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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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well documented publication. Only four strains were tested (According to OECD guideline 471 at least five strains of bacteria should be used.).

Data source

Reference Type:
Salmonella mutagenicity tests: III. Results from the testing of 255 chemicals
Zeiger, E. et al.
Bibliographic source:
Environ. Mutagen. 9, Suppl. 9, 1 - 2, 4, 11 - 18, 20, 48

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
purity 98 % (supplier: Adrich)


Species / strain
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254 induced)
Test concentrations with justification for top dose:
10 - 1666 µg/plate (at least 5 doses were tested in triplicates)
Vehicle / solvent:
ethanol 95%
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: see remarks
Positive controls in absence of metabolic activation were sodium azide (TA1535 and TA 100), 9-aminoacridine (TA 1537), and 4-nitro-o-phenylenediamine (TA98). Positive control in presence of metabolic activation was 2-aminoanthracene for all strains.
Details on test system and experimental conditions:
The substance was tested in the absence of metabolic activation and with rat and hamster S-9 fractions.
The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described by [Haworth et al, 1983]* .

The preincubation assay was performed as described by Haworth et al (1983). The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37°C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel-Bonner medium [Vogel and Bonner, 1956]* . The histidine-revertant (his+) colonies arising on these plates were counted followiñg 2 days incubation at 37°C . The plates hand-counted when a precipitate was present; otherwise automatic colony counters were used.

The substance was tested initially in a toxicity assay to determine the appropriate dose range.
The toxicity assay was performed by using TA 100 or the system developed by Waleh et al [1982]***. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn.
At least five doses of the chemical were tested in triplicate. Experiments were repeated at least 1 week following the initial trial. Each chemical was tested initially at half-log doses up to a dose that elicited toxicity; subsequent trials occasionally used narrower dose increments. Chemicals that were not toxic were tested to a maximum dose of 10 mg/plate. Chemicals that were poorly soluble were tested up to a dose defined by their solubility.
A maximum of 0.05 mL solvent was added to each plate.

* Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., Zeiger, E. (1983) : Salmonella mutagenicity results for 250 chemicals. Environ Mutagen 5 [Suppl 1]:3-142.
** Vogel, H.J., Bonner, D.M. (1956): Acetylornithinase of E. coli: Partial purification and some properties. J Biol Chem 218 :97-106 .
*** Waleh, N.S., Rapport S.J., Mortelmans K. (1982): Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains. Mutat Res 97: 247-256.

Evaluation criteria:
The data was evaluated as described by Haworth et al (1983)*. An individual trial was judged
- mutagenic (+) if a dose-related increase over the corresponding solvent control was seen,
- weakly mutagenic (+W) if a low-level dose response was seen,
- questionable (?) if a dose related increase was judged insufficiently high to justify a call of "+W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen.
A chemical was judged to be
- mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials,
- questionable (?) if a reproducible increase of his+ revertants did not meet the criteria for either a "+" or "+W," or if only single doses produced an increase in his+ revertants in repeat trials.
The chemical was decoded by the chemical repository only after a determination had been made regarding its mutagenicity or nonmutagenicity.

* Haworth, S., Lawlor, T., Mortelmans, K., Speck, W., Zeiger, E. (1983) : Salmonella mutagenicity results for 250 chemicals. Environ Mutagen 5 [Suppl 1]:3-142.

Results and discussion

Test results
Species / strain:
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
Untreated negative controls validity:
not examined
Positive controls validity:
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion