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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Test substance was considered to be non-mutagenic under the conditions of the AMES test performed.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 08th to November 07th, 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 102, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Source: the strains were obtained from the University of California at Berkeley on culture discs.
- Storage: cells were stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
- Check: prior to the master strains being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvrB mutation and the spontaneous reversion rate.
- Sub-culture: overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 °C for approximately 10 hours.
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolising system
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST: 0, 50, 150, 500, 1500 and 5000 µg/plate
EXPERIMENT 1: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9
EXPERIMENT 2: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, without S9
EXPERIMENT 2: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate, with S9
Vehicle / solvent:
Test substance was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 30 minutes at room temperature on the day of each experiment.
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
Remarks:
without S9 mix
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 1,8-dihydroxyanthraquinone (DANTHRON)
Remarks:
with S9 mix
Details on test system and experimental conditions:
EXPERIMENT 1
Seven concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

EXPERIMENT 2
The second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, up to seven concentrations of test material per bacterial culture and control solutions. The dose range for each strain was modified in the second experiment to ensure that each strain was both tested up to its toxic limit and that four non-toxic dose levels were achieved.

PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. A mixture of 0.1 ml of bacterial culture (TA 100), 2 ml of molten, trace histidine supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml phosphate buffer was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Five doses of the test material and a vehicle control (dimethyl sulphoxidel were tested in duplicate. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine supplemented, top agar was overlaid onto sterile Vogel-Bonner Minimal agar plates in order to assess the sterility of test substance. After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

MICROSOMAL ENZYME FRACTION
S9 was prepared in-house from the livers of male Sprague-Dawley rats weighing ~ 200 g. These had each received a single ip. injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation. Prior to use, all batches of S9 were checked for suitability using the indirect mutagens 2AA and DANTHRON. The S9 was stored at -196 °C.
The S9-mix was prepared at 4 °C as follows: S9 5.0 ml, 1.65 M KCl/0.4 M MgCl2 1.0 ml, 0.1 M Glucose-6-phosphate 2.5 ml, 0.1 M NADPH 2.0 ml, 0.1 M NADH 2.0 ml, 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 ml and Sterile distilled water 12.5 ml.
A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine supplemented top agar was overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. The procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride with 5 ml of 1.0 mM histidine/1.0 mM biotin solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were prepared using 1.6 % Oxoid Agar Technical No.3 with Vogel-Bonner Medium E and 20 mg/ml D-glucose.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in the revertant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic dose levels.
To be considered negative, the number of revertants at each dose level should be less than twofold that of the vehicle control frequency.
Statistics:
All of the data was analysed using the statistical methods recommended by the UKEMS (5) with Dunnett’s method of linear regression used to evaluate the result.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 102, TA 98 and TA 100
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:
The test material caused a visible reduction in the growth of the bacterial background lawn and/or a reduction in the frequency of revertant colonies to all of the tester strains except TA102. The most sensitive strain to the toxicity of the test material was TA 1537 with a weakened bacterial lawn initially observed at 500 µg/plate. The sensitivity of the bacterial strains to the toxicity of the test material varied both between strains and exposures with or without metabolic activation.
No significant increases in the frequency of revertant colonies of bacteria were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was shown to be satisfactory.
Conclusions:
Non-mutagenic under the conditions of the test.
Executive summary:

Salmonella typhimurium strains TA 1535, TA 1537, TA 102, TA 98 and TA 100 were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Extra dose levels were included in both experiments to allow for the toxicity of the test material. The dose range for each strain was modified in the second experiment to ensure that each strain was both tested up to its toxic limit and that four non-toxic dose levels were achieved. The method used follows OECD guideline 471.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.

Test substance caused a visible reduction in the growth of the bacterial background lawn and/or a reduction in the frequency of revertant colonies to all of the tester strains except TA 102. The most sensitive strain to the toxicity of the test material was TA 1537 with a weakened bacterial lawn initially observed at 500 µg/plate. The sensitivity of the bacterial strains to the toxicity of the test material varied both between strains and exposures with or without metabolic activation. Test substance was tested up to the maximum recommended dose of 5000 µg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of test substance, either with or without metabolic activation.

Conclusion

Test substance was considered to be non-mutagenic under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Salmonella typhimurium strains TA 1535, TA 1537, TA 102, TA 98 and TA 100 were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The method used follows OECD guideline 471.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a reduction in the frequency of revertant colonies to all of the tester strains except TA 102. The most sensitive strain to the toxicity of the test material was TA 1537 with a weakened bacterial lawn initially observed at 500 µg/plate. The sensitivity of the bacterial strains to the toxicity of the test material varied both between strains and exposures with or without metabolic activation. The test material was tested up to the maximum recommended dose of 5000 µg/plate. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of the test.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, on the basis of the available information, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).