Decyldimethylamine

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:

2021-01-2021 to 2021-03-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 26th June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Decyldimethylamine

EC Number:

214-302-7

EC Name:

Decyldimethylamine

Cas Number:

1120-24-7

Molecular formula:

C12H27N

IUPAC Name:

decyldimethylamine

Test material form:

liquid

Details on test material:

Chemical Name: N,N-dimethyl-decan-1-amine
CAS: 1120-24-7

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Clariant

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate
- method of preparation of S9 mix: A volume of 1 mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM Nicotinamide Adenine Dinucleotide Phosphate (NADP) disodium salt, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) of pH 7.34 for initial cytotoxicity, and pH 7.33 for plate incorporation and preincubation method to get the concentration of 10% (v/v).
- concentration or volume of S9 mix and S9 in the final culture medium: 500 µL S9 mix for direct plate incorporation method or preincubation method.
- quality controls of S9: Each batch of S9 homogenate was assessed for sterility by streaking the supernatant fluid on Nutrient Agar plates and incubated at 37±1ºC for 24 hours. It was found sterile and was further evaluated for its protein content (Modified Lowry Assay, Sword and Thomson, 1980) and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain. The results were found to be acceptable for the tested parameters.

Test concentrations with justification for top dose:

Precipitation test and initial cytotoxicity test: concentration of 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 5 µL/plate.
For plate incorporation method and preincubation method 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate were selected (with half-log dose interval) based on the results of the initial cytotoxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: based on solubility test

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate

METHOD OF TREATMENT/ EXPOSURE:
Density: Inoculum was adjusted to a density of 18×10E8 cells/mL.
- Test substance added in medium; in agar (plate incorporation); preincubation;

TREATMENT AND HARVEST SCHEDULE:
- Trial 1: Plate incorporation Method: Plate incorporation method was carried out with test concentrations of 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate of test item, vehicle, negative and positive control. The tester strains along with S9/PBS were mixed with 2 mL molten soft agar and poured on to MGA plates. Five concentrations of the test item were plated, with each of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) with and without metabolic activation. Plates were incubated at 37±1°C for 64 hours and 53 minutes.
The condition of the bacterial background lawn was evaluated for evidence of the test item cytotoxicity using the code system and revertant colonies for each strain within the test item dilution series were counted manually.
- Trial 2: Preincubation period: Preincubation method was carried out with test concentrations of 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate of the test item, vehicle, negative and positive control. The tester strains along with S9/PBS and the test constituents were transferred into sterile test tubes and incubated in an incubator shaker for 20 to 25 minutes at 37±1°C at 100±5 rpm.
- Exposure duration/duration of treatment: Post incubation, the test constituents were mixed with 2 mL molten soft agar containing histidine-biotin for Salmonella typhimurium and L-tryptophan for E.coli WP2 uvrA (pKM101) and poured on to MGA plates. Five concentrations of the test item were plated, with each of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) with and without metabolic activation. Plates were incubated at 37±1°C for 66 hours and 39 minutes.
- Harvest time after the end of treatment (sampling/recovery times): The bacterial suspension of each tester strain was diluted up to 10E-7 in PBS and 1000 µL of the diluted suspension from each tester strain was plated onto nutrient agar plates in triplicate. The plates were incubated at 37±1ºC for 64 hours and 3 minutes for plate incorporation for preincubation method. Post incubation, the number of colonies in each plate were counted manually and expressed as number of Colony Forming Units per mL (CFU/mL) of the bacterial suspension.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition. Condition of the bacterial background lawn was evaluated for evidence of the test item cytotoxicity using the code system. [0 = No lawn (absent), 1+ Very thin lawn (extremely reduced), 2+ Thin lawn (moderately reduced), 3+ Slightly thin lawn (slightly reduced), 4+ thick lawn (normal)]. Revertant colonies for each strain within the test item dilution series were counted manually.

Evaluation criteria:

The conditions necessary for determining a positive result are:
- there should be a dose related increase in the mean revertants per plate of at least that one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the limit dose tested is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.
A response will be evaluated as negative, if it is neither positive nor equivocal.

Statistics:

Values of Revertants are in Mean±SD

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at test concentrations 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate. Based on the results of initial cytotoxicity test, 0.2 µL/plate selected as highest test concentration for mutation assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

at test concentrations 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate. Based on the results of initial cytotoxicity test, 0.2 µL/plate selected as highest test concentration for mutation assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at test concentrations 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate. Based on the results of initial cytotoxicity test, 0.2 µL/plate selected as highest test concentration for mutation assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at test concentrations 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate. Based on the results of initial cytotoxicity test, 0.2 µL/plate selected as highest test concentration for mutation assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at 0.00625, 0.0125, 0.025, 0.005 and 0.1 µL/plate. Slight cytotoxicity at 0.2 µL/plate. Based on initial cytotoxicity test 0.2 µL/plate selected as highest test concentration for mutation assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

at test concentrations 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate. Based on the results of initial cytotoxicity test, 0.2 µL/plate selected as highest test concentration for mutation assay.

Applicant's summary and conclusion

Conclusions:

non-mutagenic

Executive summary:

Genamin 10 R 302 D was assayed for bacterial reverse mutation test at the concentrations of 0.002, 0.006, 0.02, 0.063 and 0.2 µL/plate for plate incorporation method and preincubation method using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains. 

The test item was tested for plate incorporation method (trail I) and for preincubation method (trail II) in the presence and absence of metabolic activation system using ethanol as vehicle and appropriate positive controls (2-nitrofluorene, sodium azide, 9-Aminoacridine and 4-nitroquinoline N-oxide for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. The trials were carried out in triplicate.

In the two trials conducted, the test item concentrations tested resulted in no appreciable increase in the number of revertant colonies over the vehicle control, while the positive controls tested simultaneously, resulted in 3.4 to 16.8 fold increase in the number of revertant colonies/plate under identical conditions.

This was observed for all five tester strains.

Based on the results of the study it is concluded that Genamin 10 R 302 D is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.2 µL/plate under the test conditions when tested on Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E.coli WP2 uvrA (pKM101) tester strains.