Propanamide, 2-hydroxy-N,N-dimethyl-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B. 49 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-N,N-dimethylpropanamide

EC Number:

609-066-0

Cas Number:

35123-06-9

Molecular formula:

C5H11NO2

IUPAC Name:

2-hydroxy-N,N-dimethylpropanamide

Details on test material:

- Name of test material (as cited in study report): Agnique AMD 3L
- Test substance No.: 11/0303-2
- Molecular weight: 117.15 g/mol
- Physical state: Liquid, yellowish, clear
- Purity: 98.6 corr. area % (see analytical report, project no. 11L00343)
- Lot/batch No.: 0007864415
- Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance solutions
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed until 6 Aug 2013 as indicated by the sponsor.
- Storage condition of test material: Room temperature; protected from light
- Stability of the test substance at room temperature in water was verified analytically (BASF study 01Y0303/11Y072)

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line is a permanent cell line derived from the Chinese hamster and has a
− high proliferation rate (doubling time of about 12 - 14 hours),
− high plating efficiency (≥ 90%),
− stable karyotype (modal number of 22 chromosomes).

Stocks of the V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for
− mycoplasma contamination,
− karyotype stability,
− plating efficiency (=colony forming ability) incl. vital staining.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

First experiment:
Tested:0; 37.5; 75; 150; 300; 600 and 1 200 μg/mL (4 hours exposure, with and without S9 mix).
Evaluated: vehicle controls and 300; 600 and 1 200 μg/mL without S9 mix, and 150; 300; 600 and 1 200 μg/mL with S9 mix.

Second experiment:
24 hours exposure, without S9 mix: tested 0; 75; 150; 300; 600 and 1 200 μg/mL, evaluated 0; 300; 600 and 1 200 μg/mL.
4 hours exposure, with S9 mix: tested 0; 150; 300; 600; 900 and 1 200 μg/mL, evaluated 0; 600; 900 and 1 200 μg/mL

Vehicle / solvent:

Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (experiment 1 with and without S9 mix, and experiment 2 with S9 mix) or 24 hours (experiment 2 without S9 mix)
- Recovery time: At the end of the exposure period, the medium was removed and the cultures were rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10% [v/v] FCS) was added and incubated at 37°C, 5% (v/v) CO2 and ≥ 90% humidity for 20 hours (experiment 1 and experiment 2 with S9 mix). In the case of continuous treatment (experiment 2 without S9 mix), the cell preparation was started directly at the end of exposure.
- Harvest time: 24 hours after start of exposure, the medium was completely removed. For hypotonic treatment, 5 mL of prewarmed 1.5% (w/v) Sodium citrate solution (37°C) was added for about 5 minutes. Then the hypotonic solution was removed and 5 mL cold fixative (ethanol:glacial acetic acid, ratio 3:1; +4°C) was added. After 5 minutes the fixative was removed and 5 mL of fresh cold fixative was added. Then the dishes were kept at room temperature for at least another 5 minutes for complete fixation.

STAIN (for cytogenetic assays): Wrights solution (modified May-Grünwald solution)

NUMBER OF REPLICATIONS: at least 2 cultures were evaluated per test group

NUMBER OF CELLS EVALUATED: at least 1000 cells per culture were evaluated for the occurrence of micronucleated cells

DETERMINATION OF CYTOTOXICITY
Proliferation index (PI):
For the assessment of test substance induced cytotoxicity the PI as measure of the proliferative activity of the cells was determined. The PI was determined in at least 1 000 cells per culture (corresponding to at least 2 000 cells per dose group) in all test groups evaluated. The number of clones (packs) containing 1, 2, 3 - 4 or 5 - 8 cells was recorded and the PI was calculated.

Relative increase in cell count (RICC):
For additional information about the cytotoxic potential of the test substance cells were seeded in flasks (2.5x10^5 cells per 25 cm2 flask) about 24 – 30 hours prior to exposure. The cells were treated similar to the slides using the same media and test substance concentrations. At the end of recovery period single cell suspensions were prepared from each test group (all test groups, except the positive controls) and the cells were counted using a cell counter. Based on these data the relative increase in cell count (RICC) was calculated.

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
- The number of cells containing micronuclei in the vehicle control was within the range of the historical negative control data.
- The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells.

Assessment criteria
A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
- The number of micronucleated cells exceeds both the value of the concurrent vehicle control and the range of the historical negative control data.

A test substance generally is considered "negative" if the following criteria are met:
- The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data.

Statistics:

The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition indicated by clearly reduced cell count (RICC 51.7%) was observed in the first valid experiment after 4 hours treatment with S9 mix at the highest applied test substance concentration. Proliferation index was not affected.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
No pretest for dose selection was performed prior to this study. Based on the observations and toxicity data of a previously performed pretest of a HPRT study (BASF project No. 50M0303/11M336) 1 200 μg/mL (approx. 10mM) Agnique AMD 3L was used as top concentration in both experiments of this cytogenetic study. In the pretest the parameters pH value and osmolarity were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no test substance precipitation in culture medium was observed up to the highest applied concentration of 1 200 μg/mL in the absence and the presence of S9 mix. Under all test conditions, in the absence and in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentration.

MICRONUCLEUS FREQUENCY
No relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system. In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.5 – 1.7% micronucleated cells) were close to the concurrent negative control values (0.8 – 1.3% micronucleated cells) and clearly within the historical negative control data range (0.1 - 1.8% micronucleated cells).
The positive control substances EMS (without S9 mix; 500 μg/mL) and CPP (with S9 mix; 2.5 μg/mL) induced statistically significantly increased micronucleus frequencies in both independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (3.0 – 10.1% micronucleated cells) was clearly above the range of the historical negative control data range (0.1 - 1.8% micronucleated cells) and within the historical positive control data range (2.3 – 26.6% micronucleated cells).

CYTOTOXICITY
In the absence and the presence of S9 mix no cytotoxicity indicated by reduced PI values was observed at the test groups scored for cytogenetic damage.
Growth inhibition indicated by clearly reduced cell counts was observed only in the first valid experiment after 4 hours treatment with S9 mix at the highest applied test substance concentration of 1 200 μg/mL (RICC 51.7%).

CELL MORPHOLOGY
Cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for the induction of micronuclei.

TREATMENT CONDITIONS
Osmolarity and pH values were not influenced by test substance treatment. No precipitation of the test substance in culture medium was observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.