N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine

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:

Experimental start date: 6 September 2016 Experimental completion date: 03 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

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:

N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine

EC Number:

930-859-5

Molecular formula:

C23-25H49-53NO3

IUPAC Name:

N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine

Test material form:

solid

Remarks:

Amber solid

Specific details on test material used for the study:

Physical state/Appearance: Amber coloured gelatinous solid
Batch: E00268-014-212
Purity: 100%
Expiry Date: 10 June 2018
Storage Conditions: Room temperature in the dark

Method

Target gene:

The purpose of the study was to evaluate EXP1505486 for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1758279 10/20) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal preparation (S9-mix) prepared from rats pre treated with a mixture known to induce an elevated level of these enzymes.

Test concentrations with justification for top dose:

Experiment 1
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
TA1537 (without S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
TA100 and TA1535 (without S9-mix) and TA1537 (with S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
TA100 and TA1535 (with S9-mix) and TA98 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

In solubility checks performed in house, the test item was noted to be insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully soluble in acetone at 100 mg/mL and dimethyl formamide at 50 mg/mL. Acetone was selected as the vehicle.

Controls

Controls 1

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

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Direct acting compounds in the absence of S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Remarks:

Indirect acting compounds in the presence of S9-mix

Details on test system and experimental conditions:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

Rationale for test conditions:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Evaluation criteria:

All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are as follows
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
A reproducible increase at one or more concentrations.
Biological relevance against in-house historical control ranges.
Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Following Sponsor confirmation, statistical analysis was excluded from the data set.

Results and discussion

Test results

Test results 1

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EXP1505486 was considered to be non-mutagenic under the conditions of this test.

Any other information on results incl. tables

Please see attached tables:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report. 

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2. 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) and in the absence of S9-mix, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains initially from 150 µg/plate (TA100, TA1535 and TA1537) and 500 µg/plate (TA98). In the presence of S9-mix reduced growth of the bacterial background lawns were noted at and above 500 µg/plate to all of the Salmonella strains. No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix. Consequently, the toxic limit or the maximum recommended dose level (5000 µg/plate) of the test item was employed in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix. In the second mutation test (pre-incubation method), the test item induced an identical toxic response with reduced growth of the bacterial background lawns noted from 150 µg/plate (absence of

S9-mix) and 500 µg/plate (presence S9-mix). No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix. A test item precipitate (particulate and greasy in appearance) was noted at and above 500 µg /plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). 

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 ¿g/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.05 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.

Eight test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Results

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) and in the absence of S9-mix, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains initially from 150 µg/plate (TA100, TA1535 and TA1537) and 500 µg/plate (TA98). In the presence of S9-mix reduced growth of the bacterial background lawns were noted at and above 500 µg/plate to all of the Salmonella strains. No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix. Consequently, the toxic limit or the maximum recommended dose level (5000 µg/plate) of the test item was employed in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix. In the second mutation test (pre-incubation method), the test item induced an identical toxic response with reduced growth of the bacterial background lawns noted from 150 µg/plate (absence of

S9-mix) and 500 µg/plate (presence S9-mix). No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix. A test item precipitate (particulate and greasy in appearance) was noted at and above 500 ¿g/plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). 

Conclusion

The test item was considered to be non-mutagenic under the conditions of this test.