Reaction product of propylidynetrimethanol, propoxylated, reaction products with ammonia and 2,2-Dimethyl-3-(4-morpholinyl)propanal

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-29 to 2014-07-23

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

(3E)-12-[(2-{2-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]propoxy}propoxy)methyl]-12-ethyl-2,2,5,8-tetramethyl-1-(morpholin-4-yl)-7,10,14-trioxa-4-azaheptadec-3-en-16-ol; 4-[(3E,20E)-12-[(2-{2-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]propoxy}propoxy)methyl]-12-ethyl-2,2,5,8,16,19,22,22-octamethyl-23-(morpholin-4-yl)-7,10,14,17-tetraoxa-4,20-diazatricosa-3,20-dien-1-yl]morpholine; 4-[(3E,20E)-12-ethyl-2,2,5,8,16,19,22,22-octamethyl-23-(morpholin-4-yl)-7,10,14,17-tetraoxa-4,20-diazatricosa-3,20-dien-1-yl]morpholine

EC Number:

700-879-7

Cas Number:

1379822-00-0

Molecular formula:

NA: UVCB substance

IUPAC Name:

(3E)-12-[(2-{2-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]propoxy}propoxy)methyl]-12-ethyl-2,2,5,8-tetramethyl-1-(morpholin-4-yl)-7,10,14-trioxa-4-azaheptadec-3-en-16-ol; 4-[(3E,20E)-12-[(2-{2-[(E)-[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino]propoxy}propoxy)methyl]-12-ethyl-2,2,5,8,16,19,22,22-octamethyl-23-(morpholin-4-yl)-7,10,14,17-tetraoxa-4,20-diazatricosa-3,20-dien-1-yl]morpholine; 4-[(3E,20E)-12-ethyl-2,2,5,8,16,19,22,22-octamethyl-23-(morpholin-4-yl)-7,10,14,17-tetraoxa-4,20-diazatricosa-3,20-dien-1-yl]morpholine

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and ß-naphtoflavone induced rat liver S9 Mix

Test concentrations with justification for top dose:

Experiment I, without S9-Mix:
1000, 1200, 1400, 1600, 1800, 2000, 2200 µg/mL
With S9-Mix:
100, 150, 200, 300, 400, 500, 600 µg/ mL

Experiment 2, without S9-Mix:
1000, 1100, 1200, 1300, 1350, 1400, 1500 µg/mL
Experiment 2, With S9 Mix:
100, 150, 200, 300, 400, 500, 600 µg/mL

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Both experiments without S9-Mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Both experiments with S9-Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours, 20 hours
- Expression time (cells in growth medium): 8 days
- Fixation time: After selection period.

SELECTION AGENT: EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL containing 3.4 μg/mL of 6-thioguanine (6-TG)).

NUMBER OF REPLICATIONS: 5

NUMBER OF CELLS EVALUATED: 200

STAIN: Giemsa

DETERMINATION OF CYTOTOXICITY
- Method: Determining the relative cloning efficiency (survival).

Evaluation criteria:

The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
• The assay is valid.
• The mutant frequency at one or more doses is significantly greater than that of the relevant control.
• Increase of the mutant frequency is reproducible.
• There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data: Mutant frequency between the negative (solvent) and the test item or positive control item treated groups.
The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See "Any other information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and Dose selection

The test item was dissolved in DMSO. A clear solution was obtained up to a concentration of 150 mg/mL. For all test item concentrations examined, no precipitation in the medium was noted. The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Main Mutation Assays (Experiments 1 and 2), both in the absence and in the presence of a metabolic activation system (rodent liver S9 mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control and results were noted as percentage of cells in relation to the negative control. The results obtained were used for dose selection of the test item in the Main Mutation Assays.

Main Experiment I

On Day 1, there was very clear evidence of toxicity with the test item in both absence and presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
In Experiment 1, in some cases the mutant frequencies of the cells were slightly above the concurrent control values. These higher values did not show biologically or statistically significant alterations compared to the concurrent control.

Main Experiment II

There was very clear evidence of toxicity with the test item in presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls and very clear evidence of toxicity in the absence of metabolic activation, confirming the response seen in the dose selection cytotoxicity assays. On the basis of Day 8 cloning efficiency data the cells had recovered during the expression period.
In Experiment 2, in some cases the mutant frequencies of the cells were slightly above the concurrent control values. These higher values did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (0.4 or 1.0 ìL/mL) and 7,12-Dimethyl benz[a]anthracene (20 ìg/mL). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.
The osmolality of test item solutions did not show any alterations compared to the concurrent control groups in Experiments 1 and Experiment 2. The pH values of test item solutions with S9 mix were similar compared to the control values in Experiments 1 and Experiment 2. The pH values of test item solutions without S9 mix were slightly above the concurrent control values in Experiments 1 and Experiment 2.

Applicant's summary and conclusion

Conclusions:

negative

The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.
Thus, the test item was not mutagenic under the conditions of this study.

Executive summary:

The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals as follows:

Experiment 1, 5-hour treatment period without S9 mix:

1000, 1200, 1400, 1600, 1800, 2000 and 2200 μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

100, 150, 200, 300, 400, 500 and 6001 μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

1000, 1100, 1200, 1300, 1350, 1400 and 1500 μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

100, 150, 200, 300, 400, 500 and 6001 μg/mL

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation, when tested up to cytotoxic concentrations. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours) up to cytotoxic concentrations. Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency even when cytotoxicity occurred.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose-response relationships were noted.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate and 7,12-Dimethyl benz[a]anthracene.

The test item tested both without and with metabolic activation, did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxic concentrations.Thus, the test item was not mutagenic under the conditions of this study.