[No public or meaningful name is available]

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

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.

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

Reaction mass of disodium 1-amino-4-[[3-[(2,3-dibromo-1-oxopropyl)amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and disodium 1-amino-4-[[3-[(2-bromo-1-oxoallyl)amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Molecular formula:

C26H20.85Br1.82Na1.97N3O9S2

IUPAC Name:

Reaction mass of disodium 1-amino-4-[[3-[(2,3-dibromo-1-oxopropyl)amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and disodium 1-amino-4-[[3-[(2-bromo-1-oxoallyl)amino]-2,4,6-trimethyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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.