N-[3-(dimethylamino)propyl]stearamide monoacetate

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:

From 2014-11-25 To 2015-01-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]stearamide monoacetate

EC Number:

236-291-8

EC Name:

N-[3-(dimethylamino)propyl]stearamide monoacetate

Cas Number:

13282-70-7

Molecular formula:

C23H48N2O.C2H4O2

IUPAC Name:

N-[3-(dimethylamino)propyl]stearamide monoacetate

Method

Target gene:

Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) prepared from male Sprague-Dawley rats induced with Aroclor 1254 was purchased commercially (Moltox Inc., Boone, North Carolina, U.S.A.). The S9 protein content was: 41.0, 42.1, and 40.1 mg/mL for lots 3315, 3327, and 3331, respectively.
The S9 was thawed and the 10% S9 mix prepared immediately prior to its use. The S9 mix was held on ice at all times.
The S9 mix contained proportionate volumes of the following components:

Molecular-grade water = 2.4 mL
0.825 M KCl/0.2 M MgCl2 = 0.4 mL
0.2 M phosphate buffer, pH 7.4 = 5.0 mL
0.25 M glucose-6-phosphate = 0.2 mL
0.04 M NADP = 1.0 mL
S9 = 1.0 mL
Total Volume = 10 mL

The sham mix was 100 mM phosphate buffer at pH 7.4.

Test concentrations with justification for top dose:

In the initial toxicity-mutation test (trial T-1) the dose levels tested were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. Tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA were tested in both the absence and presence of S9 metabolic activation. The negative control values for tester strain TA98 were outside of the acceptable limits.
In the second toxicity-mutation test (trial T-2) the dose levels tested were 5000, 3333, 1000, 667, 333, 100, 66.7, 33.3, 10, 6.67, 3.33 μg/plate with tester strain TA98 in both the absence and presence of S9 metabolic activation; and 66.7, 33.3, 10, 6.67, 3.33 μg/plate for tester strains TA100, TA1535, and TA1537 in the absence of S9 metabolic activation. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of
S9 metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:sterile water (CAS 7732-18-5, molecular grade, Mediatech Inc.)

- Justification for percentage of solvent in the final culture medium: Sterile water was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in water at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile water (CAS 7732-18-5, molecular grade, Mediatech Inc.)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene (CAS 613-13-8, Moltox Inc.)

Details on test system and experimental conditions:

The tester strains were the Salmonella typhimurium histidine auxotroph tester strains TA98, TA100, TA1535, and TA1537, and the Escherichia coli tryptophan auxotroph WP2uvrA.(1,2,3) All tester strains were obtained from Moltox Inc. (Boone, North Carolina, U.S.A.). These strains were selected due to their ability to revert to histidine and tryptophan independence when exposed to a mutagen.
In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagens. A mutation of either the uvrA or uvrB gene results in a deficient DNA excision repair system. Since the uvrB deletion extends through the bio gene, the Salmonella typhimurium tester strains also require the vitamin biotin for growth.
The Salmonella typhimurium tester strains also contain the rfa wall mutation which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide (LPS) barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded by a normal intact cell wall.
Tester strains TA98 and TA100 also contain the pKM101 plasmid, which further increases the sensitivity of these strains to some mutagens.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independent (prototrophy) by frameshift mutagens. Tester strain TA100 is reverted by both frame shift and base substitution mutagens. Tester strains TA1535 and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.

Rationale for test conditions:

The tester strain cultures must exhibit a characteristic mean number of spontaneous revertants per plate when plated along with the negative (vehicle) control under selective conditions.

Evaluation criteria:

A minimum of 3 non-toxic scorable dose levels are required to validate the study. A dose level is considered toxic if it causes:
• A >50% reduction in the mean number of revertants per plate relative to the mean negative control value and exhibits a dose-dependent drop in the revertant count, or
• A reduction in the background lawn.
In the event that less than 3 non-toxic dose levels are achieved, the affected portion of the test
will be repeated with an appropriate change in dose levels.

Data will be judged positive if the increase in mean revertants at the highest numerical dose response is  3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
Data sets will be judged positive if the increase in mean revertants at the highest numerical dose response is  2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.
A data set may be judged equivocal if there is a biologically relevant increased response that only partially meets criteria for a positive response. A response will be evaluated as negative if it is neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

with metabolic activation 667 μg/plate without metabolic activation 1000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

The positive control that was used with tester stain TA98 in the presence of S9 activation was past the supplier’s expiration date; however, the positive control performed as expected with values consistent with the historical control data.

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

The maximum dose prepared was 3333 mg/mL and a 100 μL plating aliquot was used.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation.

Any other information on results incl. tables

Trial T-2

 

Tester Strain	Metabolic Activation	Precipitation starting at (μg/plate)	Toxicity starting at (μg/plate)   >50 reduction in revertant colonies	Toxicity starting at (μg/plate)   Background lawn reduction
TA98	- +	1000 667	66.7 667	66.7 33.3
TA100	-	None*	667	3.3.3
TA1535	-	None*	None*	66.7
TA1537	-	None*	None*	33.3

* None observed at the dose levels tested.

 

 

Dose levels used in T-2 Test.

 

Tester Strain	Metabolic Activation	Dose levels (μg/plate)
TA98	- +	100, 66.7, 33.3, 10, 6.67, 3.33 333, 100, 66.7, 33.3, 10
TA100	- +	100, 66.7, 33.3, 10, 6.67, 3.33 333, 100, 66.7, 33.3, 10
TA1535	- +	100, 66.7, 33.3, 10, 6.67, 3.33 333, 100, 66.7, 33.3, 10
TA1537	- +	100, 66.7, 33.3, 10, 6.67, 3.33 333, 100, 66.7, 33.3, 10
WP2uvrA	- +	1000, 667, 333, 100, 66.7 3333, 1000, 667, 333, 100

 

 

Mutagenicity Test

 

Tester Strain	Metabolic Activation	Precipitation starting at (μg/plate)	Toxicity starting at (μg/plate)   >50 reduction in revertant colonies	Toxicity starting at (μg/plate)   Background lawn reduction
TA98	- +	None* None*	100 333	100 333
TA100	- +	None* None*	66.7 333	66.7 None*
TA1535	- +	None* None*	100 333	66.7 333
TA1537	- +	None* None*	None* None*	66.7 333
WP2uvrA	- +	667 667	None* 3333	667 1000

* None observed at the dose levels tested.

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met. Under the conditions of this study, H-31339 showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Executive summary:

The test substance, H-31339, was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9).

The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance.

Sterile water was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in water at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The toxicity-mutation test was conducted in two trials. The plate incorporation method was employed.

In the initial toxicity-mutation test (trial T-1) the dose levels tested were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. Tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA were tested in both the absence and presence of S9 metabolic activation. The negative control values for tester strain TA98 were outside of the acceptable limits; therefore, the data collected for this strain in trial T-1 were considered invalid. The invalid data is maintained with the study records but not included in this final report. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation.

In the second toxicity-mutation test (trial T-2) the dose levels tested were 5000, 3333, 1000, 667, 333, 100, 66.7, 33.3, 10, 6.67, 3.33 μg/plate with tester strain TA98 in both the absence and presence of S9 metabolic activation; and 66.7, 33.3, 10, 6.67, 3.33 μg/plate for tester strains TA100, TA1535, and TA1537 in the absence of S9 metabolic activation. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation.

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test varied depending on the tester strain and metabolic activation. The maximum dose prepared was 3333 mg/mL and a 100 μL plating aliquot was used.

In the mutagenicity test the plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation.

All criteria for a valid study were met. Under the conditions of this study, H-31339 showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.