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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay (Ames test). In addition, the test substance was negative in an in vitro HPRT assay in Chinese hamster ovary (CHO) cells, as well as in an in vitro micronucleus test in Chinese hamster V79 cells. Based on these studies, 2-hydroxy-N,N-dimethylpropanamide is considered not to be mutagenic.

Link to relevant study records

Reference

Reference 1

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

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

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

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.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

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.

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'.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO (Chinese hamster ovary) cell line is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.

Stocks of the CHO cell line (1-mL portions) are maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.
During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

1st experiment
150, 300, 600, 1200 µg/mL (4-h exposure, with and without S9 mix)

2nd experiment
150, 300, 600, 1200 µg/mL (24-h exposure, without S9 mix)
200, 400, 800, 1200 µg/mL (4-h exposure, with S9 mix)

Vehicle / solvent:

Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20-24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression time (cells in growth medium): The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with at least 20 mL Ham's F12 medium incl. 10% (v/v) FCS and left to stand in the incubator for about 3 days (4-hour treatment) or 2 days (24-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).
- Selection time (if incubation with a selection agent): For selection of the mutants, six 75 cm2 flasks with 3x10^5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT: 6-thioguanine

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

NUMBER OF CELLS EVALUATED: The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10^6 cells seeded.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should be within the historical negative control data range of 0.00 -16.43 mutants per 10^6 clonable cells.
- The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies.
- At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
- Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and the historical negative control data range.
- Evidence of the reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.

Isolated increases of mutant frequencies above the historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within the historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
In the pretest for toxicity based on the purity and the molecular weight of the test substance 1200 μg/mL (approx. 10 mM) Agnique AMD 3L was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. 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 culture medium no test substance precipitation occurred up to the highest applied concentration in the absence and presence of S9 mix. Under all test conditions, in the absence and presence of S9 mix, no cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed up to the highest applied concentration.

MUTANT FREQUENCY
No relevant increase in the number of mutant colonies was observed with or without S9 mix. In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.92 – 11.58 per 10^6 cells) were close to the respective vehicle control values (MFcorr.: 0.92 – 5.77 per 10^6 cells) and clearly within the range of the historical negative control data (without S9 mix: MFcorr.: 0.00 – 16.43 per 10^6 cells; with S9 mix: MFcorr.: 0.00 – 15.83 per 10^6 cells).
In the 1st Experiment with metabolic activation a statistically significant dose-related increase in the mutant frequency was found in cells after 4 hours of treatment (MFcorr.: 0.92 - 11.58 per 10^6 cells). However, the values obtained for the corrected mutation frequency of this experimental part were well within the historical negative control data range (MFcorr.: 0.00 – 15.83 per 10^6 cells). Therefore, this finding has to be regarded as biologically irrelevant.
The positive control substances EMS (without S9 mix; 300 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 153.20 – 462.82 per 10^6 cells; with S9 mix: MFcorr.: 380.58 – 549.85 per 10^6 cells) were clearly within the historical positive control data range (without S9 mix: MFcorr.: 47.35 – 1338.10 per 10^6 cells; with S9 mix: MFcorr.: 131.35 – 1250.00 per 10^6 cells).

CYTOTOXICITY
No cytotoxic effects (no clearly reduced cloning efficiencies of about or below 20% of the respective negative control values) were observed in all experimental parts under all test conditions up to the highest required concentration.

CELL MORPHOLOGY
There were no adverse observations on cell morphology (cell attachment).

TREATMENT CONDITIONS
Osmolarity and pH values were not influenced by test substance treatment.
In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest required test substance concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-05-18 to 2009-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

deionised water because the solubility properties of the test item

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene diamine; 4-NOPD

Remarks:

TA 1537, TA 98 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 102 without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2-AA)

Remarks:

TA 1535, TA 1537, TA 98, TA 100, TA 102 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation test (Experiment I); pre-incubation test (Experiment II)

DURATION
- Preincubation period: 60 min
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 per dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding thethreshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biolgically relevant.

Statistics:

According g to the OECD guideline 471, a statistical analysis of the data is not mandatory and was not performed.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: yes
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: yes; same as described for Experiment I;

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation test in bacteria (Ames test)

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed according to OECD Guideline 471 and EU method B13/14 in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/ Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the inductions factor of 0.5), occured in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with inceasing concentraions in the range below the generally acknowledged border of biological relevance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshift in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

In vitro mammalian cell gene mutation test (HPRT assay)

 

The potential of the test substance to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells was assessed in vitro in a GLP compliant study according to OECD guideline 476 (BASF SE, 2013). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and beta-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity acutally found in the main experiments, the following doses were tested: first experiment 0, 150, 300, 600 and 1200 µg/mL (4 -hour exposure, with and without S9 mix); second experiment: 0, 150, 300, 600 and 1200 µg/mL (24 -hour exposure without S9 mix) and 0, 200, 400, 800 and 1200 µg/mL (4 -hour exposure with S9 mix). Following attachment of the cells for 20-24 hours, cells were treated with the test substance and subsequently cultured for 6 -8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethylmethanesulphonate (EMS) and 7,12-dimethylbenzanthracene (DMBA), led to the expected increase in the frequencies of forward mutations. In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations.

 

In vitro mammalian cell micronucleus test

 

The potential of the test substance to induce micronuclei in Chinese hamster V79 cells in vitro (clastogenic or aneugenic activity) was assessed in a GLP compliant study according to OECD guideline 487 (BASF 2013). Two independent valid experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses (which was performed for the HPRT test with the test substance), the following doses were tested in the first experiment: 0, 37.5; 75; 150; 300; 600 and 1 200 μg/mL (4 hours exposure, 24 hours harvest time, with and without S9 mix). The doses tested in the second experiment were: 0, 75; 150; 300; 600 and 1 200 μg/mL (24 hours exposure, 24 hours harvest time, without S9 mix) and 0, 150; 300; 600; 900 and 1 200 μg/mL (4 hours exposure, 24 hours harvest time, with S9 mix). The vehicle controls and the highest three or four concentrations were evaluated. A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group. The negative controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, ethylmethanesulphonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) was observed only in the first valid experiment at the highest test substance concentration (1 200 μg/mL) after 4 hours treatment in the presence of metabolic activation. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Short description of key information:
The test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay (Ames test). In addition, the test substance was negative in an in vitro HPRT assay in Chinese hamster ovary (CHO) cells, as well as in an in vitro micronucleus test in Chinese hamster V79 cells. Based on these studies, 2-hydroxy-N,N-dimethylpropanamide is considered not to be mutagenic.

Endpoint Conclusion:

Justification for classification or non-classification

Based on the results of the Ames, the HPRT, and the micronucleus test, the substance does not need to be classified according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.