Benzenemethanol, 2-[(3S)-3-[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-hydroxypropyl]-.alpha.,.alpha.-dimethyl-

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:

18 May 1999 - 17 June 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD method and in accordance with GLP. Study material is well characterized.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

May 1983

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

ICH Guidelines 1996 and 1997

GLP compliance:

yes

Remarks:

OECD Principles of Good Laboratory Practice, Statutory Instrument No. 654, 1997, ISBN 0-11-064105-1

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1S)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propanol

EC Number:

608-251-3

Cas Number:

287930-77-2

Molecular formula:

C29H28NO2Cl

IUPAC Name:

(1S)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propanol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

White powder, stored in the dark at ambient temperature

Method

Target gene:

five histidine-requiring strains

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aclor 1254-induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity.

Test concentrations with justification for top dose:

5 to 2000 µ.g per plate

Vehicle / solvent:

solvent- DMSO

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulphoxide

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene:

Remarks:

The positive control substances, except sodium azide, were dissolved in dimethylsulphoxide. The Sodium Azide was dissolved in sterile, ultra pure water.

Details on test system and experimental conditions:

The inducer was the polychlorinated biphenyl mixture, Aroclor 1254. The animals used to prepare the activation system were male Fischer 344 rats

Diluted agar (0.6% Difeo Bacto-agar, 0.6% NaCl) was sterilised by autoclaving L-histidine and biotin solutions, and L-tryptophan solutions were sterilised by filtration.

For use with S. typhimurium strains, 5 ml of sterile 1.0 mM L-histidine.HC l, 1.0 mM biotin solution was added to each 100 ml of soft agar, and for E. coli WP2uvrA, 1.0 ml of 1.35 mM L-tryptophan was added to each 100 ml agar. Each of the agars (with additions) were thoroughly mixed prior to use and maintained in a water bath at 45°C.

The tests were performed using the Direct Plate method.

A toxicity test using strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. One plate of each of the following concentrations of L-744,341 was used:

17, 50, 167, 500, 1667 and 5000 µ.g per plate.

Two independent mutation tests were conducted using 5 bacterial strains (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WI"luvrA). All the tests used the direct plate method. The dose levels used for the mutation assays and repeat assay with
E. coli, based on the results of the toxicity test, were: 5, 17, 50, 167, 500 and 1667 µ.g per plate.

In a subsequent repeat assay with TA 100 (Test 6), the dose levels were amended to: 125, 250, 500, 1000, 1500 and 2000 µ.g per plate.

Triplicate plates were prepared for each bacterial strain and dose level in both the presence and absence of S9 mix .

At the times that the experiments were conducted, each strain was tested for its resistance to ampicillin (indicating the presence of pKMlOl) and sensitivity to ultraviolet (u.v.) light and crystal violet (indicating persistence of the uvrB and rfa mutations).

Evaluation criteria:

A test was considered acceptable if for each strain:

i) the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light.

ii) at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli WP2uvrA 1-60.

iii) on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean vehicle control mutant numbers per plate.

If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.

iv) no toxicity or contamination was observed in at least 4 dose levels.


v) in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
i) for S. typhimurium strains TA 1535, TA 1537 and TA 98 and for E. coli, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.

ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the S9 mix.

iii) a reproducible effect in independent tests

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

500 ug upwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test material occurred at 500 µg per plate and above. No toxicity to the bacteria was observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that the test substance was not mutagenic to Salmonella typhimurium or Escherichia coli, when tested in dimethylsulphoxide up to the limit of its solubility in the test system

Executive summary:

No mutagenic activity was observed with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 or Escherichia coli, in either activation condition. There was an indication of mutageni cactivity with TA 100, in the presence of S9 mix in the first mutation assay and in the absence of S9mix, in the second mutation assay. However, since these results were not reproducible with a subsequent assay, it was considered to be artefactual.

Precipitation of the test material was observed at 500µ.g per plate and above.There was no toxicity to the bacteria.

It was concluded that the test substance was not mutagenic to Salmonella typhimurium or Escherichia coli, when tested in dimethylsulphoxide up to the limit of its solubility in the test system.