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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:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four tester strains tested; no tester strain to detect cross-linking mutagens was included.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
None

Method

Target gene:
Histidine for Salmonella
Species / strain
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
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver post mitochondrial supernatant (S9 fraction)
Test concentrations with justification for top dose:
- Confirmatory experiment: 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
The starin cultures were stored as stock cultures in ampoules with nutrient broth +5 % DMSO in liquid nitrogen.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without Metabolic activation - strains TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene, 4-NOPD
Remarks:
WIthout metabolic activation - Strains TA 1537, TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA
Remarks:
With metabolic activation - Starins TA 1535, TA 1537, TA 98, TA 100
Details on test system and experimental conditions:
Selection Agar:
The plates with the minimal agar were obtained.
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
-corresponding background growth on both negative control and test plates.
-normal range of spontaneous reversion rates.

Range of spontaneous reversion frequencies
1535 1537 98 100
10-29 5-28 15-57 77-189

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for atleast one test concentration in induced. A test article producing neither a dose related and reproducible increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non mutagenic in this system. A biologically relevant response is described as follows:
A test article is considered mutagenic if the number of reversions is atleast twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537.

Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.
Statistics:
The colonies were counted using the AUTOCOUNT. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to the intense colour of the test article the colonies were counted manually from 2500 µg/plate upto 5000 µg/plate.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The generally accepted conditions for the evaluation of the results are:

- corresponding background growth on both negative control and test plates

- normal range of spontaneous reversion rates.

 

Range of spontaneous reversion frequencies • (3)

1535

1537

98

100

10 - 29

5-28

15-57

77 - 189

  

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced. A test article producing neither a dose related and reproducible increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A biologically relevant response is described as follows:

A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not. No toxic effects evident as a reduction in the number of revertants occurred in any of the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

In experiment I a dose dependent increase in revertant colony numbers exceeding the recommended threshold for a mutagenic response was observed in the absence of metabolic activation in strains TA 1537 (recommended factor 3.0, cf. page 17), TA 98 (recommended factor 2.0) and TA 100 (recommended factor 2.0). [n the presence of metabolic activation only strain TA reached this threshold. A dose dependent increase was also observed in strains TA 1535 (without S9 rnix) and TA 1537 (with S9 mix) but the recommended factor of 3.0 was not reached. In experiment II a dose dependent increase in revertant colony numbers was observed in strains TA 1535, TA 1537 (with and without S9 mix), TA 98 (without S9 mix) and TA 100 (with and without S9 mix). In the absence of metabolic activation, the threshold for a mutagenic response (factor 3.0 in strains TA 1535 and TA 1537 and 2.0 in strains TA 98 and TA 100) was exceeded in all strains used, whereas, in the presence of metabolic activation the recommended factor could only be reached in strain TA 100.

 

In both experiments a dose dependent and reproducible increase in revertant colony numbers was observed following treatment with Lanasol Blue 3R with and without S9 mix in all strains used. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

 

Applicant's summary and conclusion

Conclusions:
FAT 40069 induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 98, TA 100, TA 1535 and TA 1537.
Executive summary:

The study was performed to investigate the potential of Lanasol Blue 3R to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate

No toxic effects, evidenced by a reduction in the number of revertants, occured in the test groups with and without metabolic activation. The plates incubated with the test article showed normal background growth upto 5000.0 µg/plate with and without S9 mix in all strains used. In both experiments, a dose dependent and reproducible increase in revertant colony numbers was observed following treatment with the test article in the absence of metabolic activation in all strains used. In the presence of metabolic activation a relevant increase of the factor exceeding the threshold of 2.0 occured in strain TA 100 exclusively. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenecity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98 and TA 100. Therefore, FAT 40069 is considered to be mutagenic in the Salmonella typhimurium reverse mutation assay.