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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Phase: 10 August 2018 to 05 September 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Updated information. Study now available and provided ahead of extended submission date of 31/05/2019.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
There were two deviations from the study plan. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
There were two deviations from the study plan. None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates
EC Number:
271-672-2
EC Name:
Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates
Cas Number:
68603-74-7
Molecular formula:
The substance is a UVCB so there is no molecular formula
IUPAC Name:
Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates
Test material form:
solid

Method

Target gene:
Histine for Salmonella
Tryptophan for E. Coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced Sprague Dawley rat liver microsomal enzymes (S9 Homogenate).
Test concentrations with justification for top dose:
Dose Range-finder

Strains TA100 and WP2uvrA:
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence S9-mix
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the presence S9-mix
(Results of this dose-range finding test were reported as part of the first mutation assay)

First Experiment: Direct Plate Assay

Strains TA1535, TA1537 and TA98:
0.17, 0.54, 1.7, 5.4, 17 and 52 µg/plate in the absence of S9-mix
0.54, 1.7, 5.4, 17, 52 and 164 µg/plate in the presence of S9-mix.

To complete the data of the tester strains following on from severe toxicity observed in the dose range finding study, two additional concentrations (0.17 and 0.54 µg/plate) were also tested in strains TA100 and WP2uvrA in the absence and presence of S9-mix.

Second Experiment: Pre-Incubation Assay

Strains, TA1535, TA1537 and TA98:
0.083, 0.27, 0.83, 2.6, 8.2 and 26 µg/plate in the absence of S9-mix
0.83, 2.6, 8.2, 26, 80 and 250 µg/plate in the presence of S9-mix.

Strain TA100:
Without S9-mix: 0.17, 0.54, 1.7, 5.4, 17 and 52 µg/plate
With S9-mix: 0.54, 1.7, 5.4, 17, 52 and 164 µg/plate

Strain WP2uvrA:
Without S9-mix: 0.082*, 0.27*, 0.54, 1.7, 5.4, 17, 52 and 164 µg/plate
With S9-mix: 1.7, 5.4, 17, 52, 164 and 512 µg/plate

*To complete the data of tester strain WP2uvrA the dose levels, 0.083 and 0.27 µg/plate, were additionally tested.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA98
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA100
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1535
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1537
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of WP2uvrA
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA98
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA100
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1535
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of TA1537
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate of WP2uvrA
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation) for experiment 1 and preincubation for experiment 2.

DURATION
- Preincubation period: 30 ± 2 minutes (second preincubation experiment only)
- Exposure duration: 48 ± 4 hours

NUMBER OF REPLICATIONS: Triplicate for each treatment.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:
No formal hypothesis testing was done.

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.

b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.

b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be rep roducible in at least one follow up experiment

Results and discussion

Test resultsopen allclose all
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:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Information on Precipitation and Cytotoxicity

Precipitation Observed

Dose range finding test: Precipitation of the test item on the plates was observed at the start of the incubation period at a concentration of 5000 µg/plate. Precipitation of the test item on the plates was observed at the end of the incubation period at 1600 and 5000 µg/plate and 5000 µg/plate in the absence and presence of S9-mix, respectively in tester strain TA100 and at 512 µg/plate and above and 1600 and 5000 µg/plate in the absence and presence of S9-mix, respectively in tester strain WP2uvrA.

Supplemental dose range finding test conducted as part of the second mutation assay: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 512 µg/plate and no precipitate was observed at the end of the incubation period.

 

First mutation assay: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 164 µg/plate and no precipitate was observed at the end of the incubation period.

Second mutation assay: Precipitation of the test item on the plates was not observed at the start or of the incubation period. Precipitation of the test item on the plates was observed at the end of the incubation period at concentrations of 80 and 250 µg/plate.

Toxicity - First Experiment:  Direct Plate Assay

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies was observed in all tester strains, except in the additional test with the tester strains TA100 and WP2uvrA.

Toxicity - Second Experiment:  Pre-Incubation Assay

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies was observed in all tester strains, except in the additional test with tester strain WP2uvrA.

Applicant's summary and conclusion

Conclusions:
It is concluded that Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Introduction

The objective of this study was to determine the potential of Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium, and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA.The study was designed to meet the requirements of the following guidelines:

  • OECD Guideline 471. Genetic Toxicology: Bacterial Reverse Mutation Test. (Adopted July 21, 1997).
  • OEC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

 

Method

 The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. Doses for these experiments were based on the results of a dose range-finding study.

In the first mutation experiment, the test item was tested up to concentrations of 52 and 164 µg/plate in the tester strains TA1535, TA1537 and TA98 in the absence and presence of S9-mix, respectively. In addition, due to severe toxicity in the dose range finding study, two additional concentrations (0.17 and 0.54 µg/plate) were tested in tester strains TA100 and WP2uvrA in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 26 and 250 µg/plate in the tester strains TA1535, TA1537 and TA98 in the absence and presence of S9-mix, respectively, in the pre-incubation assay. In addition, since only 3 analysable concentrations where observed in tester strain WP2uvrA in the absence of S9-mix in the second dose range finding study, two additional concentrations (0.083 and 0.27 µg/plate) were tested in the second mutation assay. 

 

Results

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in either experiment.

Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Conclusion

Based on the results of this study it is concluded that Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.