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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
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-conducted study performed prior to GLP regulations. Documentation is less complete than current practice but sufficient for the study to be used and rated. Test method used is similar to current guideline.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
Study performed prior to GLP regulations
Type of assay:
bacterial forward mutation assay

Test material

Test material form:
other: clear, colorless liquid


Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9 induced with Aroclor 1245
Test concentrations with justification for top dose:
0, 0.8, 4, 20, 100, 500 µg/plate
Vehicle / solvent:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine, 4-aminobiphenyl
Details on test system and experimental conditions:
The Salmonella typhimurillin mutants used in this study, viz. S. typh. TA 1535, TA 1531, TA 1538, TA 98 and TA 100, a gift or Dr. B.N. Ames, Berkeley,California, USA, are stored as frozen cultures at - 80 °C. To obtain fresh cultures for mutagenesis testing, nutrient broth is inoculated with the frozen culture and grown up overnight with shaking at 37 °C. The reversion properties of each strain are regularly checked, using the mutagens: MMS; MNNG; 9-AA and 4-ABP. In addition, the strains are checked for histidine requirement, for sensitivity to crystal violet, deoxycholate and for resistance to ampicillin.

The procedures used in this mutagenic assay are described in detail by Ames et al. (1975). Briefly, the procedure was as follows: to 2.5 ml molten soft agar vere added 0.1 ml of a fully grown culture of one of the tester strains, 0.1 ml of an appropriate dilution/suspension of the test compound and the liver microsome system if indicated. The ingredients were thoroughly mixed and immediately poured onto minimal glucose agar plates. After the top agar had been allowed to harden, the plates vere incubated at 37 °C for three days. Then the colonies (revertants vhich are histidine-independent) were counted, and the background lawn of bacterial growth examined. Based on the results of preliminary toxicity tests, the test materials were examined at levels up to 1000 µg/plate except for TMPH which was tested at levels up to 500 µg/plate. All determinations were carried out in triplicate and appropriate controls were included in each assay.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
at 100 and 500 µg/plate
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Additional information on results:
Background lawn of bacterial growth less dense than in control plates.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

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

Not mutagenic
Executive summary:

The mutagenic activity of THPH was examined in the Salmonella/ microsome mutagenicity test, using a set of five histidine requiring mutants of Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and liver homogenate of Aroclor induced rats.

Incorporation of TMPH up to non-inhibitory levels did not induce an increase of the reversion rate to his+ prototrophy with any of the five tester strains. At higher levels, chemical toxicity interfered with the mutagenicity testing. It was concluded that the present results did not reveal any mutagenic activity of TMPH in the Salmonella/microsome mutagenicity test.