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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998-08-13 to 1998-09-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study conducted with the read-across substance (please refer to read-across statement).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
including MITI, MHW, MOL and MAFF
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
other: viscous liquid
Details on test material:
- Molecular formula (if other than submission substance): C28H62NO4P (for a representative structure: Phosphoric acid, di(C8)ester, compds with C12 amine)
- Molecular weight (if other than submission substance): 507.76
- Smiles notation (if other than submission substance): CCCCCCCCCCCCN.O=P(O)(OCCCCCCCC)OCCCCCCCC
- InChl (if other than submission substance): InChI=1/C12H27N/c1-2-3-4-5-6-7-8-9-10-11-12-13/h2-13H2,1H3
- Substance type: organic (Alkylamine salt of alkyl phosphoric acid)
- Physical state: extremely pale straw coloured viscous liquid
- Analytical purity: 91%
- Composition of test material, percentage of components: Data relating to the identity, purity and stability of the test material are the responsibility of the Sponsor
- Storage condition of test material: room temperature in the dark

Method

Target gene:
his- (Salmonella typhimurium)
trp- (Escherichia coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient broth
- Properly maintained: yes
Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB mutation and the spontaneous reversion rate.
Additional strain / cell type characteristics:
other: uvrB- and rfa mutations
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient broth
- Properly maintained: yes
Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrA mutation and the spontaneous reversion rate.
Additional strain / cell type characteristics:
other: uvrA- mutations
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 (500 mg/kg) induced rat liver S9 mix
Test concentrations with justification for top dose:
Preliminary toxicity study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Main test: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (without metabolic activation)
Main test: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone.
- Justification for choice of solvent/vehicle: the test material is well-soluble in acetone
Controls
Untreated negative controls:
yes
Remarks:
concurrent untreated controls
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 (2AA) (tested only with S9 mix): at 1 µg/plate for TA100, at 2 µg/plate for TA1535 and TA1537, at 10 µg/plate for WP2uvrA-.
Remarks:
ENNG: 3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA-; 9AA: 80 µg/plate for TA1537; 4NQO: 0.2 µg/plate for TA98; BP: 5 µg/plate for TA98 (tested only with S9 mix).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter .

DURATION
- Exposure duration: 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: growth of the bacterial background lawn.

Preliminary Toxicity Study:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. A mixture of 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test material formulation and a vehicle control (acetone) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto sterile Vogel-Bonner Minimal agar plates in order to assess the sterility of the test material. After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.


Evaluation criteria:
The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett’s method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.
Statistics:
Dunnett’s method of linear regression

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially at 500 and 1500 µg/plate without and with S9-mix, respectively. The test material was, therefore, tested up to either 5000 µg/plate or its toxic limit depending on tester strain type and presence or absence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
initially at 500 and 1500 µg/plate without and with S9-mix, respectively. The test material was, therefore, tested up to either 5000 µg/plate or its toxic limit depending on tester strain type and presence or absence of S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:An oily precipitate was observed at 5000 pg/pIate, this did not prevent the scoring of revertant colonies.


RANGE-FINDING/SCREENING STUDIES: The test material was initially toxic at 500 µg/plate in tester strain TA100 and 5000 µg/plate in E. coli strain WP2uvrA-. The number of revertant colonies for the toxicity assay is presented below ("Any other information on results incl. tables").

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, a history profile of vehicle and positive control values is presented in the study report.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Study

The number of revertant colonies for the toxicity assay were:

With ( + ) or without (-) S9-mix Strain Dose (µg/plate)
0 0.15 0.5 1.5 5 15 50 150 500 1500 5000
+ TA100 126 138 130 120 125 134 121 113 105 83S OTP
- TA 100 130 118 133 134 134 128 112 142 119S 105V OTP
+ WP2uvrA- 21 12 15 17 22 31 25 15 17 22 12SP
- WP2uvrA- 26 25 24 18 22 14 28 17 14 5 14VP

S = sparse background lawn;

V = very weak background lawn;

T = toxic, no background lawn;

P = precipitate.

Mutation Study

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

The test material caused a visible reduction in the growth of the bacterial lawn of all the tester strains, initially at 500 and 1500 µg/plate without and with S9-mix respectively. The test material was, therefore, tested up to either the maximum recommended dose of 5000 µg/plate or its toxic limit depending on tester strain type and presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation (tables are attached as pdf file to this endpoint study record). 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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate depending on bacterial tester strain type and presence or absence of S9-mix. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Extra dose levels were included in both experiments to allow for the toxicity of the test material and to ensure there were a minimum of four non-toxic doses plated out.

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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial lawn of all the tester strains, initially at 500 and 1500 µg/plate without and with %-mix respectively. The test material was, therefore, tested up to either the maximum recommended dose of 5000 µg/plate or its toxic limit depending on tester strain type and presence or absence of S9-mix. An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.