BRUGGOLITE® FF6

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:

May 1998 - July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD 471

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

none

Method

Target gene:

Strains TA 98, TA 100, TA 1535, TA 1537 were used.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

- Type and identity of media: The nutrient medium contained 15 g per litre nutrient broth (SIFIN GmbH, Berlin)
- Properly maintained: yes
- Periodically checked for histidine dependence
- Periodically checked for cell membrane permeability
- Periodically checked for ampicillin resistance

Additional strain / cell type characteristics:

other: hisD3052, rfa, uvrB, pKM101

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: The nutrient medium contained 15 g per litre nutrient broth (SIFIN GmbH, Berlin)
- Properly maintained: yes
- Periodically checked for histidine dependence
- Periodically checked for cell membrane permeability
- Periodically checked for ampicillin resistance

Additional strain / cell type characteristics:

other: hisG46, rfa, uvrB, pKM101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

- Type and identity of media: The nutrient medium contained 15 g per litre nutrient broth (SIFIN GmbH, Berlin)
- Properly maintained: yes
- Periodically checked for histidine dependence
- Periodically checked for cell membrane permeability
- Periodically checked for ampicillin resistance

Additional strain / cell type characteristics:

other: HisG46, uvrB, rfa

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

- Type and identity of media: The nutrient medium contained 15 g per litre nutrient broth (SIFIN GmbH, Berlin)
- Properly maintained: yes
- Periodically checked for histidine dependence
- Periodically checked for cell membrane permeability
- Periodically checked for ampicillin resistance

Additional strain / cell type characteristics:

other: hisC3076, rfa, uvrB

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 metabolic activation

Test concentrations with justification for top dose:

Experiment I:
Concentration range (with metabolic activation/with S9): 0.05, 0.1, 0.5, 1.0, and 5.0 mg/plate Concentration range (without metabolic activation/without S9): 0.05, 0.1, 0.5, 1.0, and 5.0 mg/plate
Experiment II:
Concentration range (with metabolic activation/with S9): 0.05, 0.1, 0.5, 1.0, and 5.0 mg/plate
Concentration range (without metabolic activation/without S9): 0.05, 0.1, 0.5, 1.0 and 5.0 mg/plate

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation system (without S9 mix) Migrated to IUCLID6: TA 1535 and TA 100

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation system (without S9 mix) Migrated to IUCLID6: TA 1537

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation system (without S9 mix) Migrated to IUCLID6: TA 98

Controls 4

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

yes

Positive controls:

yes

Remarks:

TA 1535, TA 100, TA 1537, TA 98

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

The test substance was tested at five concentration levels of 0.05, 0.1, 0.5, 1.0 and 5.0 mg/plate.

The following were added to sterile disposable tubes containing 2 ml of top agar:
- 0.1 ml test article dilution or
- 0.1 ml of solvent (negative control) or
- 0.1 ml positive control solution
- 0.1 ml of appropriate bacterial culture
- 0.5 ml of S9 mix or 0.5 ml of 0.2 M phosphate buffer

The contents of each tube were mixed and added to a Petri dish containing minimal agar.
When the top agar had set, the Petri dishes were inverted and incubated at +3 7°C for 48 hours.
Revertant colonies were scored manually at the end of the incubation period.

NUMBER OF REPLICATIONS:
Each test variant was performed as threefold replicate.

OTHER:
All used bacterial strains are defective in DNA-repair (L\uvrB). The strains also have a defective lipopolysaccharide barrier on the cell wall (rfa). These properties confer extra sensitivity to DNA damage and also greater permeability to large molecules. The strains TA 98 and TA 100 also contain a plasmid (pKM101) which enhances the effor prone repair and confers ampicillin resistance. The strains are routinely tested for histidine dependence, cell membrane permeability and ampicillin resistance.

