NO. 701 BASE

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:

1 April 1998 - 8 May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Lot/batch No.: 6090

Method

Target gene:

Histidine gene in Salmonella typhimurium
Tryptophan gene in Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix rat liver - Aroclor 1254

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:Dimethyl formamide
- Justification for choice of solvent/vehicle: not indicated

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Dimethyl formamide

Controls 2

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9-mix

Migrated to IUCLID6: 3 µg/plate for TA100; 5µg/plate TA 1535; 2 µg/plate for WP2uvrA

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Migrated to IUCLID6: 80 µg/plate for TA 1537

Controls 4

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix

Migrated to IUCLID6: 0.2µg/plate for TA98

Controls 5

Positive controls:

other: non-mutagenic in the absence of metabolic enzymes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9-mix

Migrated to IUCLID6: 5 µg/plate for TA98

Controls 6

Positive controls:

other: non-mutagenic in the absence of metabolic enzymes

Positive control substance:

other: 2-aminoanthracene, 1 µg/plate for TA100; 2 µg/plate for TA 1535 and TA 1537; 10µg/plate for WP2uvrA

Remarks:

with S9-mix

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 5000 µg/plate
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 1^9 - 9.9^9 cells/ml

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER:
precipitation of the test substance

Evaluation criteria:

The test substance should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) Significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

an amber-coloured precipitate was observed at 5000 µg/plate that did not prevent the scoring of revertant colonies

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

an amber-coloured precipitate was observed at 5000 µg/plate that did not prevent the scoring of revertant colonies

Additional information on results:

No toxicity was exhibited to any of the strains of bacteria used. An amber-coloured precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation.

All positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

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

The test substance was considered to be non-mutagenic under the conditions of this test.