2,2'-(m-tolylimino)diethanol

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

92/69/EEC

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

2,2'-(m-tolylimino)diethanol

EC Number:

202-114-8

EC Name:

2,2'-(m-tolylimino)diethanol

Cas Number:

91-99-6

Molecular formula:

C11H17NO2

IUPAC Name:

2-[(2-hydroxyethyl)(3-methylphenyl)amino]ethan-1-ol

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.