Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 October 1999 - 5 November 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
92/69/EEC
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical appearance: light-brown solid
- Storage conditions: at room temperature

Method

Target gene:
Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Liver S9-mix from rats induced with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Doses for the plate incorporation assay were determined based on the solubility of the test item at 5000 µg/plate. If no limited solubility was observed, this dose was used as the top dose with addition of at least five additional doses.
- Plate incorporation assay (all strains, with and without S9): 16, 50, 160, 500, 1600 and 5000 µg/plate
Doses in the preincubation assay were determined based on the results of the first assay.
- Preincubation assay (all strains, with and without S9): 16, 50, 160, 500, 1600 and 5000 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Remarks:
Sufficient data was available in the literature and from own experience, indicating that this solvent has no influence on the spontaneous mutant count of the used strains.
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: nitrofurantoin (for TA100, without S9), 4-nitro-1,2-phenylene diamine (for TA1537 and TA98, without S9); 2-aminoanthracene (all strains, with S9)
Remarks:
Positive controls were dissolved in DSMO
Details on test system and experimental conditions:
Two individual experiments were performed, one plate incorporation assay and one preincubation assay to confirm the results of the plate incorporation assay.

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes (only applicable to the second experiment)
- Exposure duration: 48 hours (in both experiments)

NUMBER OF REPLICATIONS: one plate with S9 and one plate without S9 per concentration

METHODS:
Plate incorporation: 0.1 mL of the test item, 0.1 mL tester strain (bacteria), 0.5 mL S9 mix or buffer and 2.0 mL soft agar were added to a petri dish with solid agar after 30 seconds in a waterbath (45 °C). The plates were incubated at 37 °C for 48 hours.
Preincubation: 0.1 mL tester strain (bacteria), 0.1 mL of the test item and 0.5 mL S9 mix or buffer was preincubated for 20 minutes in a 37 °C water bath. After the preincubation, 2.0 mL soft agar was added and the solutions were added to plates with solid agar. The plates were incubated at 37 °C for 48 hours.

DETERMINATION OF CYTOTOXICITY
- Method: colony counting was performed by an automatic counter.
Cytotoxicity was determined by assessing the background growth, determination of a dose-dependent reduction in the mutant count per plate and titer determination.
- Other: the dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased five-fold to permit the complete growth of the bacteria.

Evaluation criteria:
EVALUATION CRITERIA:
- A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
- For TA1535, TA100 and TA98 this increase should be about twice that of negative controls, whereas for TA1537, at least a threefold increase should be reached. For TA102, an increase of about 100 mutants should be reached.
- If otherwise, the result is considered to be negative.


ACCEPTABILITY CRITERIA:
- The negative controls have to be within the expected range, as defined by published data and/or the laboratories' own historical data.
- The positive controls have to show sufficient effects, as defined by the laboratories' experience.
- Titer determinations have to demonstrate sufficient bacterial density in the suspension.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the preincubation test at 5000 µg/plate only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In both experiments, at 5000 µg/plate only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In both experiments, at 5000 µg/plate only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In both experiments at and above 1600 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- None of the five strains showed a dose-related and biologically relevant increase in mutant counts compared to the negative controls in the plate incorporation test, both with and without S9. These results were confirmed in the preincubation assay.
- No precipitation was observed in any of the strains, at any dose level, with and without S9.
- The positive controls showed relevant increases in mutant counts showing that the test system functioned properly.

Applicant's summary and conclusion

Conclusions:
Based on the results of this AMES test, in which no biologically relevant effects of the test item on five Salmonella strains were observed compared to the negative controls, N,N-Bis-(hydroxyethyl)-m-toluidine was determined to be non-mutagenic.
Executive summary:

An Ames test was performed, according to OECD guideline 471 and GLP principles, to assess the mutagenic potential of N,N-Bis-(hydroxyethyl)-m-toluidine in five Salmonella strains (TA1535, TA1537, TA98, TA100 and TA102). Two individual experiments were carried out, a plate incorporation assay and a preincubation assay, in which the test item was tested up to a concentration of 5000 µg/plate in the absence and in the presence of metabolic activation (S9 -mix). Toxicity of the test item on the bacterial strains was observed only at the highest doses. No increases in mutant counts compared to the solvent controls were seen. The results of the positive controls were within the historical data range of the test facility and showed that the test system functioned properly. Based on these results, N,N-Bis-(hydroxyethyl)-m-toluidine, was determined to be non-mutagenic.