Hatcol 1760

Genetic toxicity: in vitro

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:

The experimental phase of this study was performed between 21 February 2008 and 12 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). Date of inspection: 21/08/2007. Date of signature: 29/04/2008.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

Sponsor's identification :Hatcol 1760
Description : clear colourless slightly viscous liquid
Date received : 04 February 2008
Storage conditions :room temperature in the dark

Method

Target gene:

Histidine operon for Salmonella
Tryptophan operon for Escherichia

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:

phenobarbitone/betanaphthaflavone

Test concentrations with justification for top dose:

Salmonella and E.coli strains (with and without S9):
Range finding test: 15, 50, 150, 500, 1500 and 5000 µg/plate
Main test: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran

- Justification for choice of solvent/vehicle: the test material was immiscible in sterile distilled water, dimethyl sulphoxide, acetone and dimethyl formamide at 50 mg/ml but was fully miscible in tetrahydrofuran at 200 mg/ml in solubility checks performed in-house.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: (ENNG)

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix

Migrated to IUCLID6: (9AA)

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix

Migrated to IUCLID6: (4NQO)

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: (BP)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 - 72 hours
- 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): 48 - 72 hours


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


NUMBER OF REPLICATIONS: triplicate plating


NUMBER OF CELLS EVALUATED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction


OTHER EXAMINATIONS:
- None

Evaluation criteria:

Acceptance criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmic r-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9E+09 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9 mix.
There should be a minimum of four non-toxic dose levels.
There should not be an excessive loss of plates due to contamination.

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 in 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, insome instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation.

Results and discussion

Test results

Test results 1

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

tested up to the recommended dose level of 5000 µg per plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

tested up to the recommended dose level of 5000 µg per plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not stated in report
- Effects of osmolality: not stated in report
- Evaporation from medium: not stated in report
- Water solubility: insoluble
- Precipitation: no
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test:
The test material exhibited light toxicity at 5000 µg/plate to TA100 in the absence of S9. However, this response was not repeated in either the range-finding or main tests and was therefore considered spurious and of no biological relevance. the test material was non-toxic to TA100 in the presence of S9 and WP2uvrA in both the presence and absence of S9. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all werefound 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 were shown to

ADDITIONAL INFORMATION ON CYTOTOXICITY: not applicable

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A light, globular precipitate was observed at and above 1500 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, 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:

Interpretation of results (migrated information):
negative

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

Executive summary:

Introduction.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 Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrA^-were treated with the test material using the Ames plate incorporation method at up to six 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 15 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test material formulations. The test material dose range was amended slightly, following the results of the range-finding test, and was 50 to 5000 µg/plate.

An additional dose level (15 µg/plate) was included in the range-finding test to allow for potential test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Results.The vehicle (tetrahydrofuran) 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. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A light, globular precipitate was observed at and above 1500 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.

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