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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
red powder
Batch # : 6718 Fass 10/20

Method

Species / strain
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
31.3, 62.5, 125.0, 250.0, 375.0, 500.0 µg/ml Experiment I (treatment for 4 h; with and without metabolic activation)
62.5, 125.0, 250.0, 300.0, 350.0 µg/ml Experiment Ia (treatment for 4 h; with and without metabolic activation)
31.3, 62.5, 125.0, 250.0, 375.0 µg/ml Experiment II (treatment for 24 h; without metabolic activation)
Vehicle:
DMSO
Controls
Negative controls:
yes
Remarks:
medium
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
experiment I
Metabolic activation:
without
Cytotoxicity:
yes
Remarks:
at concentrations >= 250 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
no
Remarks on result:
not determinable
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
Experiment I
Metabolic activation:
with
Cytotoxicity:
yes
Remarks:
at concentrations >= 125 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
no
Remarks on result:
not determinable
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
Experiment Ia
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at concentrations >= 300 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
Experiment Ia
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at concentrations >= 250 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
Experiment II
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at concentrations >= 250 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Applicant's summary and conclusion

Conclusions:
Hydroxyethyl-2-nitro-p-toluidine did neither in the absence nor in the presence of metabolic activation induce a biological relevant increase in mutations in the mouse lymphoma thymidine kinase locus assay in L5178Y cells under the described test conditions. Hence no indication was found in this experiment that Hydroxyethyl-2-nitro-p-toluidine might cause gene or chromosomal mutations in mammalian cells. Hydroxyethyl-2-nitro-p-toluidine is thus evaluated to be non-mutagenic in this in vitro mammalian cell mutagenicity test.
Executive summary:

Hydroxyethyl-2-nitro-p-toluidine was examined for its mutagenic activity in the L5178Y TK+/- mouse lymphoma assay in the absence and presence of metabolic activation. Mutation is detected in this test system by examining the occurrence of trifluorothymidine (TFT) resistance that is caused by forward mutation at the thymidine kinase (TK) locus. The test was performed with and without metabolic activation (S9 mix from the liver of phenobarbital/ß-naphthoflavone induced male Wistar HanIbm rats). A range-finding test (pre-test on toxicity, measuring relative suspension growth) and two independent mutagenicity xperiments were carried out. Culture medium and DMSO were used as negative and solvent controls, while cyclophosphamide (CPA; 3 µg/ml) and methyl methane sulphonate (MMS; 13 µg/ml) were used as positive controls with and without metabolic activation system, respectively. In experiment I, the cells were incubated with the test item for 4 hours in the presence and absence of S9 mix. In experiment II the incubation time was 24 hours in the absence of S9 mix. Two replicate cultures were investigated for each concentration and test group. Mutant frequency and cell survival (measured as cloning efficiency) were determined in parallel. In order to determine the ratio of small versus large colonies, the colonies were evaluated with regard to size/optical density. In the initial range-finding study, concentrations of up to 10 mM (from 15 to 2000 µg/ml) with and without S9 mix were evaluated for toxicity. Toxicity was noted at 62.5 µg/ml and above in the absence of S9 mix (relative suspension growth of 60.4 to 12.0 %) and at 250 µg/ml and above (relative suspension growth 42.8 % to 4.6 %) in the presence of S9 mix (4 hours treatment). After 24 h of treatment in the absence of S9 mix, the cell growth was distinctly reduced (relative cell growth of 9.6 % to 3.7 %) at 500 µg/ml and above. Based on these findings, 6 concentrations covering a range of 3.0 to 500 µg/ml with without S9 mix were chosen for the main experiments. Five of these 6 concentrations were evaluated in each experiment. In the first experiment an increase in mutation frequency (induction factor of 2.5) in one culture without S9 was noted at a concentration which showed toxic effects (82% toxicity). However, no evidence for an increase in mutation frequency was seen in the parallel culture. It is important to note that the next higher dose of 375 ug/ml was excessively toxic (96% toxicity) and the 500 ug/ml concentration was excluded from evaluation due to severe toxicity. No effects on the mutation rate were seen in the experiment with metabolic activation. As the positive control failed to respond in the second culture (in the presence of S9-mix), the whole experiment was repeated and was reported as experiment Ia. Due to the severe toxicity observed at 500 µg/ml and the steep toxicity gradient in experiment I, the dose range was adapted and in experiment Ia by using narrowly spaced concentrations in the critical concentration range. In experiment Ia (4 hour treatment with and without S9 mix) as well as in experiment II (24 hour treatment without S9 mix) no substantial and reproducible dose dependent increase of the mutation frequency was noted with Hydroxyethyl-2-nitro-p-toluidine. Even at highly toxic doses only very slight increases were seen which remained within the historical negative control range and were considered not to be biological relevant. The ratio of small to large colonies was not affected by the treatment with Hydroxyethyl-2-nitro-p-toluidine. The positive controls demonstrated that the system was able to detect known mutagens.