Evaluation criteria:

POSITIVE RESULT:
A test is considered to be positive if the test article induces dose related increases in numbers of revertants scored in two separate experiments and these increases are deemed to be of biological relevance. Reproducible increases at one experimental point may also indicate a positive response. For a biologically relevant response the number of revertants is expected to be at least double the spontaneous reversion rate in the S. typhimurium strains TA 98, TA 100. In the S. typhimurium strains TA 1535 and TA 1537 the number ofrevertants is expected to be at least the tripie of the spontaneous reversion rate.

NEGATIVE RESULT:
A test article producing neither a dose related and reproducible increase in the number of revertants nor a reproducible positive response at any experimental point is considered to be non-mutagenic in this test system.

Statistics:

STATISTICAL METHDODS USED:
The results are presented as individual plate counts. In addition mean values and standard deviations are calculated for each strain and experimental point.

PARAMETERS THAT WERE ANALYSED:
- dose related increase in revertant numbers (with and without S9 metabolic activation)
- reproducible increase at a single experimental point (with and without S9 metabolic activation)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.05, 0.1, 0.5, 1.0, 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.05, 0.1, 0.5, 1.0, 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
The revertant frequencies of the vehicle control were within the expected range and the positive control chemieals induced marked increases in revertant colonies. No biologically relevant increases in revertant numbers were obtained after treatment with Brüggolit FF6 in any bacterial strain and at any concentration tested. This applies to both, the presence and absence of metabolie activation.
See attached PDF-File "1998_07_03_02_FF6_Reverse Mutation Test_Results" (Table 2 -5)

TEST-SPECIFIC CONFOUNDING FACTORS:
none

RANGE-FINDING/SCREENING STUDIES:
The response of the untreated tester strains and the responses of the tester strains to the vehicle control, the test article at specified concentrations and the positive controls are shown in the attached PDF-File "1998_07_03_02_FF6_Reverse Mutation Test_Results" (Table 1).
At the chosen concentrations (5.0 to 0.01 mg/plate) no influence was observed to the number of spontaneous revertants and to the bacterial
backround lawn.

COMPARISON WITH HISTORICAL CONTROL DATA: In both independet experiments controls gave counts of spontaneous revertants within the normalranges obtained in this laboratory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Toxicity rangefinder (preleminary test)

Any other information on results incl. tables

Main experiment:

The responses of the untreated tester strains and the responses of the tester strains to the vehicle control, the test article at specified concentrations and the positive controls are presented in Tables 2 to 5 (two independent experiments without and with metabolic activation). All vehicle controls gave counts of spontaneous revertants within the expected ranges.

The positive controls markedly increased the number of revertant colonies in all bacterial strains with and without metabolie activation. No increase in revertant numbers which might indicate a mutagenic response was observed at any concentration of the test article and in any bacterial strain.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

the test item is considered to ben non-mutagenic in the bacterial reverse mutation test.

Executive summary:

The test item was tested for a possible potential to induee gene mutation in bacteria.

As test organisms the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 were used. The bacteria were exposed to the test item both, in the presence and absence of rat Iiver 89 metabolic activation. Two independent experiments were performed according to the standard plate incorporation method.

Toxicity Rangefinder: At the chosen concentrations of 5.0 to 0.01 mg/plate no influence was observed to the number of spontaneaous revertants and to the bacterial background lawn. Based on these results concentrations of 0.05, 0.1, 0.5, 1.0 and 5.0 mg/plate were chosen for the mutation experiment.

Mutation Experiment: No increase in revertant numbers which might indicate a mutagenic response was observed at

any concentration of the test article and in any bacterial strain. After treatment with the test article neither a dose related increase in revertant numbers nor a reproducible increase at a single experimental point was observed for any bacterial strain

tested. This applies to both, the presence and absence of rat liver S9 metabolic activation.

Based on the results of the reported study it is concluded that the test item does not induce gene mutation in Salmonella typhimurium under the experimental conditions described. Therefore, the test item is considered to be non-mutagenic in the bacterial reverse mutation test.