Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

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:

21 November 2013 - 20 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

EC Number:

939-981-3

IUPAC Name:

Reaction products of triethanolamine esters of polyphosphoric acids with alkyl derivatives of pyridine

Method

Target gene:

Histidine biosynthetic pathway - Salmonella
Tryptophan pathway - E.coli

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:

Lot No. PB/betaNF S9 20 October 2013 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/beta-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1 g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

S9 – 5 mL
1.65 M KCl/0.4 M MgCl2 – 1 mL
0.1 M Glucose-6-phosphate – 2.5 mL
0.1 MNADP – 2 mL
0.2 M Sodium phosphate buffer (pH 7.4) – 25 mL
Sterile distilled water – 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of the experiment. The amino acid supplemented top agar and the S9-mix used in the experiment was shown to be sterile.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Test concentrations with justification for top dose:

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.

Vehicle / solvent:

Dimethyl Sulphoxide (DMSO) due to immiscibiliy of substance in water

Controls

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

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hr

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- The plates were viewed microscopically for evidence of thinning

Evaluation criteria:

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:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. 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. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al., 1989). - MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Increases in colony frequency at 5 and 150 µg/plate were in excess of the in-house historical vehicle/untreated control range of the tester strain with a maximum increase of 5. 7-fold increase over vehicle control at 150 µg/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From the 500 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Historical Control Data

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From the 500 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Historical Control Data

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From the 500 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Historical Control Data

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From the 500 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Historical Control Data

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From the 500 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Historical Control Data

Applicant's summary and conclusion

Conclusions:

This substance is classified at mutagenic due to a positive results in the TA98 strain with metabolic activation.

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, MHL W 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 TAl00 and Escherichia coli strain WP2uvr A were treated with the test item using the Ames plate incorporation method 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 was predetermined and was 1.5 to 5000 µg/plate.

The second experiment was not performed because the OECD 471 test guideline permits non-repetition of the experiment when a clear, positive response is obtained in the first experiment.

Results

The vehicle ( dimethyl sulphoxide) 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. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 µg/plate with and without metabolic activation. A brown color was noted at and above 1500 µg/plate, however, no test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

The test item induced dose-related and statistically significant increases in the frequency of TA98 revertant colonies (presence of metabolic activation only) at sub-toxic test item dose levels. The increases in colony frequency observed between 5 and 150 µg/plate were very large and in excess of the in-house historical vehicle/untreated control range of the tester strain with a maximum increase of 5. 7-fold noted over the concurrent vehicle control at 150 µg/plate. Smaller responses were also noted for the remaining Salmonella strains, however these responses were not clearly dose-related. Therefore, the test item was considered to be mutagenic under the conditions of the test.