4-(methylamino)pentane-2-ol dibenzoate

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:

48h

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 "Bacterial Reverse Mutation Test"
EU-Guideline B. 13/14 adopted 31. May 2008 "Mutagenicity - Reverse mutation test using bacteria"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Kaiser-Friedrich-Str. 7, D-55116 Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(methylamino)pentane-2-ol dibenzoate

EC Number:

807-124-1

Cas Number:

1519029-23-2

Molecular formula:

C20H23NO3

IUPAC Name:

4-(methylamino)pentane-2-ol dibenzoate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

4-8methylamino)pentan-2-ol dibenzoate, CAS-No.: 15 19029-23-2, monoconstituent substance, stable at room temperature, solid, soluble in DMSO
batch No. STCB/226/14, purity: 96.06%, production date: Jun. 2014

Method

Target gene:

Histidine deficiency: hisD6610 (frame shift), present in TA97a / hisD3052 (frame shift) present in TA98 / hisG46 (base pair substitutrion) present in TA100 and TA1535 / hisG428 (base pair substitution) present in TA102
UV sensitivity, biotine deficiency: uvrB (deletion) present in TA97a, TA98, TA100 and TA1535
lipopolysaccharide side chain deficiency: rfa (deletion) present in TA97a, TA98, TA100, TA102 and TA1535
ampicillin resistance: pKM101 (plasmide) present in TA97a, TA98, TA100 and TA102
tetracyclin resistance: pAQ1 (plasmide) present in TA102

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: hisD6610, uvrB, rfa, pKM101

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: hisD3052, uvrB, rfa, pKM101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: hisG46, uvrB, rfa, pKM101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: hisG428, rfa, pKM101, pAQ1

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: hisG46, uvrB, rfa

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (from livers of male Sprague-Dawley rats treated with Aroclor), obtained by Trinova BioChem, Gießen

Test concentrations with justification for top dose:

first experiment (plate incorporation method): 1500 / 500 / 150 / 50 / 15 µg/plate
second experiment (pre-incubation method): 1500 / 750 / 375 / 188 / 94 / 47 µg/plate
top dose is not cytotoxic

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

solvent: DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

A genotype confirmation was performed. Also a toxicity control (maximal dose on maximal agar) ans sterility control (without bacteria) were perfomed too.

Details on test system and experimental conditions:

2 experiments were perfomed. Per strain and dose, 4 plates with and without S9-mix were used.
Spontaneous revertants were detected in 4 replicates (with or without S9 mix) for each solvent used in the test.
In the first experiment the plate incorporation method was used: All materials (100µl test solution at each dose level, negative or positive control + 500 µl S9 mix or phosphate buffer + 100 µl bacteria suspension + 2000 µl overlay agar) were gently vortexed in a test tube and then poured onto the selective agar plates.
The second experiment was performed in the pre-incubation method. All test materials (100 µl test solutions, 500 µl S9 mix or phosphate buffer and 100 µl bacteria suspension) were vortexed and incubated for 20 minutes in selective test tubes. Then the test tubes were poured onto agar plates and 2000 µl overlay agar was added.
Then all the agar plates were closed and placed in the dark incubator at 37°C for 48h. The colonies were counted visually.

Rationale for test conditions:

according to guideline

Evaluation criteria:

The genotype of the bacteria was confirmed. The highest dose of the test item showed no signs of toxicity towards the bacteria. The sterility controls and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. The second experiment confirmed result of the first experiment. So the study was considered valid.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor =/> 2) in one strain can be observed. Also a concentration-related increase can be taken as a sign for mutagenicity.

Statistics:

The colonies were counted visually and the numbers were recorded. A spreadsheet software (Microsoft Excel) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor (f) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

not mutagenic under the conditions of this test