4-[3-(1-naphthylamino)propyl]morpholine

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

4-[3-(1-naphthylamino)propyl]morpholine

EC Number:

226-033-2

EC Name:

4-[3-(1-naphthylamino)propyl]morpholine

Cas Number:

5235-82-5

Molecular formula:

C17H22N2O

IUPAC Name:

N-[3-(morpholin-4-yl)propyl]naphthalen-1-amine

Test material form:

solid: granular

Details on test material:

Colour: Dark Brown to Black
GLP Characterized: January 21, 2016

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:

a metabolically active extract of rat liver (treated with Aroclor 1254)

Test concentrations with justification for top dose:

31.25, 62.5, 125, 250, 500 and 1000 µg 4-(3-(1-Naphthylamino)propyl)morpholine/plate both in the absence and presence (10% v/v S9 mix) of metabolic activation
Basis for top dose selection was inhibition of the bacterial lawn at 1500 and 5000 µg in the initial toxicity mutation assay.

Vehicle / solvent:

Dimethyl sulphoxide

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (613-13-8)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Negative Controls

The results of the study indicate that the revertant colony numbers for the negative controls (DMSO) in all strains were within limits of historical range.

Positive Controls

2-Aminoanthracene was used as the positive control in the presence of metabolic activation for all the tester strains during the Initial Toxicity Mutation Assay and Confirmatory Mutation Assay. Historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation.

Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were within the historical ranges .This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.

An increase in the mean number of revertants was not observed in Salmonella typhimurium tester strain TA100 (Initial Toxicity Mutation Assay and Confirmatory Mutation Assay) treated with 2-aminoanthracene in the absence of metabolic activation, but a clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Initial Toxicity Mutation Assay

Bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (two plates/concentration) both in the absence and presence of metabolic activation (5% v/v S9 mix). Precipitation was not observed up to the tested concentration of 5000 µg/plate. Complete inhibition of background lawn was observed at tested concentrations 1500 and 5000 µg/plate in all the tester strains both in absence and presence of metabolic activation. Partial inhibition of background lawn was observed at tested concentration of 500 µg/plate in all the tester strains both in absence and presence of metabolic activation. No inhibition of background lawn was observed up to the tested concentration of 150 µg/plate in all the tester strains both in absence and presence of metabolic activation. Reduction in number of revertant colonies of 44-56 % in the absence of metabolic activation in all the tester strains and 48-54 % [TA1535, TA98, TA100, and Escherichia coli WP2 uvrA (pKM101)] in the presence of metabolic activation (5% v/v S9 mix) was observed at tested concentration of 500 µg/plate. In tester strain TA1537, no appreciable reduction in revertant colonies was observed at the tested concentration of 500 µg/plate in the presence of metabolic activation (5% v/v S9 mix). Normal growth was observed up to the tested concentration of 150 µg/plate in all tester strains both in the absence and presence of metabolic activation (5% v/v S9 mix). No increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control. Hence, 1000 µg 4-(3-(1-Naphthylamino)propyl)morpholine/plate was selected as the highest concentration to be tested in the Confirmatory Mutation Assay both in the absence and presence of metabolic activation (10% v/v S9 mix).

Confirmatory Mutation Assay

In the Confirmatory Mutation Assay, bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg/plate (three plates/concentration) both in the absence and presence of metabolic activation (10% v/v S9 mix). Precipitation was not observed up to the tested concentration of 1000 µg/plate. Partial inhibition of background lawn was observed at tested concentrations of 500 and 1000 µg/plate in all the tester strains both in absence and presence of metabolic activation. No inhibition of background lawn was observed up to the tested concentration of 250 µg/plate in all the tester strains both in absence and presence of metabolic activation. Reduction in number of revertant colonies of 63-72 % in the absence of metabolic activation and 52-70 % in the presence of metabolic activation (10% v/v S9 mix) was observed at tested concentration of 1000 µg/plate in all the tester strains. Reduction in number of revertant colonies of 43-50 % in the absence of metabolic activation and 47-49 % in the presence of metabolic activation (10% v/v S9 mix) was observed at tested concentration of 500 µg/plate in all the tester strains.

Normal growth was observed up to the tested concentration of 250 µg/plate in all tester strains. No increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control.

Applicant's summary and conclusion

Conclusions:

From the results of this study, under the specified experimental conditions, 4-(3-(1-Naphthylamino)propyl)morpholine was concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).

Executive summary:

The potential of 4-(3-(1-Naphthylamino)propyl)morpholine to induce reverse mutations in Salmonella typhimurium strains TA1537, TA1535, TA98 and TA100 anda tryptophan deficient strain, Escherichia coli WP2uvrA (pKM101) was evaluated in the bacterial reverse mutation test using the pre-incubation method.

 

4-(3-(1-Naphthylamino)propyl)morpholinewas tested in the absence and presence of metabolic activation using dimethyl sulfoxide (DMSO) as the solvent. In the Initial Toxicity Mutation Assay, bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (two plates/concentration). Precipitation was not observed up to the concentration of 5000 µg/plate. Complete inhibition of background lawn was observed at the tested concentrations of 1500 and 5000 µg/plate in all the tester strains both in absence and presence of metabolic activation (5% v/vS9 mix). Partial inhibition of background lawn was observed at tested concentration of 500 µg/plate in all the tester strains both in absence and presence of metabolic activation. Normal background lawn pattern without reduction in number of revertant colonies was observed up to the tested concentration of 150 µg/plate in all the tester strains in both absence and presence of metabolic activation (5% v/vS9 mix). No increase in the number of revertant colonies was observed in any of the tester strains with the test item at any of the tested concentrationsin the absence or presence ofmetabolic activationwhen compared with the concurrent negative control. 

 

In the Confirmatory Mutation Assay, bacterial cultures were exposed to 4-(3-(1-Naphthylamino)propyl)morpholine at concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg/plate (three plates/concentration) both in the absence and presence of metabolic activation (10% v/vS9 mix). Precipitation was not observed up to the concentration of 1000 µg/plate. Partial inhibition of background lawn was observed at the tested concentrations of 500 and 1000 µg/plate in all the tester strains both in absence and presence of metabolic activation. No inhibition of background lawn was observed up to the tested concentration of 250 µg/plate in all the tester strains in both absence and presence of metabolic activation. No increase in the number of revertant colonies was observed in any of the tester strains with the test item at any of the tested concentrations in the absence or presence of metabolic activation when compared with the concurrent negative control.

 

All the values for the negative controls were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

 

The 4-(3-(1-Naphthylamino)propyl)morpholine stock concentrations 312.5, 625, 1250, 2500, 5000 and    10000 µg/mL were found to be within acceptable range of ± 15% of nominal concentrations during the Confirmatory Mutation Assay. The 0 hour concentrations of 4-(3-(1-Naphthylamino)propyl)morpholine in the dose formulation were found to be 108%, 106%, 107%, 103%, 97.4%, 96.9% of the nominal concentrations of dose level T1 (312.5 µg/mL), T2 (625 µg/mL), T3 (1250 µg/mL), T4 (2500 µg/mL), T5 (5000 µg/mL) and T6 (10000 µg/mL), respectively. The concentrations of 4-(3-(1-Naphthylamino)propyl)morpholine in the dose formulation after 4 hours were found to be 99.3% and 99.1% of the 0 hour concentrations of dose level T1 (312.5 µg/mL) and T6 (10000 µg/mL), respectively. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

 

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, 4-(3-(1-Naphthylamino)propyl)morpholine is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2uvrA (pKM101).