Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Salmonella typhimurium strains TA 1535, TA 1537, TA100 and TA 98 and Escherichia coli were exposed to upto 500µgper plate of the test substance. negative results were obtained with TA1535, TA1537, TA98 and E.coli; equivocal results were obtained with TA100.
Link to relevant study records
Reference
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.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
May 1983
Qualifier:
according to
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 information:
Composition 1
Target gene:
five histidine-requiring strains
Species / strain:
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- DMSO
Solvent 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 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

Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
500 ug upwards
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Precipitation of the test material occurred at 500 µg per plate and above. No toxicity to the bacteria was observed.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

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 mutagenic activity 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 500µg 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.

The test material was also tested for clastogenic potential in human peripheral blood lymphocyte cultures with and without metabolic activation. The maximum tolerated concentration achieved in the testing was 100 µg/ml for 5 hour treatment (with and without metabolic activation) and 40µg/ml for 25 hours treatment; precipitation of the test compound was noted above 62.5µg/ml but was not considered to adversely affect the study. No evidence was found that the test substance induced structural or numerical aberrations in this assay.

It was concluded that the substance was not clastogenic when tested for such effects in vitro with human peripheral blood lymphocytes.

The test substance was evaluated for mutagenic activity in anin vitromammalian cell gene mutation test with L5178Y mouse lymphoma cells. It was tested up to concentrations of 52 µg/ml in the absence and presence of S9-mix with an incubation time of 3 hours. Relative total growth (RTG) was reduced to 12% and 32% in the absence and presence of S9-mix, respectively. The substance precipitated in the culture medium at this dose level. The spontaneous mutation frequencies in the solvent-treated control cultures and the observed mutation frequencies of the positive control substances were within the acceptability criteria defined for this assay. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence and presence of S9-mix, the test substance did not induce a significant increase in the mutation frequency. It is concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

The test substance has been evaluated in three in vitro test systems: bacterial reverse mutation assay; human peripheral blood lymphocyte chromosome aberration assay and mouse lymphoma forward mutation assay. All the results obtained were negative with the exception of equivocal results obtained with the TA100 strain in the reverse mutation assay which were deemed to be artefactual in nature.

No further in vivo testing is mandated according to the integrated testing strategy and no classification is justified on the basis of the in vitro study results.