutagenic in the bacterial systems with and without an exogenous metoblizing system. However it should be noted that the specific method according to Privall - Mitchell for azo dyes was not utilised in this study; hence the results may be due to redundancies within the method employed. This statement is further enforced by the negative mutagenicity results observed in the other in vitro studies conducted.