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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:

experimental study

Adequacy of study:

key study

Study period:

Between 19 November 2009 and 23 December 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, without deviations from standard test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

Date of inspection 19/08/2008. Date of signature 11/26/2009.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: Pale amber colored liquid
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

Not required

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from the livers of male rats which were treated with 3 doses of phenobarbitone/b-naphthoflavone.

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: the test material was fully soluble in dimethyl sulphoxide at 50 mg/ml in solubility checks.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Positive controls include: Benzo(a)pyrene, N-ethyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-N-oxide, 9-aminoacridine and 2-Aminoanthracene (2AA).

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2: preincubation method.

DURATION
- Preincubation period: in experiment 1, preincubation was not conducted ; in experiment 2, the test material and bacterial cultures were preincubated for 20 minutes at 37°C.
- Exposure duration: approximately 48 hours incubation at 37°C
- Expression time (cells in growth medium): Not applicable.
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable.

SELECTION AGENT (mutation assays): Not applicable.
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable.

NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

NUMBER OF CELLS EVALUATED: 1~ 9.9X108 cells.


DETERMINATION OF CYTOTOXICITY
- Method: Not applicable.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.
- Other:

OTHER:
Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material.
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.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgment about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical methods recommended by the UKEMS.

Results and discussion

Test results

Test results 1

Key result

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect.
- Effects of osmolality: no effects.
- Evaporation from medium: no effects.
- Water solubility: no effects.
- Precipitation: no effects.


RANGE-FINDING/SCREENING STUDIES: yes.

COMPARISON WITH HISTORICAL CONTROL DATA: see attachment Appendix 3.

ADDITIONAL INFORMATION ON CYTOTOXICITY: mp cutptpxocoty observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

1. Preliminary Toxicity Test:

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

 The numbers of revertant colonies for the toxicity assay were:

 

With (+) or without (-) S9­mix	Strain	Dose (mg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	122	111	107	109	108	126	151	128	102	84	92P
+	TA100	99	98	110	102	112	117	101	101	101	88	93P
-	WP2uvrA"	30	19	20	14	20	26	25	22	22	24	25P
+	WP2uvrA"	37	32	42	47	30	32	44	23	36	35	44P

 

2. Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

A history profile of vehicle and positive control values is presented in Appendix 3.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. An oily precipitate was observed at and above 1500 μg/plate and at 5000 μg/plate, without and with S9-mix, respectively. An associated oily film was also observed at 5000 μg/plate in all strains without S9 only. These observations 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, at any dose level either with or without metabolic activation.

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:

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

Executive summary:

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of 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 (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

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 test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. An oily precipitate was observed at and above 1500 μg/plate and at 5000 μg/plate, without and with S9-mix, respectively. An associated oily film was also observed at 5000 μg/plate in all strains without S9 only. These observations 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 or exposure method.

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