Amines, C12-14-alkyl, isooctyl phosphates

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:

25.11.1999 to 06.12.1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japan Ministry of Agriculture, Forestry and Fisheries (1985) Notifcation of Director General, Agricultural Production Bureau. NohSan No. 4200

Deviations:

not specified

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Joint Directives of J EPA, J MHW and J MITI (31 October 1997) Kanpoan No.287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C12-14-alkyl, isooctyl phosphates

EC Number:

269-119-5

EC Name:

Amines, C12-14-alkyl, isooctyl phosphates

Cas Number:

68187-67-7

Molecular formula:

C19H42NPO4 - C29H63NPO4

IUPAC Name:

Amines, C12-14-tert-alkyl, isooctyl mono phosphates

Test material form:

liquid

Method

Target gene:

Histidine for Salmonella. Tryptophan for 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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver, S9

Test concentrations with justification for top dose:

Test 1: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Test 2: :0, 5, 15, 50, 150, and 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solubility of the test item was assessed at 50mg/l in acetone, in which it was dissloved.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide

Remarks:

Without S9. : Sodium Azide 5μg/plate for TA1535 and TA100. 9-Aminoacridine 30μg/plate for TA1537. 2-Nitrofluorene 1μg/plate for TA98. 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide 0.05μg/plate for WP2uvrA/pKM101

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

with S9: 2-Aminoanthracene 2μg/plate for TA1535 and 10μg/plate for WP2uvrA/pKM101. Benzopyrene 5μg/plate for TA1537, TA98 and TA100

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 72 hrs


NUMBER OF REPLICATIONS: Triplicate plating.

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:

Evaluation Criteria:
The mutagenic activity was assessed by applying the following criteria:
1. If treatment with the test item produces an increase in revertant colony numbers of at least twice the concurrent control, with some evidence of a positive dose relationship, in two experiments, with any bacterial strain either in the presence or absence of S9 mix, the test item will be considered to show evidence of mutagenic activity in this test system..
2.If treatment with the test item does not produce reproducible increases of at least 1.5 times the concurrent control in either mutation test, the test item will be considered to show no evidence of mutagenic activity in this test system.

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:

not applicable

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:

not applicable

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:

not applicable

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:

not applicable

Positive controls validity:

valid

Test results 5

Key result

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:

not applicable

Positive controls validity:

valid

Additional information on results:

The absence of colonies on sterility check plates confirmed the absence of microbial contamination.

The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.

The mean revertant colony counts for the solvent controls were within 99% confidence limits of the current historical control range for the laboratory. Appropriate positive controls induced substantial increases in revertant colony numbers with all strains confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

First Test (Range finding) - There were no substantial increases in revertant colony numbers over control counts in any of the tester strains following exposure to the test substance at any concentration in either the presence or absence of S9 mix.
Toxicity observed as a thinning of the background lawn of non revertant cells and reduced revertant colony counts, ocurred in all strains following exposure to the the test substance at concentrations of 500µg/plate and above.
A top concentration of 500µg/plate was therefore selected for use in the second test.

Second Test - There were no substantial increases in revertant colony numbers over control counts in any of the tester strains following exposure to the test substance at any concentration in either the presence or absence of S9 mix.
Toxicity observed as a thinning of the background lawn of non revertant cells and reduced revertant colony counts, ocurred in all strains following exposure to the the test substance at concentrations of 500µg/plate and above.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

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 MITI, MHW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", EEC Annex to Directive 92/69/EEC Part B Method For Determination OfToxicity B13 and B14 and the USA, EPA (TSCA) OPPTS harmonized guidelines.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA/pKM101 were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The dose range for the first test (range-finding) ranged between 5 and 5000µg/plate. The second test used a pre-incubation method and a dose range 5 to 500µg/plate.

 

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 test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies in the absence and presenc of S9-mix at concentrations of 500µg/plate and above.

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 or exposure method.

Conclusion

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